Proc Natl Acad Sci U S A, 1976 Jan, 73(1), 109 - 12 Energy-dependent binding of dansylgalactoside to the lac carrier protein: direct binding measurements; Schuldiner S et al.; High specific activity 6'-N-{3H}dansyl)aminohexyl 1-thio-beta-D-galactopyranoside (Dns6-Gal) has been synthesized, and its binding to Escherichia coli membrane vesicles measured directly by flow dialysis . With ML 308-225 vesicles containing the lac carrier protein, specific binding is not detected in the absence of D-lactate or reduced phenazine methosulfate . In the presence of these electron donors, binding is observed, and the binding constant and number of binding sites are approximately 4 muM and 1.5 nmol/mg of membrane protein, respectively . These values are in excellent agreement with those obtained by fluorescence titration . p-Chloromercuribenzenesulfonate, which directly inactivates the lac carrier protein, and carbonylcyanide m-chlorophenylhydrazone, which collapses the membrane potential, cause release of bound Dns6-Gal . Moreover, significant binding is not observed with membrane vesicles that are devoid of the lac carrier protein . The results provide qualitative and quantitative confirmation of previous studies which indicate that changes in dansylgalactoside fluorescence observed on "energization" of membrane vesicles reflect binding of the probe to the lac carrier protein. J Gen Microbiol, 1976 Jan, 92(1), 125 - 32 Mutations in Escherichia coli that relieve catabolite repression of tryptophanase synthesis . Mutations distant from the tryptophanase gene; Yudkin MD; Two mutants are described in which the synthesis of tryptophanase is unusually insensitive to catabolite repression . Neither mutation is linked by transduction to the tryptophane structural gene, neither mutation renders the synthesis of beta-galactosidase insensitive to catabolite repression, and the mutations do not permit tryptophanase to be synthesized in strains deficient in adenyl cyclase . During growth in glucose-minimal medium the mutants maintained a similar intracellular concentration of cyclic AMP to their wild-type parent; but since in the wild type the concentration of cyclic AMP was the same in glycerol-minimal medium as in glucose-minimal medium, it is doubtful whether catabolite repression is mediated by measurable changes in the concentration of this nucleotide. J Bacteriol, 1976 Jan, 125(1), 136 - 41 Specific inhibition of phospholipid synthesis in plsA mutants of Escherichia coli; Ray TK et al.; plsA mutants of Escherichia coli are temperature-sensitive strains which possess two enzymes of abnormal thermolability, sn-glycerol 3-phosphate acyltransferase and adenylate kinase . Phospholipid synthesis is inhibited after shift of plsA mutants to temperatures at the lower end of the nonpermissive temperature range . This inhibition is not due to inactivation of the adenylate kinase activity since nucleic acid (and hence adenosine 5'-triphosphate) synthesis is inhibited only slightly . These results show that in vivo inactivation of the sn-glycerol 3-phosphate acyltransferase can be observed under conditions which allow normal adenylate kinase function. Int Arch Allergy Appl Immunol, 1976, 50(1), 95 - 102 Effect of exogenous cyclic AMP and other adenine nucleotides on neutrophil chemotaxis and motility; Rivkin I et al.; Cyclic AMP placed in the bottom of the modified Boyden chemotaxis chamber is weakly chemotactic for rabbit peritoneal neutrophils . Dibutyryl-cyclic AMP under similar conditions gives a biphasic response, being weakly chemotactic at low concentrations and inhibiting cell movement at higher concentrations . When mixed with the cells in the upper compartment of the chemotaxis chamber, cyclic AMP and dibutyryl-cyclic AMP enhance neutrophil spontaneous motility; on the other hand, dibutyryl-cyclic AMP mixed with the cells in the upper compartment consistently inhibits neutrophil chemotactic responsiveness, while cyclic AMP has a variable effect on chemotaxis . The effect of ADP and ATP was to inhibit chemotaxis and spontaneous motility . AMP is totally without effect. Physiol Chem Phys, 1976, 8(4), 349 - 87 Biological ion exchanger resins . X . The cytotonus hypothesis: biological contractility and the total regulation of cellular physiology through quantitative control of cell water; Minkoff L et al.; Actin-like (A-L) fraction from normal E . coli was compared with the protein from a potassium-transport mutant strain, and the cell-swelling reaction of both strains was studied . Findings were: (a) The membrane fraction of the mutant by SDS electrophoresis is deficient in the A-L fragment relative to normal whereas the soluble supernatant contains an excess . (b) Important catalytic differences exist between the A-L fractions of the two strains . The parent strain accumulates potassium in low K+ and the A-L fraction polymerizes in low K+ . But the A-L fraction from the mutant fails to polymerize in low K media in the K+ concentration region where the mutant fails at K+ uptake . (c) The parent cell swells during low K+ uptake whereas the mutant does not . It is constructed from this that the differences in the characterization of A-L fraction relative to normal are related to the loss of cell-swelling in the mutant and hence to the loss in alkali cation selectivity . Thus two physical mechanisms, one macroscopic and dependent on the Gregor relation for swelling equilibria in ion exchange resins, and one more microscopic based on the dielectric dependence of the coulomb force between ion pairs, could underly regulation of ion selectivity by cell swelling . A similar proposal is made for the regulation of electron transport and oxidative phosphorylation in mitochondria . These findings and interpretations justify a new hypothesis to the effect that cell hydration is regulated by contractile proteins . The hypothesis fits together important observations hitherto unexplained, to wit: (1) The "missing link" as to the role of intermediate metabolism in biological ion exchange . (2) The swelling of bacterial protoplasts and its relation to (Mg2 + Ca2+)ATPase activity . (3) The swelling-contraction cycles of mitochondria and their role in electron transport . (4) The role of ATPase's in transport . (5) The significance of actomyosin fibers in nerve endings . (6) The significance of altered actomyosin structures in the cancerous cell. Rev Ig Bacteriol Virusol Parazitol Epidemiol Pneumoftiziol Bacteriol Virusol Parazitol Epidemiol, 1976 Jan-Mar, 21(1), 29 - 32 {Possibilities of correcting some metabolic disorders in experimental endotoxic shock by treatment with a combination of alpha-lipoic acid and epsilon aminocaproic acid}; Dragomirescu M et al.; Alpha-Lipoic acid associated with xi-aminocaproic acid proves to have a protector effect in the posttreatment of endotoxinic shock in rabbits, produced by the administration of E . coli O111, in doses of 1.5 mg/kg body weight . This effect was observed when administered early in the stage of energy privation, and was manifested by a longer survival, diminution of lactacidemia and protection of labile phosphate reserves. Prep Biochem, 1976, 6(2-3), 123 - 31 Preparation of enterochelin from Escherichia coli; Young IG; A convenient method has been developed for the preparation of enterochelin, the natural iron carrier produced by Escherichia coli . The method employs a mutant strain which is unable to transport the ferric-enterochelin complex into the cell and which excretes large quantities of enterochelin into the culture medium . The addition of excess iron to the medium allows the enterochelin to accumulate as the ferric-enterochelin complex which is purified by ion-exchange chromatography and then dissociated and the free enterochelin further purified by differential extraction and crystallization . The enterochelin is isolated in good yield and appears to be of high purity as judged by a number of criteria. Arzneimittelforschung, 1976, 26(1), 89 - 93 {Spontaneous Peripheral Proteolysis/1st Communication: method and results of clinical examinations (author's transl)}; Greuer W et al.; With a modificated Astrup-fibrin plate-method also an inhibition of proteolysis can be registrated . In various medical areas a spontaneous peripheral proteolysis had been found, especially so in chronical bacterial infections. J Bacteriol, 1976 Jan, 125(1), 142 - 8 Sucrose-dependent spectinomycin-resistant mutants of Escherichia coli; Miyoshi Y et al.; Spectinomycin-resistant (Spcr) mutants of Escherichia coli were isolated from nutrient agar plates containing 20% sucrose and 100 mug of spectinomycin per ml . About one-third of the Spcr mutants thus obtained were sucrose dependent (Sucd) and were classified into two types: I, those unable to grow on sucrose-free medium in the presence of spectinomycin; and II, those unable to grow on sucrose-free medium irrespective of the presence of spectinomycin . Most of these mutants were hypersensitive to antibiotics, dyes, and detergents and were abnormal in cell morphology, suggesting changes in cell envelopes . Reversion experiments indicated that the sucrose-dependent spectinomycin resistance and hypersensitivity to various chemicals were not independently induced properties . The Sucd-Spcr mutations of type I mutants were transducible by phage P1 and were mapped at the strA-aroE region. Genetika, 1976, 12(7), 150 - 73 {Successes and prospects for genetic engineering}; Alikhanian SI; The review of literature (1970-1976) on problems of gene engineering is given . Gene engineering is pointed out to be a new method of modern biology and a new page of modern molecular genetics . Gene engineering detected a real possibility of artificial creating living hybrid organisms, i.e . constructing functional recombinant DNA molecules according to a project of investigator, but not to possibilities of crossing . The determination of gene engineering (in contrast with genetical engineering) is given in the first division of the article . Genetical engineering is a construction of hybrid organisms on the basis of recombination between non-homologous chromosomes cy crossing . Genetical engineering is based on sex crossing, thus the application of this method is restricted by crossability (i.e . experiments in vivo), which possibilities are determined by taxonomical limits . Gene engineering is a new method of operating directly with genes . It permits constructing in vitro any hybrid genomes desirable . There is no limits of combining ability for gene engineering . Three main stages of constructing hybrid genomes should be taken into account for the proper determination of gene engineering as a method of genome constructing: 1) the gene isolation; 2) their cross-linking in vitro; 3) the transfer of hybrid DNA into recipient cell or its genome . The cardinal stage of gene engineering is the construction of hybrid DNA, cross-linking any initial DNAs from any remote animals, plants and bacteria . All the methods known of gene isolation are described . The chemical method of gene isolation is based on that case, when DNA of some gene differs in its physico-chemical characteristics from total DNA, for example, DNAs of genes coding ribosomal RNAs or sea urchine histone DNA . Isolation of promotors and operators using DNA dependent RNA polymerase, which recognizes promotors, repressor and operator DNA, should also be considered as the chemical method of gene isolation . Restrictase method, which is also well known, is convenuent because the restricts have long enough sticky ends, which is important for the following gene cross-linking . The method of total restriction, reported by Lederberg et al . and Debabov et al., is described . The phage method (in particular, Shimada method) is given, permitting the direct integration of lambda phage into a number of sites of Escherichia coli chromosome . Gene engineering method of gene isolation is mentioned, in particular, the data of Kameron et al . on hybrid phages carrying DNA ligase gene, and Clark a . Carbon on hybrid plasmids carrying triptophane and arabinose operons genes . These methods are called "shot gun" . Methods of gene isolation from higher organisms are less developed . A method of gene isolation using so called colony hybridization (according to Grunstein and Hognes) is also given... Ciba Found Symp, 1976, (42), 181 - 92 The agglutinating antibody response in the duodenum of infants with enteropathogenic Escherichia coli gastroenteritis; McNeish AS; The agglutinating antibody response in duodenal fluid and serum was measured serially in 15 infants with enteropathogenic Escherichia coli gastroenteritis . Peak levels of duodenal agglutinins were recorded 8-18 days after the onset of symptoms, and the titres fell within the next 7-14 days . The immunoglobulin (Ig) class of these antibodies was mainly IgA, but IgM antibodies were detected early in the response, especially in the younger infants . Late antibodies showed less cross-reactions with other strains of E . coli than did early antibodies . Serum antibody responses were detected in eight infants, but they correlated poorly with the titres of intestinal antibodies . No rise in serum antibodies was detected in six infants . It is not known whether these differences are host-derived, or whether they are the result of the invasive properties of some of the infecting organisms. Acta Microbiol Acad Sci Hung, 1976, 23(2), 129 - 35 Immunological cross reactivity of four enzymes involved in the biosynthetic pathway of lysine, methionine and threonine in Escherichia coli K12; Truffa-Bachi P; In Escherichia coli K12 the biosynthetic pathway of lysine, methionine and threonine is characterized by three isofunctional aspartokinases and two homoserine dehydrogenases . A single polypeptide chain carries the threonine-sensitive aspartokinase and homoserine dehydrogenase (AK I-HDH I), and a different polypeptide chain carries the methionine-repressible aspartokinase and homoserine