J Bacteriol, 1980 Oct, 144(1), 451 - 3 Incorporation of acyl moieties of phospholipids into murein lipoprotein in intact cells of Escherichia coli by phospholipid vesicle fusion; Lai JS et al.; The biosynthesis of the acyl moieties in murein lipoprotein was studied by fusion of {3H}palmitate-labeled phospholipid vesicles with intact cells of an fadD mutant of Escherichia coli . A linear increase in the incorporation of {3H}palmitate radioactivity into both the ester- and amide-linked fatty acids in lipoprotein was observed during a 3-h chase after the fusion . Addition of chloramphenicol completely prevented the incorporation of {3H}palmitate from phospholipids to lipoprotein . These results strongly support our hypothesis that the acyl moieties in phospholipids are the precursors for the fatty acids in murein lipoprotein of E . coli . Among the major glycerophosphatides in E . coli, no specificity was observed regarding the efficacy of the donor. J Bacteriol, 1980 Oct, 144(1), 45 - 52 Two different species of murein transglycosylase in Escherichia coli; Mett H et al.; We demonstrated that Escherichia coli murein transglycosylase exists in two forms . After mechanical disruption of the cells, one form was found in the soluble fraction and the other, in the cell envelope . The two enzymes differed with respect to molecular weight, isoelectric point, solubility in aqueous buffers, and to some extent in their requirements for maximal catalytic activity . The molecular weight of the membrane-bound transglycosylase (35,000) was half that of the soluble enzyme . Whether the high-molecular-weight soluble protein is a precursor of the membrane-bound enzyme species remains to be elucidated. J Bacteriol, 1980 Oct, 144(1), 438 - 40 Identification of new genes in a cell envelope-cell division gene cluster of Escherichia coli: cell envelope gene murG; Salmond GP et al.; We report the identification, cloning, and mapping of a new cell envelope gene, murG . This lies in a group of five genes of similar phenotype (in the order murE murF murG murC ddl) all concerned with peptidoglycan biosynthesis . This group is in a larger cluster of at least 10 genes, all of which are involved in some way with cell envelope growth. J Bacteriol, 1980 Oct, 144(1), 435 - 7 Identification of new genes in a cell envelope-cell division gene cluster of Escherichia coli: cell division gene ftsQ; Begg KJ et al.; We report the identification, cloning, and mapping of a new cell division gene, ftsQ . This gene formed part of a cluster of three division genes (in the order ftsQ ftsA ftsZ) which itself formed part of a larger cluster of at least 10 genes, all of which were involved in some step in cell division, cell envelope synthesis, or both . The ftsQAZ group was transcribed from at least two independent promoters. J Bacteriol, 1980 Oct, 144(1), 425 - 7 Comparison of the polypeptide composition of Escherichia coli outer membranes prepared by two methods; Chopra I et al.; Escherichia coli outer membranes were prepared by centrifugation to equilibrium in sucrose gradients and then treated with Sarkosyl in the presence of ethylenediaminetetraacetate . The polypeptide profiles of the two outer membrane preparations were compared by two-dimensional polyacrylamide gel electrophoresis . The patterns obtained were not identical, and Sarkosyl removed several minor proteins from the outer membrane. J Bacteriol, 1980 Oct, 144(1), 366 - 74 Effect of arsenate on inorganic phosphate transport in Escherichia coli; Willsky GR et al.; The effect of arsenate on strains dependent on the two major inorganic phosphate (Pi) transport systems in Escherichia coli was examined in cells grown in 1 mM phosphate medium . The development of arsenate-resistant Pi uptake in a strain dependent upon the Pst (phosphate specific transport) system was examined . The growth rate of Pst-dependent cells in arsenate-containing medium was a function of the arsenate-to-Pi ratio . Growth in arsenate-containing medium was not due to detoxification of the arsenate . Kinetic studies revealed that cells grown with a 10-fold excess of arsenate to Pi have almost a twofold increase in capacity (Vmax) for Pi, but maintained the same affinity (Km) . Pi accumulation in the Pst-dependent strain was still sensitive to changes in the arsenate-to-Pi ratio, and a Ki (arsenate) for Pi transport of 39 microM arsenate was determined . The Pst-dependent strain did not accumulate radioactive arsenate, and showed only a transient decrease in intracellular adenosine triphosphate