dehydrogenase (AK II-HDH II) . Immuno-adsorbants prepared with rabbit antibodies against AK I-HDH I bind the lysine-sensitive aspartokinase (AK III), the AK II-HDH II, and the homoserine kinase (HSK), an enzyme of the threonine biosynthetic pathway . Saturation of the immunoadsorbant with AK I-HDH I results in a decreased binding capacity for the other enzymes . Displacement of bound AK III or HSK can be obtained with pure AK I-HDH I, showing that the affinity of the antibodies to homologous antigens is higher than to heterologous ones . Immunoadsorbants prepared with anti-HSK antibodies show the same type of recognition: binding of the three aspartkinases and a capacity to displace the heterologous antigens bound . Accordingly, the same antibodies, implicated in the binding of the homologous antigen, bind the other enzymes . None of the other enzymes of the pathway, or the other kinases tested are recognized by the two immunoadsorbants . It can be postulated that in E . coli K12, duplication of a common ancestor gene gave rise to the three aspartokinases and to the homoserine kinase; two of the genes coding for the aspartokinases fused with those coding for the homoserine dehydrogenases . Indicating that only few epitopes are shared by these enzymes, by conventional immuno-diffusion techniques no precipitation lines appeared with antibodies against AK I-HDH I and the other proteins. Ann Med Sect Pol Acad Sci, 1976, 21(4), 185 - 96 The effect Escherichia coli lipopolysaccharide (endotoxin) on the graft-versus-host reaction in mice (IV); Skopinska E; Lipopolysaccharide from Escherichia coli (LPS) administered to mice during the graft-versus-host (GvH) reaction in a single dose of 100 mug stimulated the reaction if it was given on the same day as the parental spleen cells, or inhibited it if given four days before injection of the cells . The GvH reaction was intensified when LPS was injected in the dose of 20 mug on the fourth day after injection of the cells . The parental spleen cells responsible for the intestifying effect on GvH were sensitive to hydrocortisone, nonadherent, and probably develop in the course of the first week outside the donor spleen and settle in the spleen in a later stage of the GvH reaction. Int J Pept Protein Res, 1976, 8(6), 533 - 42 Thermal fragmentation of Escherichia coli beta-galactosidase . Isolation and characterization of an alpha-complementing and two non-complementing polypeptide fractions; Marinkovic DV et al.; Carboxymethylated Escherichia coli beta-galactosidase EC 3.2.1.23 could be broken to polypeptides of fairly uniform size (average molecular weight about 22,000 daltons) by heating for less than or equal to 8 h at 100 degrees C and pH 7.5 IN 8 M-urea . Using phosphocellulose chromatography in NaCl-urea gradients, the resulting polypeptide mixture could be resolved in three fractions essentially homogeneous by disc gel electrophoresis in urea at several pH values, and by isoelectric focusing . One of these fractions was active as alpha-donor in in vitro complementation of beta-galactosidase activity with Escherichia coli mutant M15; this activity was largely retained after CNBr cleavage . All three fractions carried arginine as carboxyl-terminal amino acid . No significant amount of any specific amino could be detected in NH2-terminal position. Biochem Soc Symp, 1976, (41), 205 - 23 Adaptations of enzymes for regulation of catalytic function; Atkinson DE; 1 . Enzymes must not only be extremely effective catalysts, but must also be the operating components of very sensitive and sophisticated regulatory systems . Appropriate evolutionary adjustment of the properties of different enzymes causes the sites that bind typical metabolic intermediates to be only partially saturated in vivo, thus allowing flexibility for control by variation in ligand concentration . In contrast, sites that bind such coupling agents as the pyridine and adenine nucleotides seem to be virtually saturated . Thus the ratios, rather than the absolute concentrations, of these compounds are important in metabolic regulation . This type of response is esstial to the function of these compounds simultaneously as thermodynamic energy transducers and modifiers in the kinetics regulatory system . 2 . Reaction orders of two to four are frequently encountered . They appear to be essenital to biochemical homoeostasis, which is the maintenance of nearly constant substrate concentrations at the expense of wide variation in flux rates . 3 . The strategies of enzyme adaptation are general, but the actual adaptations of enzymes are highly specific, reflecting the place of the enzyme in a metabolic sequence, the place of the sequence in the metabolism of the cell and, in complex organisms, the function of the cell, and of the organ or tissue of which it is a part, in the organism . Patterns of adaptation must be almost infinitely varied . A few are presently known in outline, but probably none as yet in detail . Several examples are discussed. Acta Microbiol Acad Sci Hung, 1976, 23(2), 121 - 8 Regulation of lysine biosynthesis in Escherichia coli K12; Patte JC et al.; A general survey of the regulation in lysine biosynthesis in Escherichia coli K12 is presented . No polygenic operon exists for the genes that code for enzymes of the lysine biosynthetic pathway . Lysyl-tRNA is not directly involved as a co-repressor in the pathway . Different regulation mechanisms must exist for the different enzymes . In the case of the last enzyme, diaminopimelate decarboxylase, its synthesis is induced in vivo by the lysine-sensitive aspartokinase under its non-inhibited allosteric conformation. Acta Biol Med Ger, 1976, 35(3-4), 285 - 99 {Intracellular protein breakdown . VII . Cathepsin L and H; two new proteinases from rat liver lysosomes}; Kirschke H et al.; Some properties (molecular weight, pI, temperature stability, action of selected inhibitors, substrate specificity and pH-activity dependence) of two not yet known cathepsins from rat liver lysosomes are compared with the properties of the known cathepsin B1 . Cathepsin L is a thiolproteinase, has a molecular weight of 23--24000 and a pI of 5,8--6,1 . By disc electrophoresis and isoelectric focusing there appear several protein bands which all have enzymatic activity . Leupeptin behaves as a strong inhibitor . The pH-optimum for digestion of proteins is close to 5,0 . Cathepsin L does not hydrolyse esters and splits synthetic low molecular substrates only to a low degree . Cathepsin L stored in presence of glutathion and EDTA in liquid nitrogen kept its activity for some months . Cathepsin H is an aminopeptidase as well as an endopeptidase . An enzyme with these bifunctional properties was detected up to now only in E . coli but not in animal cells . Cathepsin H is a thiol-enzyme with a molecular weight of 28000 and a pI of 7,1 . Strong inhibitors are leucyl-chlormethan and SH-blocking substances . Leupeptin shows only a weak inhibitory effect to this enzyme compared to its action on cathepsins L and B1 . The pH-optimum for hydrolysis of all substrates is 6.0 . Cathepsin H splits proteins, amino acid derivatives and selected N-protected amino acid derivatives . Cathepsin H compared to cathepsin L and B1 is quite temperature stable. Zh Mikrobiol Epidemiol Immunobiol, 1976, (3), 45 - 50 {Resistance to levomycetin and activity of several enzymes in Escherichia coli and the agent of plague}; Korobeinik NV et al.; The authors compared the activity of acetyl-CoA-synthetase and of the enzymes belonging to the group of asparaginic acid in levomycetin sensitive and resistant strains of Y . pestis and E . coli . There were revealed marked differences in the activity of aspartase, fumarase, synthetase and desamidase of L-asparagin, and also of the enzyme activated by acetate in the E . coli strains with plasmide resistance . Transmission of R-factor to the pestis was accompanied by decomposition of L-asparadein, formation of AC-CoA . At the same time transformation of L-asparaginic acid catalyzed by aspartase remained on the same low level in the sensitive pestis cultures and their variants with the R-factor . When the resistance was controlled by chromosomal resistance markers, the activity of the enzymes providing formation of L-asparagic acid, its amide and L-malic acid showed no significant change . In chromosomal type of resistance in the mutants of pestis and E . coli the acetyl-CoA-synthetase reaction was as a rule somewhat increased. Biochimie, 1976, 58(1-2), 35 - 49 Mechanistic studies of glutamine synthetase from Escherichia coli . An integrated mechanism for biosynthesis, transferase, ATPase reaction; Rhee SG et al.; The mechanism of biosynthetic, transferase, ATPase, and transphosphorylation reactions catalyzed by unadenylylated glutamine synthetase from E . coli was studied . Activation complex(es) involved in the biosynthetic reaction are produced in the presence of either Mg2+ or Mn2+ ; however, with the Mn2+-enzyme inhibition by the product, ADP, is so great that the overall forward biosynthetic reaction cannot be detected with the known assay methods . Binding studies show that substrates (except for NH3 and NH2OH which are not reported here) can bind to the enzyme in a random manner and that binding of the ATP-glutamate, ADP-Pi or ADP-arsenate pairs is strongly synergistic . Inhibition and binding studies show that the same binding site is utilized for glutamate and glutamine in biosynthetic and transferase reactions, respectively, and that a common nucleotide binding site is used for all reactions studied . Studies of the reverse biosynthetic reaction and results of fluorescent titration experiments suggest that both arsenate and orthophosphate bind at a site which overlaps the gamma-phosphate site of nucleoside triphosphate . In the reverse biosynthetic and transferase reactions, ATP serves as a substrate for the Mn2+-enzyme but not for the Mg2+-enzyme . The ATP supported transferase activity of Mn2+-enzyme is probably facilitated by the generation of ADP through ATP hydrolysis . When AMP was the only nucleotide substrate added, it was converted to ATP with concomitant formation of two equivalents of glutamate, under the reverse biosynthetic reaction conditions, and no ADP was detected . The reversibility of 180 transfer between orthophosphate and gamma-acyl group of glutamate was confirmed . ATPase activity of Mg2+ and Mn2+ unadenylylated enzymes is about the same . Both enzymes forms catalyze transphosphorylation reactions between various purine nucleoside triphosphates and nucleoside diphosphates under biosynthetic reaction conditions . The data are consistent with the hypothesis that a single active center is utilized for all reactions studied . Two stepwise mecanisms that could explain the results are discussed. Biochimie, 1976, 58(1-2), 213 - 8 Role of lysyl-tRNA in the regulation of lysine biosynthesis in Escherichia coli K12; Boy E et al.; A mutant of lysyl-tRNA synthetase has been isolated in Escherichia coli K12 . With this strain the Kmapp for lysine is 25 fold higher than with the parental strain . The percentage of charged tRNAlys in vivo is only 7 per cent (as against 65 per cent with HFR H) . Under these conditions no derepression of synthesis is observed for three lysine biosynthetic enzymes (AK III, ASA-dehydrogenase, DAP-decarboxylase) ; a partial derepression is obtained in the case of the dhdp-reductase . Thus lysyl-tRNA does not act as the only corepressor molecule in the lysine regulon. Biokhimiia, 1976 Jan, 41(1), 149 - 52 {Relationship between the magnitude of Km and pH for L-asparaginase}; Libinson GS et al.; The dependency of L-asparagine hydrolysis rate on L-asparagine concentration in the presence of L-asparaginases from E . coli and Erw . carotovora is studied in a broad pH range . Km values are calculated from the data obtained . It is found that Km insignificantly depends on pH value with the pH range of 5-9 for both asparaginases . Sharp Km maximum is observed at pH greater than 9 in both cases . The maximum position does not coinside with enzyme isoelectric points and with the region of the substrate transition from zwitterionic form into anionic one. Proc Soc Exp Biol Med, 1976 Jan, 151(1), 28 - 31 Myocardial substrate utilization in anaphylactic shock; Spitzer JJ; Changes in myocardial substrate utilization were studied in anesthetized dogs following the production of anaphylactic shock . Mean arterial blood pressure, cardiac output, and pH decreased significantly during this form of shock . Myocardial FFA oxidation was greatly diminished especially within the first hour following challenge and lactate uptake more than doubled during the same time . Thus, it is concluded that myocardial substrate utilization shifted away from FFA and towards lactate during anaphylactic shock and these changes resembled those observed following an acute, severe hemorrhage, or the administration of E . coli endotoxin. J Bacteriol, 1976 Jan, 125(1), 324 - 31 Purification and properties of beta-N-acetylglucosaminidase from Escherichia coli; Yem DW et al.; beta-N-acetylglucosaminidase (EC 3.2.1.30) has been purified from Escherichia coli K-12 to near homogeneity based on polyacrylamide gel electrophoresis in both 0.5% sodium dodecyl sulfate and in 6 M urea at pH 8.5 . The purified enzyme shows a pH optimum of 7.7 and the Km for p-nitrophenyl-beta-D-2-acetamido-2-deoxyglucopyranoside is 0.43 mM . The molecular weight of this enzyme, determined by both Sephadex gel filtration and by sodium dodecyl sulfate gel electrophoresis, is equivalent to 36,000 . It is shown to be a soluble cytoplasmic enzyme . Studies on the substrate specificites of the purified enzyme indicate that this enzyme is an exo-beta-N-acetylglucosaminidase. J Bacteriol, 1976 Jan, 125(1), 19 - 24 Microcalorimetric study of the anaerobic growth of Escherichia coli: measurements of the affinity of whole cells for various energy substrates; Belaich A et al.; Microcalorimetry has been used to determine the affinity of whole cells of Escherichia coli for glucose, galactose, fructose, and lactose . Anaerobic growth thermograms were analyzed, and the Km and Vmax values for these energy substrates were measured at pH 7.8 . Results obtained with this technique using various organisms growing anaerobically on different sugars are compared . This comparison shows that in practically all cases the cellular rate of catabolic activity is a hyperbolic function of the energy substrate concentrations at low sugar concentrations . In some cases this technique also allows determination of kinetics at high sugar concentrations. Biochemistry, 1975 Dec 30, 14(26), 5548 - 53 Endonuclease II of Escherichia coli: DNA reacted with 7-bromomethyl-12-methylbenz{alpha}anthracene as a substrate; Kirtikar DM et al.; An endonuclease II preparation from Escherichia coli makes single strand breaks in DNA which has been treated with the carcinogen 7-bromomethyl-12-methylbenz{alpha}anthracene . In addition, the enzyme preparation excises N6-(12-methylbenz{alpha}anthracenyl-7-methyl)adenine and N2-(12-methylbenz{alpha}anthracenyl-7-methyl)guanine residues from the DNA . These are relased as the modified purine bases, not as purine nucleoside derivatives . The rate of release of the adenine derivative is three to four times that of the guanine derivative. Ann N Y Acad Sci, 1975 Dec 30, 264, 358 - 72 Binding proteins and membrane transport; Oxender DL et al.; The recent studies have clearly established two types of active transport systems . One type is membrane-bound and can be observed in membrane vesicles and the other type is osmotic-shock-sensitive and requires binding proteins to produce active transport . It appears that the membrane-bound systems derive cellular energy from the energy-rich membrane state which can be formed from respiration or ATP-hydrolysis, while the binding protein systems are more directly coupled to phosphate bond energy derived from glycolysis or oxidative phosphorylation . The following conclusions concerning the role of the binding proteins are offered: 1 . The binding proteins are present in relatively large amounts (approximately 10(-6) or 10%-5) M) and appear to reside in the periplasmic space . 2 . They do not appear to be involved in solute translocation steps, although they cantain a second binding site that could interact with membrane components . 3 . The binding proteins appear to increase the affinity of the transport system for the solute by interacting with a membrane component . This may substrate for the membrane transport system. Mol Gen Genet, 1975 Dec 30, 142(3), 193 - 208 Localization of Escherichia coli ribosomal protein S4 on the surface of the 30S ribosomal subunit by immuno electron microscopy . I . Distribution of antibody-binding sites as obtained with immunoglobulins and monovalent antibody fragments from various S4-specific antisera; Tischendorf GW et al.; The location of the ribosomal protein S4 on the surface of the 30S subunit of E . coli ribosomes was determined by immuno electron microscopy . Immunoglobulins from six separate S4-specific antisera were investigated . In accordance with earlier findings protein S4 was shown to have an elongated conformation within the antisera . Protein S4 has therefore definitely an extended or fibrous shape in the intact ribosome (Lake et al., 1974; Stoffler and Tischendorf, 1975; Tischendorf et al., 1975) . S4 specific antibodies bind at four distinct sites of 30S ribosomal subunits, designated A, B, C and D . Two antibody binding sites (A and B) are located on the "head", they were shown to be separated by 70-85 A . The distance between these sites and the two sites C and D on the "body" of the subunit amounts to at least 90-125 A; hence protein S4 should be extended to a total length of approximately 160-200 A . At least three of the four S4-specific antibody binding sites were observed with antibodies from each of the six investigated . These sites were observed independently of whether isolated S4 protein, an S4-16S rRNA complex or 30S ribosomes were used as the antigen . They could also be visualized with monovalent antibody fragments (Fab) . S4-specific antibodies enriched by affinity chromatography bound at identical sites which conclusively ensures that antibody binding at these sites is specific for protein S4 and is not due to the presence of contaminating antibodies in the intact ribosome . The implications of these results with respect of the correlation of each of the sites to their respective antigenically active fragments within protein S4 are discussed. Mol Gen Genet, 1975 Dec 30, 143(1), 85 - 91 Location on the chromosome of Escherichia coli of genes governing purine metabolism . Adenosine deaminase (add), guanosine kinase (gsk) and hypoxanthine phosphoribosyltransferase (hpt); Jochimsen B et al.; Genes coding for enzymes functioning in purine salvage pathways have been located on the chromosome of Escherichia coli . The gene add encoding adenosine deaminase was located by transduction at 31 min, the gene order was established to be man-uidA-add-aroD . A deletion covering man-uidA-add was obtained . The gene gsk encoding guanosine kinase was cotransducible with purE and shown to be located at 13 min . The gene hpt encoding hypoxanthine phosphoribosyltransferase was cotransducible with tonA indicating a location at 3 min . The location of the gene gpt encoding guanine (xanthine) phosphoribosyltransferase in the proA-proB region was confirmed. Mol Gen Genet, 1975 Dec 30, 143(1), 79 - 83 Induction of RNA polymerase synthesis in Escherichia coli; Glass RE et al.; Studies on the rate of synthesis of the beta and beta' subunits of RNA polymerase in haploid strains of Escherichia coli K12 containing poorly-suppressed rif degrees am mutations provide conclusive evidence that synthesis of at least these two subunits is regulated. Mol Gen Genet, 1975 Dec 30, 143(1), 71 - 7 A mutant of Escherichia coli auxotrophic for organic phosphates: evidence for two defects in inorganic phosphate transport; Sprague GF Jr et al.; An inorganic phosphate transport mutant has been isolated as a sn-glycerol-3-phosphate auxotroph and characterized genetically . Two lesions are responsible for the transport defect . One lesion, pst, is located at minute 74 of the E . coli genetic map while the other lesion, pit, is located at minute 68 . All "K10" strains that were examined carry the pit lesion . Evidence is presented that the glycerol phosphate and hexose phosphate transport systems are not important inorganic phophate transport systems . The mapping data indicate that the genetic distance between malA and xyl is greater than that now allowed. Mol Gen Genet, 1975 Dec 30, 143(1), 35 - 41 The role of Chi mutations in the Spi- phenotype of phage lambda: lack of evidence for a gene delta; Malone RE et al.; Lambda red- gam- makes a small plaque on P2 lysogens (the partial Spi- phenotype) . It has been proposed that inactivation of an additional gene delta, mapping in the recombination region, makes the plaque bigger (the full Spi- phenotype) (Zissler et al., 1917b) . The present paper demonstrates that the Chi mutation in lambda (Stahl et al., 1975) confers upon red- gam- phage the full Spi- phenotype and that the deletion of the region of the chromosome attributed to delta does not . It appears unnecessary to invoke a gene delta in the Spi- phenotype. Mol Gen Genet, 1975 Dec 30, 143(1), 105 - 11 Strains of Escherichia coli diploid for the chromosomal origin of DNA replication; Masters M; F' strains of E . coli have been isolated which are merodiploid for various chromosomal segments between 66 and 78 minutes . Strains diploid for the chromosomal DNA between the genes bgl and mtl grow slowly, have a reduced DNA/mass and an increased cell size . These properties could result if the chromosomal replication origin and a second, extrachromosomal, copy of the origin (located in this case on the F') were to compete for a substance required to initiate replication . We therefore suggest that these strains are diploid for the chromosomal origin of replication and that, therefore, the origin is located between bgl and mtl, that is between 71 and 73 minutes on the E . coli chromosome. Med Microbiol Immunol (Berl), 1975 Dec 30, 162(1), 1 - 7 Passive oral immunization with bovine immunoglobulins: enterpathogenic Escherichia coli from infants and bovine anti-E . coli lactoserum assayed in the rabbit ileal loop model; Zinkernagel RM et al.; The effect of immune bovine lactoserum (BLS) antipolyvalent enteropathogenic Escherichia coli on bacterial growth, viability and bacteria-induced fluid accumulation was examined in rabbit ileal loops . Human enteropathogenic E . coli strains 0125:K70 (B15), 0111:K58 (B4) and 055:K59 (B5) (1-3 X 10(9) per inoculum) induced secretion of 4-6 ml fluid per 10 cm loop . This effect was inhibited effectively by BLS (corresponding to 50 mg IgG 1 per loop) while the viability of bacteria counts decreased 2-25 fold compared with bovine serum albumin . E . coli 026:K60 (B6), 0126:K71 (B16) and 0127:K63 (B8) caused moderate secretion (2-3 ml/10 cm loop) that was significantly neutralized by BLS . E . coli 086:K61 (B7) and 0128:K67 (B12) did not give positive loops . The fluid secretion was shown to be dose dependent for E . coli 0125:K70 (B15) over the range from 2.5 X 10(9) to 8 X 10(7) bacteria/loop . The titration of the effect of BLS on fluid secretion caused by the same strain revealed a dose dependent decrease . The best inhibition was obtained with 100 mg BLS/loop, the highest dose tested. Mol Gen Genet, 1975 Dec 29, 142(2), 87 - 103 Persistence and decay of thermoinducible error-prone repair activity in nonfilamentous derivatives of tif-1, Escherichia coli B/r: the timing of some critical events in ultraviolet mutagenesis; Witkin EM; Ultraviolet (UV) mutagenesis in E . coli is associated with a UV-inducible type of error-prone postreplication repair ("SOS" repair) which, in tif-1 strains, is thermo-inducible in coordination with other recA+ lexA+-dependent inducible functions, including filamentous growth . Mutants of E . coli B/r tif-1 strains have been isolated which retain thermoinducibility of SOS repair activity, but lack the thermosensitivity caused by filamentous growth at 42 degrees C . These strains have been used to determine: the kinetics of decay at 30 degrees C of thermally induced ability to enhance UV mutagenesis; the kinetics of thermal enhancement of spontaneous and UV-induced mutability at 42 degrees C, and the kinetics of decay at 30 degrees C of susceptibility to thermal enhancement of spontaneous and UV-induced mutability . Mutations from tryptophane requirement to prototrophy (Trp- to Trp+) were scored . UV doses were 0.2 J/M2 for excision repair-deficient (Uvr-) and 2J/m2 for Uvr+ strains . The results support the following conclusions . 1) thermally induced SOS repair activity decays at 30 degrees C to about 25% of its maximum level in 45 min, and is no longer detectable after 90 min . 2) Thermal enhancement of UV mutability occurs at sites produced primarily (perhaps exclusively) before completion of the first post-irradiation cell division . 3) UV-induced sites susceptible to thermally induced SOS repair are stable at 30 degrees C in cells not containing the error-prone repair system, and are refractory to constitutive error-free repair for at least 2-3 hours . 4) UV produces a potentially mutagenic type of photoproduct in DNA which can, without interacting with another UV lesion, provide a site susceptible to SOS repair, but which is not a sufficient signal for SOS induction . 5) 50-70% of the SOS-mutable SOS-noninducing UV photoproducts are photoreversible pyrimidine dimers . The results are discussed in relation to current models of UV mutagenesis and induction of UV-inducible functions. Wien Klin Wochenschr, 1975 Dec 26, 87(24), 809 - 15 {Irradiated cases of cervical and breast cancer II . Comparative investigation of the immune status of irradiated cases with stage III cancer of the cervix and operated, non-irradiated cases (author's transl)}; Palencia C et al.; Eleven patients with stage-III cancer of the cervix were investigated before, during and after radio-therapy in regard to their state of humoral immunity on the basis of determinations of the serum IgG, IgM and IgA concentration, of hetero- and isoagglutinins, of tetanus antitoxin before and after vaccination with toxoid, of measles antibodies and of the percentage of lymphocyte membrane fluorescence . The cellular immunity of the same patients was investigated by determination of the percentage of spontaneously-rosetting lymphocytes, of skin-test reactivity with DNCB before and after sensitization, of skin-test reactivity with candida, trichophyton, varidase, OT and staphylo antigen . The function of polymorpho-nuclear leucocytes was investigated by means of the NVT test and St . aureus, E . coli and latex particles . All investigations were performed both before, and 3, 6, 9 and 12 weeks after the commencement of radiotherapy and the results were compared with those of an operated, non-irradiated group (stages I b and II a) . Two types of noteworthy results were observed: 1 . A decrease in immunological reactivity, probably in connection with cancer, since this reaction was observed both in irradiated and in non-irradiated cases, characterized by lowered or absent immune answer to tetanus toxoid, lymphopenia, decrease in sensitization to DNCB and less positive skin tests to old tuberculin and varidase . 