levels after arsenate was added to the medium . The Pi transport-dependent strain ceased growth in arsenate-containing media . This strain accumulated 74As-arsenate, and intracellular adenosine triphosphate pools were almost completely depleted after the addition of arsenate to the medium . Arsenate accumulation required a metabolizable energy source and was inhibited by N-ethylmaleimide . Previously accumulated arsenate could exchange with arsenate or Pi in the medium. J Bacteriol, 1980 Oct, 144(1), 36 - 44 Genetic and biochemical studies of transport systems for branched-chain amino acids in Escherichia coli K-12: isolation and properties of mutants defective in leucine-repressible transport activities; Yamato I et al.; The characteristics of a mutant (hrbA) of Escherichia coli K-12 that is defective in a leucine-nonrepressible transport system, the LIV-3 system, for branched-chain amino acids were described previously (I . Yamato et al., J . Bacteriol 138:24-32, 1979) . New mutants requiring a high concentration of isoleucine for growth were isolated from strain B763 (hrbA ileA) after mutagenesis with ethyl methane sulfonate . These mutants had a defect of the leucine-repressible transport activities for branched-chain amino acids of the parental strain . One of these mutants, strain B7634, had defects of two independent genetic loci (hrbBC and hrbD) . The genes hrbBC were mapped at min 76 near malT, and the gene hrbD mapped at min 77 near xyl on the E . coli genetic map . The substrate specificity, kinetic properties, and source of coupling energy of the transport system coded for by each of these genes were studied using cytoplasmic membrane vesicles and intact cells . The results identified three transport systems with characteristic features other than the LIV-3 system . The hrbB and hrbC systems are responsible for the uptake activites of the LIV-2 system, with a high Km value, and the LIV-1 system, with a low Km value, respectively . Both activities are repressed by leucine and inhibited by threonine and the b(--) isomer of 2-aminobicycloheptyl-2-carboxylic acid . They both utilize adenosine 5'-triphosphate as coupling energy and are not detected in cytoplasmic membrane vesicles . The hrbD system is responsible for the LIV-4 system, with a high Km value . Its activity is repressed by leucine and partially inhibited by threonine . It is detected in cytoplasmic membrane vesicles with a proton motive force as the driving energy. J Bacteriol, 1980 Oct, 144(1), 356 - 65 Characterization of two genetically separable inorganic phosphate transport systems in Escherichia coli; Willsky GR et al.; Inorganic phosphate (Pi) transport by wild-type cells of Escherichia coli grown in excess phosphate-containing media involves two genetically separable transport systems . Cells dependent upon the high affinity-low velocity Pst (phosphate specific transport) system have a Km of 0.43 +/- 0.2 microM Pi and a Vmax of 15.9 +/- 0.3 nmol of Pi (mg {dry weight}-1min-1) and will grow in the presence of arsenate in the medium . However, cells dependent upon the low affinity-high velocity Pit (Pi transport) system have a Km of 38.2 +/- 0.4 microM and a Vmax of 55 +/- 1.9 nmol of Pi (mg {dry weight}-1min-1), and these cells cannot grow in the presence of an arsenate-to-Pi ratio of 10 in the medium . Pi transport by both systems was sensitive to the energy uncoupler 2,4-dinitrophenol and the sulfhydryl reagent N-ethylmaleimide, whereas only the Pst system was very sensitive to sodium cyanide . Evidence is presented that Pi is transported as Pi or a very labile intermediate and that accumulated Pi does not exit through the Pst or Pit systems from glucose-grown cells . Kinetic analysis of Pi transport in the wild-type strain containing both the Pst and Pit transport systems revealed that each system was not operating at full capacity . In addition, Pi transport in the wild-type strain was completely sensitive to sodium cyanide (a characteristic of the Pst system). J Bacteriol, 1980 Oct, 144(1), 327 - 36 Pattern of meso-dl-2,6-diaminopimelic acid incorporation during the division cycle of Escherichia coli; Verwer RW et al.; The topography of meso-DL-2,6-diaminopimelic acid incorportion into the cell envelope of Escherichia coli W7 (doubling time, tau, = 70 min) has been studied by autoradiography . To follow the incorporation pattern during the division cycle, cells have been classified according to length and the silver grain distributions have been determined in the two cell halves . In particular, the