2 . An additional inhibition (although in one investigation stimulation of the immune answer was also seen), probably in connection with radiotherapy, characterized by an additional decrease in immune answer to tetanus toxoid, in skin sensitivity to DNCB sensitization and in tests with old tuberculin, and an augmented lymphopenia, as well as an increase in positive skin tests with varidase . No significant changes were observed with any other method. J Biol Chem, 1975 Dec 25, 250(24), 9434 - 6 Cross-linking studies on the 50 S ribosomal subunit of Escherichia coli with methyl 4-mercaptobutyrimidate; Kenny JW et al.; The 50 S ribosomal subunits of Escherichia coli were incubated with methyl 4-mercaptobutyrimidate and the formation of intermolecular protein:protein disulfide bonds was promoted by oxidation . Cross-linked proteins were analyzed by diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis . Three pairs of cross-linked proteins were identified: L2-L7,L12; L5-L7,L12; and L17-L32 . The significance of the results in relation to the location of sites for factor binding, peptidyltransferase, and GTP hydrolysis is discussed. J Biol Chem, 1975 Dec 25, 250(24), 9270 - 5 Transport of purines and deoxyadenosine in Escherichia coli; Roy-Burman S et al.; The characteristics of adenine, guanine, hypoxanthine, xanthine, and uracil uptake in Escherichia coli B show that each base is transported by a specific system . The data support the concept that the transport of guanine, hypoxanthine, xanthine, and uracil function without direct involvement of the respective purine or pyrimidine phosphoribosyltransferase enzymes . Uracil phosphoribosyltransferase is not demonstrable in E . coli B, and large differences are observed in the inhibitory effects of heterologous purines on the uptake of guanine, hypoxanthine, and xanthine as compared to the corresponding inhibitory effects reported for the soluble purine phosphoribosyltransferase enzymes of E . coli B . Additional evidence is provided by the low Km values determined for the transport of adenine, guanine, hypoxanthine, and xanthine relative to the corresponding Km values for the phosphoribosyltransferase enzymes . Data are presented indicating that adenine may be transported without participation of adenine phosphoribosyltransferase . The stimulatory effect of glucose, the inhibitory effect of KCN, and the high intracellular to extracellular concentration gradients of the bases produced in the presence of glucose provide evidence that the transport processes are energy-dependent . The Km values for transport of the purines and uracil range from 10(-7) M to 5 X 10(-7) M . Characteristics of adenine and uracil uptake are similar in E . coli B, E . coli K-12, and a showdomycin-resistant mutant of E . coli B . Adenosine and deoxyadenosine are transported in E . coli B by independent transport systems . Adenine or hypoxanthine does not share the adenosine or deoxyadenosine transport systems as evidence by the mutual lack of competition of free bases and nucleosides on transport . The transport systems for deoxyadenosine and adenosine are defective in the mutant. J Biol Chem, 1975 Dec 25, 250(24), 9238 - 49 Interaction of tetraiodofluorescein with aspartate transcarbamylase and its isolated catalytic and regulatory subunits; Jacobsverg LB et al.; The interaction of the dye tetraiodofluorescein with native aspartate transcarbamylase and its isolated subunits has been investigated by both binding and activity measurements at 4 and 23 degrees . At room temperature low concentrations of tetraiodofluorescein activate the native enzyme, but high concentrations inhibit the enzyme's activity . At the low temperature the native enzyme is inhibited by all concentrations of dye . Isolated catalytic subunit is very effectively inhibited at both temperatures . For the native enzyme these results are explained by 18 tetraiodofluorescein sites of approximately equal affinity (K = 7.3 X 10(-5) M) on each enzyme hexamer: one class of six sites at the nucleoside triphosphate effector binding sites is responsible for the activation, a second class which competes with the substrate carbamylphosphate causes the inhibition, and a third class does not interact with either the effectors or the substrates . Measurements of tetraiodofluorescein binding to isolated regulatory, catalytic, and p-hydroxymercuribenzoate-inactivated catalytic subunits support the above assignments . This scheme of tetraiodofluorescein binding sites successfully predicts the changes in the tetraiodofluorescein-aspartate transcarbamylase difference spectrum induced by the addition of various ligands . The activity changes induced by the dye are explained if the binding of a single tetraiodofluorescein molecule to one of the six regulatory sites activates all six of the catalytic sites, while while a dye molecule binding to any one of the catalytic sites inactivates only that catalytic site. J Biol Chem, 1975 Dec 25, 250(24), 9421 - 7 Energy transduction in Escherichia coli . Genetic alteration of a membrane polypeptide of the (Ca2+,Mg2+)-ATPase; Simoni RD et al.; Recent genetic analyses of the membrane components involved in energy transduction in Escherichia coli have concentrated on the (Ca2+, Mg2+)-ATPase complex (EC 3.6.1.3) . Many mutants have been described with altered biochemical properties and defects in energy-requiring processes such as oxidative phosphorylation, transhydrogenase activity, and active transport of several solutes . This report describes the isolation of a mutant strain of E . coli that is defective in several energy-requiring processes . The strain BG-31 was obtained by "localized mutagenesis" using phage P1c1 . The mutation maps at approximately 73.5 min on the E . coli chromosome . Reversion and suppression analyses indicate that the defect is the result of a single amber mutation . This strain is unable to utilize succinate, D-lactate, or malate for growth . Mutant cells are unable to couple the energy derived from the hydrolysis of ATP to the active transport of proline, although coupling of energy derived from electron transport to solute transport appears normal when examined in both cells and isolated membrane vesicles . Isolated membranes of the mutant are unable to couple the energy derived from the hydrolysis of ATP to transhydrogenase activity while they can utilize the energy generated from electron transport to drive transhydrogenase activity . Extracts of strain BG-31 have normal levels of (Ca2+, Mg2+)-ATPase activity . The ATPase portion of the complex, bacterial F1 (BF1), is poorly attached to the membrane portion of the complex . In vitro reconstitution of transhydrogenase activity with stripped membrane fractions and crude preparations of BF1 localize the defect in strain BG-31 to the membrane portion of the complex . Analysis of membranes of the strain BG-31 by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate demonstrate the absence of a single polypeptide of molecular weight about 54,000 and the appearance of a new polypeptide of lower molecular weight, about 25,000 . Analysis of a spontaneous revertant of BG-31 shows complete restoration of the parental phenotype including the gel patterns . The characterization of this mutant provides the first demonstration of the consequences of a structural gene mutation on a polypeptide in the membrane portion of the complex and represents the initial stages in what we hope will be the biochemical definition and functional characterization of this important energy-transducing system. Mol Gen Genet, 1975 Dec 23, 142(1), 67 - 84 Biosynthesis of RNA polymerase in Escherichia coli . II . control of RNA polymerase synthesis during nutritional shift up and down; Iwakura Y et al.; As an effort to elucidate the control of quality and quantity of the DNA-dependent RNA polymerase in Escherichia coli, the rate of synthesis of the individual subunits was determined during shift-up and -down of nutrients . When the strain B/r grown in a succinate medium was imposed to a shift-up by adding a mixture of glucose and amino acids, rapid rise was observed of the differential rates of the synthesis of alpha, beta and beta' subunits, the constituents of core enzyme, leading to the increase of core polymerase concentration . The differential rates decreased thereafter to the level characteristic of the post-shift rate of cell growth . Compared to the strain B/r, the adaptation was slow in the strain K12 W3350 . On the other hand, upon transfer of the strain B/r from a glucose-amino acids medium to a glucose medium lacking amino acids, the differential rate of core polymerase synthesis decreased rapidly and then regained the rate characteristic of the new growth rate . Similar control was also observed on the rate of ribosomal protein synthesis suggesting the coordinate expression of genes for the core polymerase subunits and ribosomal proteins . Thus, the intracellular concentration of RNA polymerase as well as of ribosomes might be one of the most important factors that affect the rate of bacterial growth . The rate of alpha subunit synthesis, however, exhibited little change during the shift-up but a considerable decrease was observed during the shift-down. Biochim Biophys Acta, 1975 Dec 19, 414(3), 341 - 8 Evidence for the participation of a host enzyme in the activation of poly (A)-Qbeta RNA as an infectious agent; Gilvarg C et al.; It has been reported earlier that phage Qbeta RNA (Gilvarg, C., Bollum, F.J . and Weissmann, C . (1975) Proc . Natl . Acad . Sci . U.S . 72, 428-432) elongated at its 3' terminus with up to 100 or more AMP residues retained its full infectivity for Escherichia coli spheroplasts, and that the resulting progeny did not inherit the poly (A) appendage . We now show that while poly (A)-Qbeta RNA appears to function normally as messenger for the synthesis of virus-specific proteins it has lost its capacity to serve as template for Qbeta replicase . Template function could be restored by phosphorolysis with polynucleotide phosphorylase . Taken in conjunction, these results imply that after poly (A)-Qbeta RNA enters the spheroplast a host enzyme (perhaps polynucleotide phosphorylase) removes part or all of the adenylate residues prior to replication of the RNA. Biochim Biophys Acta, 1975 Dec 19, 414(3), 326 - 40 Activities of guanosine triphosphate analogues in reactions catalyzed by elongation factor Tu and initiation factor 2 of Escherichia coli; Hamel E; In earlier studies two natural analogues of GTP, guanosine 3'-diphosphate 5'-triphosphate (pppGpp) and dGTP, were found to substitute for GTP in reactions catalyzed by initiation factor 2 (IF-2) and elongation factor Tu (EF-Tu), while only dGTP could replace GTP with elongation factor G . These observations with IF-2 and EF-Tu have been extended to two analogues of GTP modified at the 3' ribose hydroxyl position, 3'-deoxyguanosine 5'-triphosphate (3'dGTP) and 3'-deoxy-3'-aminoguanosine 5'-triphosphate (3'dNH2GTP) . These compounds were found to be similar to GTP, dGTP, and pppGpp in IF-2-dependent formation of N-formylmethionyl-puromycin and EF-Tu-dependent formation of N-acetyl-Phe-Phe-tRNA . The apparent Km values for the five guanosine nucleotides were 2 - 10(-6)-4 - 10(-6)M in the former reaction and 2-10(-7)--6-10(-7) M in the latter . These reactions did not have an absolute requirement for either an intact pentose ring or for the guanine base in the nucleotide . Although substantially less active than the guanine nucleotides, ITP and the dialcohol derived from GTP by periodate oxidation and borohydride reduction (ox-redGTP) were partially active in both the IF-2 and EF-Tu-dependent reactions, with apparent Km values about 40-100 times those of GTP. Biochim Biophys Acta, 1975 Dec 16, 413(3), 371 - 93 In vitro reassembly of the membranous vesicle from Escherichia coli outer membrane components . Role of individual components and magnesium ions in reassembly; Nakamura K et al.; A method was developed for the reassembly of membranous vesicle from the sodium deoxycholate-dissociated outer membrane components of Escherichia coli . The removal of the detergent by dialysis and the presence of Mg2+ were essential for the reassembly . Membrane protein alone did not form any membranous structure . Closed membranous vesicles similar to the native outer membrane were reassembled only when protein was mixed with both lipopolysaccharide and phospholipid in deoxycholate solution and subsequently dialized . The membrane showed a distinct trilaminar structure with a center-to-center distance between two dark lines of 53 A, which is a characteristic of the native outer membrane . This characteristic trilaminar structure was shown to be due to the presence of lipopolysaccharide . Phospholipd was required for the vesicularization of membrane . Lipopolysaccharide and/or phospholipid formed a membranous structure in the absence of protein, while the morphology of their negatively stained sample was quite different from that of the native outer membrane unless the outer membrane protein was added to the reassembly mixture . The protein from the cytoplasmic membrane was unable to reform membranous vesicle with lipopolysaccharide and phospholipid, indicating that the reassembly system discriminated outer membrane proteins from cytoplasmic proteins. Biochemistry, 1975 Dec 16, 14(25), 5529 - 34 Studies on the cooperative binding of the Escherichia coli DNA unwinding protein to single-stranded DNA; Ruyechan WT et al.; The cooperative binding of the Escherichia coli DNA unwinding protein to single-stranded DNA has been studied by electron microscopy . Analysis of the electron microscopic data by means of a simple statistical mechanical model has yielded a value of 3.8-7.6 X 10(10) l./mol for the cooperative binding constant in 0.15 M NaCl . Studied under elevated salt conditions have shown that the average DNA protein complex length is 50% of the length found at 0.04 or 0.15 M NaCl. Eur J Biochem, 1975 Dec 15, 60(2), 513 - 23 The tryptophan synthase from Escherichia coli . An improved purification procedure for the alpha-subunit and binding studies with substrate analogues; Kirschner K et al.; An improved method is described for the purification of the alpha-subunit of tryptophan synthase from Escherichia coli . The standard manganese chloride and acid-precipitation steps have been replaced by rapid and efficient chromatographic procedures . Indoleethanol phosphate, indoleprapanol phosphate and indolebutanol phosphate have been synthesized . They are not cleaved by tryptophan synthase and are strictly competitive inhibitors versus indoleglycerol phosphate . The inhibition constant decreases as the number of methylene groups in the side chain increases . This may reflect an improved accommodation of the indole and phosphate moienerated by binding indole, indoleglycerol phosphate and indolepropanol phosphate to the alpha-subunit are very similar . This reflects the transfer of the indole moiety to an hydrophobic environment within the active center . The binding of indolepropanol phosphate to the alpha2beta2-complex perturbs the spectrum of pyridoxal 5'-phosphate located in the beta2-subunit . This demonstrates direct or indirect interactions between the component active sites . Bind studies by spectrophotometric titration and equilibrium dialysis with indolepropanol {32P}phosphate show that there is only one binding site per equivalent of alpha-subunit . Complex formation with the beta2-subunit increases the affinity of the alpha-subunit for indolepropanol phosphate, It is a general consequence of protein-protein interaction in this system. Eur J Biochem, 1975 Dec 15, 60(2), 445 - 9 The binding of maltose to 'virgin' maltose-binding protein is biphasic; Hazelbauer GL; The biphasic binding properties of the galactose-binding and maltose-binding proteins of Escherichia coli may be important in the functioning of these proteins as recognition components of chemoreceptors . However, Richarme and Kepes {Eur . J . Binding curve of the galactose-binding protein may be the result of isotopic dilution, during equilibrium dialysis, by unlabeled ligand retained by the binding throughout purification . Here the binding of maltose to maltose-binding protein which has never previously been exposed to sugar ('virgin' binding protein) is shown to be biphasic . This implies that the unusual binding properties are attributable to the maltose-binding protein itself. Eur J Biochem, 1975 Dec 15, 60(2), 395 - 8 Biosynthesis of a specific lipoprotein of the Escherichia coli outer membrane on polyribosomes; Hirashima A et al.; With use of polyribosomes isolated from Escherichia coli, biosynthesis of a specific lipoprotein of the E . coli outer membrane was investigated . The products of the cell-free system were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoprecipitation with anti-lipoportein serum, followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis . It was found that one of the major products was the lipoprotein of the E . coli outer membrane . The lipoprotein was also found to be preferentially synthsized on smaller polyribosomes. Eur J Biochem, 1975 Dec 15, 60(2), 371 - 7 The first enzyme of the gal operon in inducible and operator-constitutive strains of Escherichia coli . A comparison of the porperties and amino-terminal sequences of UDP galactose 4-epimerase; Deeley R et al.; 1 . UDPgalactose 4-epimerase, the product of the first structural gene of the gal operon in Escherichia coli K-12, has been purified from strain HfrH and from its gal operator-constitutive mutant, HfrH 81-2 . 2 . The two enzymes are purified by the same procedure, behaving identically throughout . They are identical in sedimentation coefficient, sioelectric point, electrophoretic mobility at pH 8.8 and amino-terminal sequence for the first 30 residues . 3 . Slight but reporducible differences were observed between the amino acid compositions and peptide maps of the two proteins . These differences may indicate real differences in the primary sequences of the proteins, but if so they are unlikely to be due to the operator-constitutive mutation because of the identy of the sequences for the first 30 residues . 4 . No evidence was found to indicate that the gal operator might overlap with the first structural gene. Eur J Biochem, 1975 Dec 15, 60(2), 349 - 55 Evidence for an aminoendopeptidase localized near the cell surface of Escherichia coli . Regulation of synthesis by inorganic phosphate; Lazdunski A et al.; An enzyme capable of hydrolyzing the substrate L-alanine p-nitroanilide has been found in the various Escherichia coli strains tested . This enzyme has been called aminoendopeptidase since it shows both activities (see accompanying paper) . It is released from the cells by osmotic shock and by lysozyme -- EDTA spheroplasting treatment, and 50% of the total activity is directly detectable with suspensions of intact cells . However, the release by osmotic shock or spheroplasting is not as efficient as it is for alkaline phosphatase . This periplasmic aminoendopeptidase is constitutively produced but the differential rate of synthesis is increased 4-fold when the cell growth is limited by Pi . The occurrence of this 'derepression' is simultaneous with that of alkaline phosphatase . Increasing the concentration of inorganic phosphate in the medium has no effect on the constitutive aminoendopeptidase synthesis . The effect of phosphate starvation is specific since starvation for neither nitrogen nor carbon and energy source are effective in derepressing aminoendopeptidase. Eur J Biochem, 1975 Dec 15, 60(2), 437 - 43 Kinetics and effect of salts and polyamines on T4 polynucleotide ligase; Raae AJ et al.; The kinetics of T4 polynucleotide ligase has been investigated at pH 8,20 degrees C and using the double-stranded DNA substrate (dA)n - {(dT)10}n/10 . Double-reciprocal plots of initial rates vs substrate concentrations as well as product inhibition studies have indicated that the enzyme reacts according to a ping-pong mechanism . The overall mechanism was found to be non-processive . The true Km for the DNA substrate was 0.6 muM and that of ATP 100 muM . Several attempts were made to reverse the T4 polynucleotide ligase joining reaction using 32-p-labelled (dA)n - {(DT)40}n/40 as substrate . No breakdown of this DNA could be detected . The joining reaction was inhibited by high concentrations, i.e . above approximately 70mM, of salts such as KCl, NaCl, NH4Cl and CsCl . At a concentration of 200 mM almost 100% inhibition was observed . Polyamines also caused inhibition of the enzyme, the most efficient inhibitor being spermine followed by spermidine . At a concentration of 1 mM spermine, virtually no joining took place . Addition of salts or polyamines resulted in a large increase in the apparent Km for the DNA substrate whereas the apparent Km for ATP remained unchanged . It is suggested that the affinity of the enzyme for the DNA substrate is decreased in the presence of inhibiting agents. Eur J Biochem, 1975 Dec 15, 60(2), 363 - 9 Purification and properties of a periplasmic aminoendopeptidase from Escherichia coli; Lazdunski C et al.; A periplasmic aminoendopeptidase from Escherichia coli has been purified to hemogeneity . It is a monomer of molecular weight 45000 and containing one -- SH group that is necessary for catalytic activity . The study of its substrate specificity indicated that the enzyme has both aminopeptidase and endopeptidase activity . The pH optimum for L-alanine p-nitroanilide hydrolysis is between 7 and 7.5 and that for 125I-labeled casein proteolysis between 7.3 and 7.6 . The activation energy for the hydrolysis of L-anine p-nitroanilide was calculated to be 5.3 kcal X mol-1 (22.2 kJ X mol-1). Biochim Biophys Acta, 1975 Dec 15, 412(2), 262 - 72 Hybrids of chemical derivatives of Escherichia coli alkaline phosphatase; Meighen E et al.; The activities of hybrid dimers of alkaline phosphatase containing two chemically modified subunits have been investigated . One hybrid species was prepared by dissociation and reconstitution of a mixture of two variants produced by chemical modification of the native enzyme with succinic anhydride and tetranitromethane, respectively . The succinyl-nitrotyrosyl hybrid was separated from the other members of the hybrid set by DEAE-Sephadex chromatography and then converted to a succinyl-aminotyrosyl hybrid by reduction of the modified tyrosine residues with sodium dithionite . A comparison of the activities of these two hybrids with the activities of the succinyl, nitrotyrosyl and aminotyrosyl derivatives has shown that either the subunits of alkaline phosphatase function independently or if the subunits turnover alternately in a reciprocating mechanism, then the intrinsic activity of each subunit must be strongly dependent on its partner subunit. Chromosoma, 1975 Dec 10, 53(3), 191 - 208 Studies on Macronuclear DNA from Paramecium aurelia; Cummings DJ; Macronuclear DNA was isolated from purified macronuclei of Paramecium aurelia and the size distribution was determined with regard to growth phase and method of extraction . DNA molecules as long as 105 microns and as short as 0.2 microns were observed . It was concluded that the method of extraction affected the observed length of DNA extracted and that macronuclear DNA isolated from cells in balanced growth was less susceptible to nuclease degradation than was DNA isolated from cells in stationary phase . Renaturation studies were performed on macronuclear DNA and a kinetic complexity of 22-times E . coli DNA was determined . This value was similar to those values reported for Tetrahymena and Stylonychia macronuclear DNA . Correcting for GC base content yielded a kinetic complexity for Paramecium macronuclear DNA of 11-times E . coli DNA which corresponded to 3 X 10(10) daltons . There would be about 1400 copies of a unit genome of this complexity within each newly replicated macronucleus . Density gradient analysis indicated that the genes coding for ribosomal RNA had a greater density in CsCl than the bulk DNA . Molecular hybridization studies indicated that the genes coding for 25 S RNA represented 0.14 percent of the total macronuclear DNA . Correcting for GC base content, this corresponded to 30-35 25 S RNA genes per unit genome . These results on Paramecium are discussed in relationship to other ciliate macronuclear DNA. J Biol Chem, 1975 Dec 10, 250(23), 9112 - 20 Kinetic analysis of ribonucleic acid chain initiation by Escherichia coli Ribonucleic acid polymerase bound to DNA; Rhodes G et al.; The kinetics of the RNA chain initiation reaction carried out by RNA polymerase bound to the initiator region of a DNA template have been analyzed . Initiation proceeds in a two-substrate reaction in which the initial binary complex (enzyme-DNA) is transformed into a ternary complex (enzyme-DNA-RNA) by formation of a dinucleoside tetraphosphate and release of inorganic pyrophosphate . In this reaction RNA polymerase serves as a reactant rather than acting catalytically . The concentration of the reacting binary complex decreases throughout the reaction; hence steady state approximations cannot be used . Kinetic equations for an ordered two-substrate reaction are derived . These are most useful for the special case of reaction in the presence of an inhibitor of initiation, such as rifampicin . Equations for the latter instance are solved exactly with recourse to the steady state approximation . It is found that measurements of the extent of the initiation reaction determined at different inhibitor and substrate concentrations can give information about the initiation reaction analogous to that obtained in standard steady state kinetic analysis . This theory is applied to the experimental study of the initiation reaction carried out by Escherichia coli RNA polymerase . It is found that the inhibitor rifampicin, which blocks the initiation reaciton, acts by binding to the same form of the binary complex as the nucleoside triphosphate substrate (ATP or GTP) which is incorporated into the 5' terminus of nascent RNA molecule . The binding of the 5'-terminal nucleoside triphosphate to the enzyme appears to be rate-limiting for the initiation reaction under standard assay conditions . Initiation appears to follow an ordered reaction mechanism; however, the order of addition of the two substrates is still uncertain. J Biol Chem, 1975 Dec 10, 250(23), 9076 - 82 Binding of MET-TRNAf and GTP to homogeneous initiation factor MP; Safer B et al.; Homogeneous initiation factor MP forms a stable complex with Met-tRNAf which binds to nitrocellulose filters in the absence of ribosomal subunits . Complex formation is rapid at 0 degrees and the rate of reaction is stimulated 20-fold by GTP when freshly prepared initiation factor MP is used . Under optimal assay conditions, a 1:1:1 stoichiometry for initiation factor MP, GTP, and Met-tRNAf is indicated, based on a molecular weight for initiation factor MP of 180,000 . Kinetic analysis of ternary complex formation suggests an ordered reaction sequence with binding of GTP followed by binding of Met-tRNAf . However, binding of GTP appears to produce an unstable state which leads to rapid inactivation of initiation factor MP in the absence of Met-tRNAf . Formation of a stable binary complex of initiation factor MP and Met-tRNAf occurs in the absence of GTP . The binary complex cannot subsequently bind GTP . While storage of initiation factor MP at 0 degrees for several weeks has no effect on the rate or extent of Met-tRNAf binding in the presence of GTP, the rate of binary complex formation is increased 10-fold . The binary and ternary complexes appear to bind to 40 S ribosomal subunits with equal efficiency. J Biol Chem, 1975 Dec 10, 250(23), 9053 - 9 Mutants of Escherichia coli defective in membrane phospholipid synthesis . Effect of cessation of net phospholipid synthesis on cytoplasmic and outer membranes; McIntyre TM et al.; The effect of cessation of net phospholipid synthesis on the cytoplasmic and outer membranes of Escherichia coli was investigated in a mutant strain defective in the first enzyme of phospholipid synthesis, the sn-glycerol-3-phosphate (glycerol-P) acyltransferase . The glycerol-P (glycerol) auxotropic phenotype of this strain resulted from an altered membranous glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than that of the parental activity . When net phospholipid synthesis was halted during glycerol deprivation, both soluble and cell envelope protein synthesis continued . Fractionation of the membranes derived from glycerol-supplemented and glycerol-deprived cultures by isopycnic banding in sucrose gradients revealed that both the cytoplasmic and outer membranes of the deprived culture banded at higher buoyant densities . The protein/phospholipid ratio of both the cytoplasmic and outer membranes increased approximately 60% during the period of glycerol deprivation . The distribution of two cytoplasmic membrane activities, NADH oxidase and 1-acylglycerol-P acyltransferase, and an outer membrane activity, phospholipase A1, showed that the total membranes derived from glycerol-deprived cultures were separated cleanly into cytoplasmic and outer membrane fractions . Both cytoplasmic and outer membrane proteins were synthesized and integrated into their respective membranous structures when net phospholipid synthesis was halted . Hence, the biosynthesis of membrane phospholipid and membrane protein are not tightly coupled . Further, these data suggest that cellular control mechanisms exist which maintain the protein content of both membranous structures below the point where they are saturated with protein. J Biol Chem, 1975 Dec 10, 250(23), 8931 - 7 Active transport in Excherichia coli B membrane vesicles . Irreversible uncoupling by chloropyruvate; Kaczorowski G et al.; In the accompanying report (Kaczorowski, G., Shaw, L., Laura, R., and Walsh, C . (1975) J . Biol . Chem . 