question of equivalence (with respect to the grain distributions) of the two cell halves has been investigated statistically . The grain localizations have been determined separately for the left cell halves (highest number of grains) and right cell halves . The highest probability of finding grains was in the central area for cells of all length classes . In the longest cells (dividing or nondividing) incorporation occurred in the future septal regions of the prospective daughter cells . Autoradiography of tritiated thymidine-labeled cells indicated the presence of an atypical deoxyribonucleic acid replication cycle (at tau = 70 min) . Initiation of deoxyribonucleic acid replication occurred during the latter part of the division cycle, and its termination occurred in the next cycle. J Bacteriol, 1980 Oct, 144(1), 312 - 21 Identification of a novel genetic element in Escherichia coli K-12; Greener A et al.; Induction of the SOS repair processes of Escherichia coli K-12 caused a 14.4-kilobase species of circular deoxyribonucleic acid, called element e14, to be excised from the chromosome . To aid further characterization of this species, an 11.6-kilobase segment of e14 was inserted into the HindIII site of plasmid pBR313 . To map e14 on the E . coli K-12 chromosome, the recombinant plasmid, pAG2, was used to transform a polA recipient, an event which required integration of pAG2 into the recipient chromosome . This recombinational event was dependent upon the region of homology between the incoming plasmid and the chromosome, as no transformants were scored when either a strain cured of the element was the recipient or pBR313 was the transforming deoxyribonucleic acid . Using these transformants, we have shown that e14 maps between the purB and pyrC loci near min 25 . Several strains of E . coli K-12 were found to contain e14; however, one strain, Ymel trpA36, did not . In addition, e14 was found to be absent in both E . coli B/5 and E . coli C . The approach to mapping developed for this work could be used to map other fragments of E . coli deoxyribonucleic acid which have no known phenotype. J Bacteriol, 1980 Oct, 144(1), 28 - 35 Isolation and mapping of a mutation in Escherichia coli with altered levels of ribonuclease H; Carl PL et al.; A mutant of Escherichia coli with altered levels of ribonuclease (RNase) H was isolated after mutagenesis with ethyl methane sulfonate . A procedure for assaying RNase H in partially purified extracts was used to screen approximately 1,500 colonies for variations in RNase H activity . Confirmation of a lower level of RNase H in the mutant was accomplished by analysis of RNase H in sodium dodecyl sulfate-polyacrylamide gels . By Hfr, F', and P1 transduction mapping, the genetic locus responsible for the lower levels of RNase H was located at 5.1 min on the E . coli chromosome . This mutation (rnh) represents a new locus on the E . coli chromosome . The only phenotypic characteristic of this mutation which has been observed to date is the lower level of RNase H (30% of parental values). J Bacteriol, 1980 Oct, 144(1), 274 - 8 Substrate specificity and transport properties of the glycerol facilitator of Escherichia coli; Heller KB et al.; The specificity of the glycerol facilitator (glpF) of Escherichia coli was studied with an osmotic method . This transport system allowed the entry of polyols (glycerol and erythritol), pentitols, and hexitols . The analogous sugars were not transported . However, urea, glycine, and DL-glyceraldehyde could use this pathway to enter the cell . The glpF protein allowed the rapid efflux of preequilibrated xylitol . Glycerol surprisingly did not inhibit the uptake of xylitol, and xylitol only slightly reduced the uptake of glycerol . The observation and the insensitivity of the xylitol transport to low temperature suggest that the facilitator behaves as a membrane channel. J Bacteriol, 1980 Oct, 144(1), 185 - 91 Further characterization of sfiA and sfiB mutations in Escherichia coli; Huisman O et al.; The sfiA and sfiB mutations, originally isolated in thermoresistant ultraviolet-resistant revertants of a tif lon strain, also suppressed filamentation in tsl strains (mutated at the lexA locus) . When deoxyribonucleic acid synthesis was arrested, however, sfi-independent filamentation occurred . Other SOS functions were not affected by sfiA and sfiB mutations; in particular, ultraviolet-induced repair and mutagenesis of bacterial deoxyribonucleic acid were normal, as was tsl-tif-induced synthesis of recA protein . Genetic