250, 8921-8930), we have shown that the oxidation of beta-chloro-D-alanine by a membrane-bound D-alanine dehydrogenase results in the inactivation of both dehydrogenase-coupled and P-enolpyruvate-dependent active transport in membrane vesicles . We have also demonstrated that chemically prepared chloropyruvate has the same inactivating effects on transport . In this report, we show that in addition to abolishing hexose and proline uptake, chloropyruvate inhibits lactose and several other amino acid uptake systems to different extents, although proline transport is the most severely inhibited . The degree of transport inactivation also depends on whether the keto acid is added exogenously or is generated by the D-alanine dehydrogenase . Chloropyruvate treatment does not inhibit D-alanine dehydrogenase, D-lactate dehydrogenase of the passage of electrons to oxygen by the membrane cytochrome chain . However, alanine racemase and pyruvate oxidase (to a lesser extent) are inactivated by this keto acid . Treatment of vesicles with chloropyruvate does not affect the establishment of maintenance of a membrane potential, however, this does inhibit solute transport in response to an artificially induced potential . If chloropyruvate is added at any point during a time course of proline transport, there is an instantaneous blockade of amino acid uptake suggesting that the proline carrier can no longer translocate solute across the membrane . Upon examining the functionality of the carrier protein after exposure to chloropyruvate, there is no appreciable difference in efflux or exchange properties as compared to untreated controls . Therefore chloropyruvate does not block proline passage through the membrane, but rather appears to interfere with the ability of the proline carrier to sense the membrane potential . The beta-halo keto acid does not then uncouple respiration from energization of the membrane but does interfere with the ability of the energized membrane state to be used for the transport of most solutes. J Biol Chem, 1975 Dec 10, 250(23), 8921 - 30 Active transport in Escherichia coli B membrane vesicles . Differential inactivating effects from the enzymatic oxidation of beta-chloro-L-alanine and beta-chloro-D-alanine; Kaczorowski G et al.; Isolated membrane vesicles from Escherichia coli B grown on DL-alanine and glycerol carry out amino acid active transport coupled to a membrane-bound D-alanine dehydrogenase (Kaczorowski, G., Shaw, L., Fuentes, M., and Walsh, C . (1975) J . Biol . Chem . 250, 2855) . Certain L-amino acids can also energize solute transport by conversion to their D isomers via an alanine reacemase . Both D-chloroalanine and L-chloroalanine initially drive amino acid and methyl-beta-thiogalactose uptake . The D isomer however causes rapid inactivation of both dehydrogenase-coupled transport and the phosphotransferase system . Transport functions can be protected by dithiothreitol which is postulated to act as a scavenging nucleophile . This inactivation by the D isomer is time-dependent and irreversible not only for proline transport but also for alpha-methylglucoside uptake . Unlike the D isomer, beta-chloro-L-alanine does not inactivate transport . L-Chloroalanine is not racemized to the D isomer but rather undergoes a racemase catalyzed beta elimination of chloride ion to produce pyruvate . Pyruvate can subsequently be oxidized to stimulate active transport . This pyridoxal phosphate-dependent racemase is inactivated by low concentrations of D-chloroalanine but the L isomer can only cause inactivation at a 40-fold higher concentration and longer times of exposure . The D-alanine dehydrogenase-catalyzed oxidation product of D-chloroalanine is chloropyruvate, and this keto acid is hypothesized to be the inactivating species of transport for the following reasons . Chloropyruvate has been isolated from D-chloroalanine oxidation but not from oxidation of the L isomer . Chlorolactate which can be oxidized to chloropyruvate (via membrane-bound lactate dehydrogenases) also causes inactivation of transport in E . coli K-12 membrane vesicles . Mutants having diminished lactate dehydrogenase activity show a slower rate of inactivation with chlorolactate . Moreover, synthetic chloropyruvate irreversibly inactivates both active transport of proline and phosphotransferase system-dependent group translocation of alpha-methylglucoside . The effects of D- and L-chloroalanine and chlorolactate on transport in membrane vesicles are also seen in whole cells. J Biol Chem, 1975 Dec 10, 250(23), 8913 - 20 Conformation of deoxynucleoside triphosphate substrates on DNA polymerase I from Escherichia coli as determined by nuclear magnetic relaxation; Sloan DL et al.; A unique conformation of deoxynucleoside triphosphate substrates bound to Escherichia coli DNA polymerase I has been determined by nuclear magnetic resonance techniques . The effects of Mn(II) bound at the active site of the enzyme on the longitudinal (T1p-1) and transverse (T2p-1) relaxation rates of the alpha, beta, and gamma phosphorus atoms and 5 protons of enzyme-bound thymidine 5'-triphosphate (dTTP) were measured at 40.5 MHz (31P), 100 and 220 MHz (1H) . From frequency dependence of T1p-1, a correlation time of 7 X 10(-10) s and Mn(II) to proton distances of 10.4, 9.9, 10.3, 10.8, and 8.4 A were calculated for the --CH3, H6, H'1, H'2, and H'4 protons . The calculated Mn(II) to phosphorus distances of 4.2, 4.8, and 3.2 A for the alpha, beta, and gamma phosphorus atoms indicates that Mn(II) corrdinates directly only with the gamma-phosphoryl group and that a puckered triphpsphate conformation exists for the enzyme-bound dTTP . This differs from the binary Mn(II)-dTTP complex in which alpha, beta, and gamma phosphoryl coordination occurs, and a thymine-deoxyribose torsion angly (chi) about the glycosidic bond of 40 degrees is detected . The eight manganese-substrate distances on the enzyme are fit by a unique Mn-dTTP conformation, with a torsion angle equal to 90 degrees, indistinguishable from that found for a deoxynucleotidyl unit in double helical DNA-B . Hence, binding to DNA polymerase appears to adjust the conformation of dTTP for Watson-Crick basepairing . Similarly, the binding of Mn-dATP to DNA polymerase I increased the distances from Mn(II) to the H2, H8, H'1, and H'4 protons of dATP but the adenine-deoxyribose torsion angle of 90 degrees was preserved . Such preorientation of substrates could facilitate incorporation of the complementary nucleotide . When positioned within the DNA structure, the conformation of enzyme-bound Mn-dTTP requires an inline nucleophilic attack on the alpha phosphorus with Mn(II) promoting pyrophosphate departure. J Biol Chem, 1975 Dec 10, 250(23), 8893 - 6 Lifetime and rotational relaxation time of dansylgalactoside bound to the lac carrier protein; Schuldiner S et al.; The results presented in this paper demonstrate that the excited state lifetime, anisotropy, and rotational relaxation time of 2'-(N-dansyl)aninoethyl 1-thio-beta-D-galactopyranoside (DG2) increase when the probe is bound specifically to the lac carrier protein in "energized" Escherichia coli membrane vesicles . Although the probe also binds nonspecifically to the vesicle membrane, such binding is independent of the lac carrier protein and is unaffected by "energization" of the vesicles . The experiments provide further evidence that the dansylgalactosides are useful probes for the beta-galactoside transport system and support the hypothesis that the changes in dansylgalactoside fluorescence observed on "energization" of membrane vesicles reflect changes in the binding of the probe. Mol Gen Genet, 1975 Dec 9, 141(4), 343 - 55 Specific cross-linking of proteins S7 and L4 to ribosomal RNA, by UV irradiation of Escherichia coli ribosomal subunits; Moller K et al.; Radioactive 30S and 50S subunits from E . coli ribosomes were irradiated with UV light, under conditions giving rise to approximately 10% cross-linking of protein to ribosomal RNA . Irradiation to levels of cross-linking higher than 10% caused unfolding of the ribosomal subunits, even in the presence of 5 mM magnesium . The specificity of the cross-linking reaction at this low level was found to be extremely high . Cross-linked RNA-protein complexes, freed from unbound protein, were treated with nuclease and then analysed on Sarkosyl gels . S7 was found to be the primary target of the cross-linking reaction in the 30S particle . This was proven by using subunits from both E . coli MRE 600 and A19, whose respective S7 species differ markedly . In the 50S particle, L4 was the primary target, although L2 was also cross-linked to a small extent . Ambiguity in the identification of L4 in the Sarkosyl system was resolved by two-dimensional electrophoresis, which was also used to demonstrate a genuine linkage to RNA in the case of both S7 and L4; proteins spots containing 32P were observed, derived from these two proteins, when subunits containing 32P-RNA were irradiated, treated with nuclease, and applied to the electrophoresis . The identities of S7, L4 and L2 were finally confirmed by Ouchterlony tests with protein-specific anti-sera. Mol Gen Genet, 1975 Dec 9, 141(4), 317 - 29 Alteration of ribosomal proteins in revertants of a valyl-tRNA synthetase mutant of Escherichia coli; Wittmann HG et al.; The work described in this paper was done to see whether the partial suppression of temperature-sensitive aminoacyl-tRNA synthetase mutations by ribosomal mutations is restricted to the aminoacyl-tRNA synthetase mutation which was used for the selection of the suppressor strains or whether the ribosomal mutations can also suppress mutations of other aminoacyl-tRNA synthetases . It is shown that a mutation in ribosomal protein S5 which was selected for suppression of an alanyl-tRNA synthetase mutation (alaS-3) can also partially compensate the temperature-sensitivity of two valyl-tRNA synthetase mutants and of another alanyl-tRNA synthetase mutant . Furthermore, revertants of a temperature-sensitive valyl-tRNA synthetase mutant were isolated and screened for alterations in ribosomal proteins by electrophoretic and immunochemical methods . Alterations in at least two proteins, S8 and S20, were clearly observed among the mutants . The alteration in protein S8 renders the growth of this strain severely cold-sensitive . Presence of the mutation in protein S8 is strictly correlated with suppression of temperature-sensitivity . The S8 mutation maps between strA and spc on the Escherichia coli chromosome . Five suppressor strains have quantitatively or qualitatively altered ribosomal proteins S20 . In one strain no S20 protein could be detected at all, employing different electrophoretic and immunological methods . All five suppressor mutations map in the thr-leu region of the E . coli chromosome, i.e . in an area where the alteration of protein S20 in two alaS suppressor strains has been localized previously. Lancet, 1975 Dec 6, 2(7945), 1113 - 6 Role of heat-labile toxigenic Escherichia coli and Reovirus-like agent in diarrhoea in Boston children; Echeverria P et al.; 61 Boston children aged five years or less with acute diarrhoea were studied for evidence of infection with Escherichia coli strains that produce heat-labile enterotoxin (L.T.) or with a reovirus-like agent associated with childhood gastroenteritis . This represented the first evaluation of the prevalence of disease produced by these two agents in the same population . E . coli, isolated from acute-phase stool specimens, were tested in adrenal-cell tissue-culture and adult-rabbit ileal-loop assays for L.T . Acute and convalescent phase sera, collected from 31 children, were tested by the adrenal-cell assay for anti-L.T . activity . None of the 61 children demonstrated evidence of infection with L.T.-positive E . coli . Paired sera from 31 of the children studied were also tested for evidence of recent infection with the reovirus-like agent by determining titres of immunofluorescent-staining antibody to the serologically related Nebraska calf diarrhoea virus . 11 of the children (35%) had evidence of recent infection . These results suggest that an important proportion of endemic acute diarrhoea of young children in Boston is caused by the reovirus-like agent, and that disease caused by L.T.