studies (i) established the identity of map location of the sfiA and sulA loci, (ii) showed that the two sfiB mutations are recessive, and (iii) showed that of six independent sfiA mutations, three are recessive and three are dominant . One sfiB strain was shown to have a 6% growth disadvantage relative to a sfi+ or sfiA strain . It is proposed that the sfiA locus may define the structural gene of a hypothetical inducible SOS-associated division inhibitor. J Bacteriol, 1980 Oct, 144(1), 179 - 84 Acetaldehyde coenzyme A dehydrogenase of Escherichia coli; Clark DP et al.; Mutants of Escherichia coli (adh) in which alcohol dehydrogenase is derepressed under aerobic conditions were also found to overproduce acetaldehyde coenzyme a dehydrogenase . However, acetaldehyde coenzyme A dehydrogenase was induced by ethanol or acetaldehyde and subject to strong catabolite repression, whereas alcohol dehydrogenase was little affected by these conditions . Mutants no longer able to use ethanol as carbon source were isolated from an adh strain . Some of these mutants were revertants at the adh locus and no longer produced either alcohol dehydrogenase or acetaldehyde coenzyme A dehydrogenase . Others, designated acd, were found to lack only acetaldehyde coenzyme A dehydrogenase . The acd mutation was located at min 62 of the E . coli genetic map, the gene order being thyA-lysA-acd-serA-fda . Isolation of Tn10 insertions cotransducible with acd greatly simplified the mapping procedure. J Bacteriol, 1980 Oct, 144(1), 149 - 58 Biochemical and immunological characterization of an R plasmid-encoded protein with properties resembling those of major cellular outer membrane proteins; Ferrazza D et al.; MRB, a major R222 plasmid-encoded protein previously described by us, is synthesized in large amounts in host Escherichia coli cells, where it is located principally in the outer membrane . Most of this protein is also bound to the peptidoglycan layer in a form which is trypsin resistant . Its monomeric molecular weight is about 29,000, but it is isolated from cell membranes in aggregate molecular weights of more than 100,000 . These properties demonstrate a strong similarity between MRB and porins, major outer membrane proteins of host E . coli cells . They suggest that MRB may have an as-yet unidentified transport function, as do cellular outer membrane proteins with similar biochemical properties . By using antiserum specific for MRB, we demonstrated identity between MRB and the product of the traT gene, one of the surface exclusion proteins on the F plasmid . The synthesis of MRB was found to be constitutive, in contrast to other tra genes, which appear to be under more rigid regulation by the tra operon . These findings suggest that on R222 and other F-like R plasmids this protein has its own promoter. J Bacteriol, 1980 Oct, 144(1), 114 - 23 Are growth rates of Escherichia coli in batch cultures limited by respiration? Andersen KB, von Meyenburg K. Batch cultures of Escherichia coli were grown in minimal media supplemented with various carbon sources which supported growth at specific growth rates from 0.2 to 1.3/h . The respiration rates of the cultures were measured continuously . With few exceptions, the specific rate of oxygen consumption was about 20 mmol of O2/h per g (dry weight), suggesting that the respiratory capacity was limited at this value . The adenosine triphosphate (ATP) required for the production of cell material from the different carbon sources was calculated on the basis of known ATP requirements in the biochemical pathways and routes of macromolecular synthesis . The calculated ATP requirements, together with the measured growth rates and growth yields on the different carbon sources, were used to calculate the rate of ATP synthesis by oxidative phosphorylation . This rate was closely related to the respiration rate . We suggest that aerobic growth of E . coli in batch cultures is limited by the rate of respiration and the concomitant rate of ATP generation through oxidative phosphorylation. Surg Gynecol Obstet, 1980 Oct, 151(4), 477 - 80 Effects of cortisone on decrease of serum albumin secondary to experimental infections; Deysine M et al.; The observation that severe clinical bacterial infections are associated with decreases in total serum albumin levels was confirmed in experiments involving dogs and rats . In rodents, the injection of methylprednisolone sodium succinate after the onst of the infection significantly reduced the decrease in the serum albumin values . However, this protective or corrective effect did not occur if the steroid was given prior to the infection . The effect of cortisone may be due to its capacity to stimulate albumin synthesis by the parenchyma of the liver. J Parasitol, 1980 Oct, 66(5), 730 - 4 Responses of B-cells to mitogens and antigen in mice receiving isogenic splenocytes from animals treated with Trichinella extract; Barriga OO; Splenocytes of C57BL/6J mice injected with a Trichinella spiralis larval extract for 7 consecutive days were transferred in two doses into isogenic, immunocompetent mice . On the 3rd day, some recipients were immunized with 10(9) sheep red blood cells and others were killed to investigate blastogenic response of their splenocytes to concanavalin A (Con A), Escherichia coli lipopolysaccharide (LPS), and Mycobacterium's purified protein derivative (PPD) . On the 8th day of immunization, the corresponding mice were killed to study rosette-forming cells (RFC) and direct and indirect plaque-forming cells (D- and I-PFC) in their spleens . Transfer of 10(6) cells depressed the Con A reactivity and the number of RFC and 1-PFC, but increased the PPD reactivity and the number of D-PFC in the recipients, as compared to control mice receiving splenocytes from donors injected with a saline solution . Ten million cells inhibited only the Con A reactivity, but enhanced the number of LPS- and PPD-responding cells and of D-PFC in the recipients over the controls . Inoculation of cells from mice injected with bovine serum albumin did not reproduce the same effects . Splenocytes of mice treated with T . spiralis extract simultaneously inhibit and enhance diverse functions of the immune system . Stimulation is exerted on IgG antibody production and appears to be mediated by suppressor T-cells . Stimulation is exerted mainly on IgM antibody formation . Depression seems to be antigen-specific; it is partially compensated by the concurrent suppression, and it is probably a result of macrophage activation. Can J Biochem, 1980 Oct, 58(10), 885 - 97 Localization of the dicarboxylate binding protein in the cell envelope of Escherichia coli K12; Bewick MA et al.; Examination of the localization of the dicarboxylate binding protein (DBP) in the cell envelope of Escherichia coli K12 reveals that this protein is present on the cell surface, and also in the inner and outer regions of the periplasmic space . The cell surface DBP is release by treating the cells with EDTA . This protein can be surface labeled by lactoperoxidase radioiodination, and by diazo{125I}iodosulfanilic acid in whole cells . It also binds tightly, but not covalently, to lipopolysaccharide . The DBP located in the outer region of the periplasmic space is released when the outer membrane is dissociated by EDTA-osmotic shock treatment . The DBP located in the inner region of the periplasmic space is released only when the EDTA-osmotic shocked cells are subjected to lysozyme treatment . At the moment, it is not certain whether this protein is bound to or trapped by the peptidoglycan network . This protein cannot be surface labeled in whole cells or in EDTA-osmotic shock treated cells; and it is not associated with lipopolysaccharide . Analysis of transport mutants indicates that these DBP are coded by the same gene. Can J Biochem, 1980 Oct, 58(10), 1172 - 8 The effect of amphipaths on the flavin-linked aerobic glycerol-3-phosphate dehydrogenase from Escherichia coli; Robinson JJ et al.; When assayed by the phenazine methosulphate coupled 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, the flavin-linked aerobic glycerol-3-phosphate dehydrogenase from Escherichia coli strain 7 showed a marked loss of activity if depleted of detergent, and this activity could be reconstituted with amphipaths . However, enzyme activity, as measured by the ferricyanide reduction assay, showed no loss of activity upon detergent depletion . Cross-linking studies indicated that amphipath was not inducing any significant quaternary structural change in the enzyme . Flavin fluorescence titration experiments indicated a single type of binding site for glycerol 3-phosphate whose affinity was unaffected by the presence of amphipath; apparent Kd values of 19.5 and 17.1 mM were measured in the presence and absence of amphipath, respectively . The Michaelis-Menten constants for DL-glycerol 3-phosphate, as measured by the phenazine methosulphate coupled MTT and ferricyanide reduction assays in the presence of amphipath, were found to be 1.9 and 10.2 mM, respectively . Cupric