-producing E . coli is uncommon. Biochim Biophys Acta, 1975 Dec 5, 411(2), 236 - 49 Enzymatic epimerization of D-erythro-dihydroneopterin triphosphate to L-threo-dihydroneopterin triphosphate; Heine MC et al.; An enzyme has been discovered in Escherichia coli that catalyzes the conversion of the triphosphate ester of 2-amino-4-hydroxy-6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropteridine, (i.e . D-erythro-dihydroneopterin triphosphate) to an epimer of this compound, L-threo-dihydroneopterin triphosphate . The enzyme, which is here named "D-erythro-dihydroneopterin triphosphate 2'-epimerase," needs a divalent cation (Mg2+ or Mn2+ is most effective) for maximal activity . Its molecular weight is estimated at 87 000-89 000 . Little or no activity can be detected if either the monophosphate or the phosphate-free form of the substrate is incubated with the enzyme . Evidence is presented to establish that all three phosphate residues of the substrate are retained in the product and that the product is of the L-threo configuration. Biochemistry, 1975 Dec 2, 14(24), 5299 - 303 The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: chemiluminescence and peroxidation; Hodgson EK et al.; Reaction of bovine erythrocyte superoxide dismutase with H2O2 was accompanied by a luminescence whose intensity was a function of the concentration of H2O2 and whose duration was coincident with the inactivation of the enzyme by this reagent . Oxygen, which protected against inactivation, also diminished the luminescence . Several other compounds which prevented the inactivation by H2O2 also modified the luminescence . Thus urate, formate, and triethylamine inhibited luminescence whereas imidazole and xanthine augmented it . These seemingly contrary effects can be explained by assuming that the compounds which protected the enzyme were peroxidized in competition with the sensitive group on the enzyme . The luminescence arises because that group on the enzyme was oxidized to a product in an electronically excited state, which could return to the ground state by emitting light . Imidazole and xanthine gave electronically excited products whose quantum efficiency was greater than that of the group on the enzyme, whereas urate, formate, and triethylamine gave products with much lower luminescent efficiencies . This superoxide dismutase could catalyze the peroxidation of a wide range of compounds, including ferrocytochrome c, luminol, diphenylisobenzofuran, dianisidine, and linoleic acid . In control experiments, boiled enzyme was inactive . This peroxidative activity can lead to unexpected effects when superoxide dismutase is added to H2O2-producing systems, as a probe for the involvement of O2- . Several examples from the literature are cited to illustrate the misinterpretations which this previously unrecognized peroxidative activity can generate. Biochemistry, 1975 Dec 2, 14(24), 5267 - 73 Purification and properties of Escherichia coli dihydrofolate reductase; Baccanari D et al.; Dihydrofolate reductase has been purified 40-fold to apparent homogeneity from a trimethoprim-resistant strain of Escherichia coli (RT 500) using a procedure that includes methotrexate affinity column chromatography . Determinations of the molecular weight of the enzyme based on its amino acid composition, sedimentation velocity, and sodium dodecyl sulfate gel electrophoresis gave values of 17680, 17470 and 18300, respectively . An aggregated form of the enzyme with a low specific activity can be separated from the monomer by gel filtration; treatment of the aggregate with mercaptoethanol or dithiothreitol results in an increase in enzymic activity and a regeneration of the monomer . Also, multiple molecular forms of the monomer have been detected by polyacrylamide gel electrophoresis . The unresolved enzyme exhibits two pH optima (pH 4.5 and pH 7.0) with dihydrofolate as a substrate . Highest activities are observed in buffers containing large organic cations . In 100 mM imidazolium chloride (pH 7), the specific activity is 47 mumol of dihydrofolate reduced per min per mg at 30 degrees . Folic acid also serves as a substrate with a single pH optimum of pH 4.5 . At this pH the Km for folate is 16 muM, and the Vmax is 1/1000 of the rate observed with dihydrofolate as the substrate . Monovalent cations (Na+, K+, Rb+, and Cs+) inhibit dihydrofolate reductase; at a given ionic strength the degree of inhibition is a function of the ionic radius of the cation . Divalent cations are more potent inhibitors; the I50 of BaCl2 is 250 muM, as compared to 125 mM for KCl . Anions neither inhibit nor activate the enzyme. J Immunol Methods, 1975 Dec, 9(2), 157 - 64 Radioimmunoassay for nanogram quantities of DNA; Leon SA et al.; A direct competitive binding radioimmunoassay for DNA has been developed, using 125I-iododeoxyuridine-labelled DNA as the antigen and the serum from a patient with systemic lupus erythematosus . The assay is sensitive in the range of 25 to 1000 ng/ml of DNA . The sensitivity is determined by the affinity of the antibody: this SLE serum contains a component with an association constant of 9.6 X 10(-5) l/mol active at high dilution (1/10,000) . Any biological material, such as serum, synovial fluid or tissue extracts can be tested directly . No interference has been found by DNAse in normal serum, or inhibition by mononucleotides or RNA . Native or denatured DNA from different sources (Escherichia coli, salmon sperm, calf thymus and human placenta) either purified or not, competes equally well for the antibody in this system. Eur J Biochem, 1975 Dec 1, 60(1), 103 - 7 Affinity labelling to - SH groups in adenosine - triphosphate - phosphoribosyl transferase with the dinitrophenyl group from S-dinitrophenyl-6-mercaptopurine-riboside 5'-phosphate; Dall-Larsen T et al.; Adenosine-triphosphate-phosphoribosyl transferase from Escherichia coli reacts with S-dinitrophenyl-6-mercaptopurine-riboside 5'-phosphate . In this reaction the dinitrophenyl group becomes attached to the enzyme, while the nucleotide is split off . Most aliphatic high and low-molecular-weight-SH compounds react with the thioether in the opposite way, i.e . bind the nucleotide and split off dinitrothiophenol . It appears that the dinitrophenyl moiety of the thioether interacts with the enzyme in a specific way, and that this interaction activates the bond between the dinitrophenyl group and the sulfur atom . In support of this it was found that dinitrophenol inhibits the transferase reaction with half maximal effect at 0.4 mM . The inhibition is competitive with ATP . Dinitrophenol also competes with ATP in binding studies. Proc Soc Exp Biol Med, 1975 Dec, 150(3), 755 - 8 Effect of acute infection and endotoxemia on zinc absorption in the rat; Pekarek RS et al.; Intestinal zinc absorption was found to be significantly increased during acute bacterial infection and endotoxemia in the rat . Although serum zinc concentrations were depressed, there was a significant accumulation of 65Zn in the livers of the stressed animals . This study demonstrates that acute inflammation produces a redistribution of zinc within the host, which results in both increased zinc absorption and retention. Proc Natl Acad Sci U S A, 1975 Dec, 72(12), 5145 - 9 Alterations in envelope structure of heptose-deficient mutants of Escherichia coli as revealed by freeze-etching; Bayer ME et al.; The surface of freeze-etched E . coli strain GR467, a heptose-deficient ("deep rough") mutant derived from CR34, was studied by electron microscopy . The outer membrane of GR467 has an increased ratio of phospholipid to protein, mainly due to a decreased protein content . Freeze-etched CR34 showed structural features indistinguishable for wild-type E . coli, i.e., the primary cleavage occurring in the inner membrane with only minor appearance of cleavage within the outer membrane . In contrast to this, in mutant GR467 most of the freeze-cleavages had taken place along a new plane, presumably in a hydrophobic region of the outer membrane . In this cleavage plane numerous particles were seen . Often the cleavage extended over the entire exposed cell surface; occasionally only a few large plateaus were visible, around which the next deeper cleavage plane, that of the protoplasmic or inner membrane, was discernible . Two spontaneous revertants (R11 and R16) with protein and lipid A levels similar to wild-type cells showed mostly freeze fractures with wild-type characteristics, and only a few cells had retained fracturing properties of GR467 . A partial revertant revealed intermediate characteristics . Thus, there appears to be a morphological correlation with the chemical data relating the amount of outer membrane protein with the heptose content of the lipopolysaccharide. Proc Natl Acad Sci U S A, 1975 Dec, 72(12), 5036 - 40 Identification of a gene for the alpha-subunit of RNA polymerase at the str-spc region of the Escherichia coli chromosome; Jaskunas SR et al.; A structural gene for the alpha-subunit of RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) has been identified and mapped between spcA and trkA, near 64 min on the E . coli chromosome . It appears to be coordinately expressed and possibly cotranscribed with the genes for ribosomal proteins S11, S4, and L17. Proc Natl Acad Sci U S A, 1975 Dec, 72(12), 4835 - 9 Strand-selective transcription of globin genes in rabbit erythroid cells and chromatin; Wilson GN et al.; In order to investigate the symmetry of globin gene transcription, complementary RNA (cRNA) was synthesized using rabbit globin complementary DNA (cDNA) as a template for Escherichia coli DNA-dependent RNA polymerase (RNA nucleotidyltransferase) . The cRNA hybridized specifically to its own cDNA template but not to sheep cDNA, rabbit globin mRNA, or poly(dT) . Hybridization studies with cRNA demonstrated that RNA sequences transcribed from the DNA strand complementary to the globin gene region (anti-strand) were not present in cellular, total nuclear, or fractionated nuclear RNA from rabbit marrow . Such sequences were detected in RNA transcribed from rabbit marrow chromatin by E . coli or sheep liver RNA polymerases, but amounted to less than 50% of the globin mRNA sequences present in the same transcript . The evidence indicates that globin mRNA transcription is predominantly DNA strand specific. Proc Natl Acad Sci U S A, 1975 Dec, 72(12), 4820 - 4 Architecture of the Escherichia coli ribosome as determined by immune electron microscopy; Tischendorf GW et al.; Binding sites for antibodies specific to nineteen of the twenty-one ribosomal proteins from the 30S subunit of E . coli ribosomes have been localized on the surface of the 30S ribosomal subunit by immune electron microscopy . The locations of 13 ribosomal proteins from the 50S subunit were similarily assessed . The arrangement of these proteins is illustrated in three-dimensional models of the 30S and 50S ribosomal subunits and of 70S ribosomes . With specific antibodies to six proteins of the 30S subunit we found only one attachment point for each protein . Antibodies against each of nine of the proteins attached at two separate sites that were separated by various distances . Four further proteins were exposed at three or four sites for antibody binding . Altogether eight to ten of the 19 proteins of the 30S subunit have shown antibody attachment sites at remote points on the surface of the ribosome, at distances which are incompatible with globular shapes; these proteins must therefore have elongated or fibrous structures within the ribosome . On the other hand, only two proteins of the 50S subunit, namely L11 and L18, have so far revealed two separated antibody binding sites; proteins L7/L12 occurred, however, at multiple sites. Proc Natl Acad Sci U S A, 1975 Dec, 72(12), 4815 - 9 Histone gene arrangement in the sea urchin, Strongylocentrotus purpuratus; Weinberg ES et al.; The DNA coding for histones from Strongylocentrotus purpuratus, purified up to 100-fold with the use of Hg+2-CS2-SO4 and actinomycin-CsC1 equilibrium density gradients, has been used to study the clustering of genes coding for different histones and the size of the repeating multigene cluster . When digested with EcoRI restriction endonuclease, the histone DNA is identified in two classes of fragments with molecular weights of 1.15 X 106 and 2.8 X 106, whereas after treatment of the DNA with HindIII restriction endonuclease, histone gene sequences can be identified only in a fragment of 3.95 X 106 . Treatment of the DNA with both enzymes simultaneously shows that there is a HindIII site within the smaller EcoRI fragment . Partial digests with HindIII give fragment sizes that appear to be simple multiples of a 3.95 X 106 repeat . Individual histone mRNAs all hybridize to the 3.95 X 106 fragment but only to one or the other EcoRI fragments . The evidence strongly suggests a repeating unit of 3.95 X 106 containing the genes for most, if not all, the histonrs. Proc Natl Acad Sci U S A, 1975 Dec, 72(12), 4805 - 9 Studies on nucleic acid reassociation kinetics: reactivity of single-stranded tails in DNA-DNA renaturation; Smith MJ et al.; The reassociation kinetics of Escherichia coli DNA were measured by S1 nuclease resistance and hydroxyapatite binding . While the reaction assayed by hydroxyapatite displays second order kinetics, the S1 nuclease measurements follow a non-second order from, as previously reported by Morrow (Ph.D . Dissertation, Stanford University . 1974) . Much of the reaction measured with S1 nuclease occurs between single stranded regions of fragments already bearing duplex structures from previous collisions, and between such regions and totally free single strands . Experimental determinations indicate that the nucleation rate of single stranded regions on fragments also containing duplexes is inhibited by an average factor of 2 to 4. Proc Natl Acad Sci U S A, 1975 Dec, 72(12), 4790 - 4 A method for the isolation of specific tRNA precursors; Vogeli G et al.; tRNA affinity chromatography, based on complex formation between tRNAs with complementary anticodons, has been applied to the isolation of specific tRNA precursors . When {32P}RNA, isolated from an Escherichia coli strain containing a thermolabile ribonuclease P, was chromatographed on resin-bound yeast phenylalanine tRNA, precursor tRNAGlu (possessing the complementary anticodon) was specifically retained . Likewise, precursor tRNAPhe was isolated from a column of resin-bound E . coli glutamate tRNA . Both precursor tRNAs isolated were monomeric and may be processed products of an originally larger RNA precursor . Both tRNA precursors contain additional nucleotides beyond the 5'-end of the mature tRNA and have all modified bases found in mature tRNA . The method can be extended to isolate other tRNA precursors by affinity chromatography with different tRNAs . Since the principle of complementary anticodon interaction is not restricted to any particular organism, specific precursor tRNAs from other sources may also be isolated in this way. Proc Natl Acad Sci U S A, 1975 Dec, 72(12), 4780 - 4 Content of elongation factor Tu in Escherichia coli; Furano AV; The content of elongation factor Tu in E . coli B has been determined both by radioimmune assay and by GDP binding . The two assays gave comparable results: cells growing at 2 doublings per hour contained about 8 molecules of Tu per ribosome, whereas those growing at 0.22 doublings per hour contained about 14 molecules per ribosome . These levels resemble those reported for tRNA, in contrast with the 1:1 ratio of factor to ribosomes reported for elongation factors Ts and G. Mol Gen Genet, 1975 Dec 1, 141(3), 263 - 7 Compatibility properties of P1 and PhiAMP prophages; Hedges RW et al.; Although phages P1 and PhiAmp are heteroimmune (Chesney and Scott, 1975 and Yarmolinsky, unpublished), their plasmid prophages are incompatible . Thus, the immunity and compatibility systems are two distinct regulators of phage replication . The two prophages, and plasmid P15B (Ikeda, Inzuka and Tomizawa, 1970) constitute a new compatibility group, designated Y. Mol Gen Genet, 1975 Dec 1, 141(3), 251 - 62 Increased spontaneous reversion of certain frameshift mutations in DNA polymerase I deficient strains of Escherichia coli; Vaccaro KK et al.; A tenfold increase in the spontaneous reversion frequency of two of six lacZ frameshift mutations tested was observed in strains containing the following DNA polymerase I mutations--polA1, polA5, polA6, polAex1, res-3 and resA1 . Reconstruction experiments indicated that this increase was not the result of a selective effect . Only a fourfold increase in frameshift mutations was found in strains containing a polA107 mutation . Both the polAex1 and polA107 mutations result in defective 5' to 3' exonuclease activity and do not affect polymerizing activity, but have different effects on frameshift mutation . A polA mutation on the chromosome induced frameshift mutations in a gene on an F episome . None of three auxotrophic mutations studied showed high frequency reversion in the presence of the polA1 or polA6 mutations. Mol Gen Genet, 1975 Dec 1, 141(3), 189 - 205 The effect of lexA and recF mutations on post-replication repair and DNA synthesis in Escherichia coli K-12; Ganesan AK et al.; We have examined lexA1 uvrA6 and recF143 uvrBdelta derivatives of Escherichia coli K-12 for post-replication repair and DNA synthesis after UV irradiation . Compared to corresponding lex+ rec+ strains, we found that the lexA and recF cells were defective in (1) converting short DNA segments synthesized after irradiation to DNA of normal size; (2) synthesizing high molecular weight DNA after irradiation; (3) transferring pyrimidine dimers from irradiated DNA into unirradiated daughter strands . Our results support the hypothesis that after UV irradiation the formation of large DNA molecules in excision-deficient cells of E.coli depends directly or indirectly upon joining short DNA segments into longer strands, concomitant with the transfer of DNA from irradiated tamplates into unirradiated daughter strands . This process appears to require the activity of lexA and recF genes. J Gen Microbiol, 1975 Dec, 91(2), 369 - 75 Mutagenic DNA repair in Escherichia coli: conditions for error-free filling of daughter strand gaps; Eyfjord JE et al.; Two situations have been observed in which daughter strand gaps in DNA synthesized after exposure of excision-deficient Escherichia coli to ultraviolet light are filled but in which no mutations are formed as judged by loss of photoreversibility: (i) during the first 20 min of growth after u.v . irradiation, and (ii) when repair is allowed to occur in buffer . We suggest as an explanation that the majority of daughter strand gap-filling is error free and that mutations arise through a minor error-prone repair pathway which is inoperative under these conditions. J Gen Microbiol, 1975 Dec, 91(2), 307 - 14 A timing control of cell division in Escherichia coli; de Pedro MA et al.; The effect of heat treatment at 42degreeC on a thermosensitive division-defective strain of Escherichia coli K12, MAC1, has been studied under conditions which support a generation time of about 50 min . Synchronous cells gained simultaneously the ability to divide at 42degreeC and to divide in the presence of nalidixic acid or chloramphenicol, 20 min before physical separation of daughter cells . When synchronous cells of different ages (between 0 to 20 min after elution from an absorbent membrane) were subjected to a heat shock, division always took place 55 to 60 min after the shock . A similar treatment of an exponential culture resulted in synchronous cell division after a lag of 55 to 60 min during which no division occurred . Division is probably controlled for 40 to 45 min by the gene mutated in MAC1 . Thus MAC1 cells of different ages appear to return to the same point of their division cycle when they are heated at 42degreeC . We propose that the gene mutated in MAC1 has a role in the timing control of E . coli cell division . Progress to division appears to require a fixed period in which the function controlled by the gene is performed: this period ends, under physiological conditions, when division does not require further protein or DNA synthesis. J Antibiot (Tokyo), 1975 Dec, 28(12), 982 - 7 A spectrophotometric assay for gentamicin; Williams JW et al.; A rapid and accurate spectrophotometric assay has been developed for the determination of blood werum levels of gentamicin and related antibiotics . The assay uses a purified enzyme from Escherichia coli JR88/C600 that acetylates gentamicin with the production of coenzyme A, linked to a chemical reaction with a sulfhydryl reagent to produce stoichiometric amounts of a sensitive chromophore, monitored in the visible spectrum . The system provides advantages of speed, cost, convenience, accuracy, and enzyme stability to the desirable characteristics encountered with previous enzymatic methods. Infect Immun, 1975 Dec, 12(6), 1271 - 5 Induction of heat-labile enterotoxin synthesis in enterotoxigenic Escherichia coli mitomycin C; Isaacson RE et al.; Introduction of heat-labile toxin (LT) synthesis in enterotocigenic strains of Escherichia coli by mitomycin C (MTC) was demonstrated . Six enteropathogenic strains which produce LT were inducible, exhibiting an 896-fold increase in LT when compared to ininduced cultures . On the other hand, four nonenteropathogens and three other pathogens which produce only the heat-stable toxin were not induced to produce LT . Gel filtration chromatography, antibody neutration, and heat lability studies suggest that the toxin synthesized in the presence of MTC is the same as the toxin synthesized in the absence of MTC. Infect Immun, 1975 Dec, 12(6), 1475 - 7 Antibodies to heat-labile Escherichia coli enterotoxin in Apaches in Whiteriver, Arizona; Sack RB et al.; Antitoxin titers to heat-labile Escherichia coli enterotoxin were measured in Apache children hospitalized with acute diarrhea and in Apaches of different age groups without diarrhea in Whiteriver, Ariz . The study suggests that in this locale, exposure to enterotocigenic E . coli is probably widespread and occurs early in life . Antitoxin titer rises after idarrheal disease associated with enterotocigenic E . coli infection, however, were not regulary found. Eur J Biochem, 1975 Dec 1, 60(1), 57 - 66 Studies of intracellular thymidine nucleotides . Thymineless death and the recovery after re-addition of thymine in Escherichia coli K 12; Ohkawa T; In a thymine-deprived culture, the mutant cells (deficient in dTDP-glucose pyrophosphorylase activity and named Ter-15) lose viability at a faster rate, form longer filaments for the first 60 min and lose thymidine nucleotides and dTDP-sugar pools at a faster rate for the first 15 min than those of the parent cells, but the dTDP-sugar pool in the parent cells is maintained at high concentration for the first 90 min during thymine starvation . In the recovery of cell growth after re-addition of thymine into the thymine-deprived culture, parent cells recommence growth immediately, but the mutant cells (Ter-15) show a lag-phase for 45 min after which time their growth recommences . The rate of dTTP synthesis for the first 10 to 15 min after re-addition of thymine to thymine-deprived cultures of parent and mutant (Ter-15) cells is three-fold higher than that of thymine nondeprived culture (control), but the rates of dTMP and dTDP-sugar syntheses are the same as those of the control . The total DNA synthesis after re-addition of thymine is equal to that of the control, and the period of thymine starvation other than the number of viable cells during thymine starvation plays an important role . After separation of the filament cells from normal-sized cells by sucrose gradient centrifugation, the initial rate of DNA synthesis of filament cells is three-fold faster than that of normal-sized cells . These results show that the dependency of DNA synthesis upon dTTP concentration is maintained after re-addition of thymine into thymine-deprived culture. Eur J Biochem, 1975 Dec 1, 60(1), 51 - 5 Rapid isolation of highly active RNA polymerase from Escherichia coli and its subunits by matrix-bound heparin; Sternbach H et al.; 1 . RNA polymerase from Escherichia coli is selectively and strongly retained by a heparin-substituted agarose and can be eluted therefrom by a neutral buffer containing 0.6 M salt . The method is applicable to relatively crude preparations of the enzyme on a preparative scale giving highly purified RNA polymerase in excellent yield . The enzyme obtained by this procedure shows the highest specific activity so far reported and is pure and enriched in factor sigma as indicated by dodecylsulfate gel electrophoresis . 2 . Based on the differential affinity of the subunits of the enzyme for the heparin-carrying gel matrix, a method for separation of alpha, beta' + beta and sigma subunits by application of urea and salt-containing buffers is described . Upon recombination and dialysis with urea-free buffer 40-50% of the enzyme activity is restored. Eur J Biochem, 1975 Dec 1, 60(1), 303 - 7 The major proteins of the Escherichia coli outer cell-envelope membrane . Cyanogen bromide fragments of protein I, composition and order; Garten W et al.; The cyanogen bromide fragments of protein I, a major protein of the Escherichia coli outer cell envelope membrane, have been isolated and characterized . There appear to be two methionine-serine or methionine-threonine sequences causing incomplete cleavage but complete conversion of methionine to homoserine . Largely due to the existence of these overlapping fragments the order of 5 of the 6 fragments present could be deduced . None of the fragments exhibits any remarkable low degree of polarity, and the tryptic fingerprint of the largest fragment (comprising about 60% of protein I) also does not show any conspicuous large fraction of lipophilic peptides . It is concluded that the domain of protein I that may be buried in the lipid phase of the outer membrane in all likelihood is not very large, and there is, in fact, no definite proof yet that protein I is a membrane protein sensu stricto. Eur J Biochem, 1975 Dec 1, 60(1), 289 - 94 Recognition of initiation codons in modified f2 RNA by Escherichia coli ribosomes; Szkopinska A et al.; f2 phage RNA treated with O-methylhydroxylamine under denaturing conditions loses its ordered structure with consequent exposure of the normally hidden initiation codons . In the presence of Escherichia coli ribosomes and crude initiation factors modified f2 RNA binds about 50 times more f-{3H}Met-tRNA than native f2 RNA . The interaction of native f2{14C}RNA with ribosomes requires initiation factors . The binding of O-methylhydroxylamine-modified f2 {14C}RNA to E . coli 70-S or 20-S ribosomes does not depend on the presence of initiation factors . A significant number of ribosomes deficient in initiation factors interact with a molecule of modified f2 {14C}RNA . Treatment of the resultant polysomal complex with pancreatic RNase yields ribosomes with f2 RNA fragments protected against RNase . Almost all AUG/GUG codons in the f2 RNA are located on the RNase-insensitive ribosome-bound fragments, constituting only 25% of the entire molecule . Addition of crude initiation factors to such ribosomes with fragments of modified f2 RNA promotes binding of f-{3H}Met-tRNA . The resultant complex is fully reactive with puromycin . No binding of Ac-Phe-tRNA takes place under similar conditions. Biotechnol Bioeng, 1975 Dec, 17(12), 1797 - 1804 Engineering analysis of continuous production of L-aspartic acid by immobilized Escherichia coli cells in fixed beds; Sato T et al.; The reaction mechanism and decay behavior of aspartase activity for immobilized Escherichia coli cells were investigated by using a sectional packed col