ions were found to specifically inhibit the phenazine methosulphate coupled MTT reducing activity while zinc ions inhibited the ferricyanide reducing activity . Circular dichroic studies provided initial evidence for a conformation change in the presence of the nonionic detergent Brij 58 and this was substantiated by quenching of tryptophan fluorescence at concentrations of Brij 58 equivalent to those needed to stimulate activity . We conclude from these studies that amphipaths may specificity induce a phenazine methosulphate binding site thus permitting reduction of this electron acceptor. Infect Immun, 1980 Oct, 30(1), 231 - 43 Localization of electron-dense tracers during entry of Rickettsia tsutsugamushi into polymorphonuclear leukocytes; Rikihisa Y et al.; The invasion of Rickettsia tsutsugamushi, Gilliam strain, into guinea pig polymorphonuclear leukocytes (PMNs) and the localization and distribution of tracers were followed during the process by electron microscopy . The seven tracers used were: cationized ferritin, ferritin, thorium dioxide (ThO2), carbon particles, latex spheres, paraffin oil, and Escherichia coli . These markers were added to the incubation medium containing the PMNs before or simultaneously with R . tsutsugamushi-infected BHK-21 cells . Both morphologically intact and degenerating rickettsiae were present in the phagosomes in PMNs, but only the viable-appearing rickettsiae were free in the cytoplasm . The intact rickettsiae were singly and selectively phagocytized in tightly enclosed phagosomal membranes which usually excluded the tracers, except when ThO2 or ferritin was used . When ThO2, which labels the plasma membrane of PMNs, was used . ThO2-labeled phagosomal membranes enclosing rickettsiae were observed and short membrane fragments still labeled with this tracer were found in the vicinity of rickettsiae in the cytoplasmic matrix of PMNs . When ferritin or ThO2 was used as a tracer, some of the phagosomes contained rickettsiae still enclosed in an envelope of BHK-21 cytoplasm and cell membrane . Phagolysosomes preloaded with electron-dense markers fused with subsequently formed phagosomes containing degenerated rickettsiae but not with those containing intact rickettsiae . These results support our interpretation that viable rickettsial entry into PMNs is by selective phagocytosis and escape from these phagosomes. Cancer Res, 1980 Oct, 40(10), 3508 - 11 Genetic factors in Escherichia coli that affect cell killing and mutagenesis induced by benzo(a)pyrene-7,8-dihydrodiol 9,10-oxide; Ivanovic V et al.; In the present study, the induction of base-pair reversion mutations from trp to Trp+ in the wild type and in uvrA, recA, and lexA mutants of Escherichia coli B/r was used to analyze the mechanism of benzo(a)pyrene-7,8-dihydrodiol 9,10-oxide (BPDE) mutagenesis . BPDE mutagenesis was compared to that of two well characterized mutagens: ethyl methanesulfonate and 4-nitroquinoline 1-oxide . We found that a uvrA mutant was about 20 times more sensitive to BPDE killing and BPDE-induced mutations than was wild-type E . coli . This suggests that BPDE-DNA lesions are removed by an excision repair mechanism similar to that which excises pyrimidine dimers . Although strains carrying either recA or lexA mutations were also more sensitive to BPDE killing, BPDE was not mutagenic in these strains . The recA+ and lexA+ gene products coordinately control the regulation of a set of genes that are expressed in response to DNA-damaging agents (SOS functions) . Therefore, our results indicate that in E . coli BPDE is an indirect mutagen that acts through host-mediated functions, i.e., the SOS pathway, in the process of mutation fixation . The possible significance of these findings to carcinogenesis are discussed. J Natl Cancer Inst, 1980 Oct, 65(4), 759 - 68 Immunosuppressive factors from mastocytoma cells cultured in serum-free medium; Nakamura M et al.; Immunosuppressive factors were isolated from culture fluids of DBA/2 mouse mastocytoma cells grown in serum-free RPMI-1640 medium by measurement of inhibitory activity on tritiated thymidine uptake of DBA/2 spleen cells responding to Escherichia coli lipopolisaccharide (LPS) and concanavalin A (ConA) and by comparison of the number of hemolytic antibody-forming cells in vitro after simultaneous addition of the factors with sheep red blood cells (SRBC) . The culture fluids were separated into four fractions with immunosuppressive activities: F-I (mol wt, > 30,000), and F-II (mol . wt, 10,000-30,000), F-III (mol wt, 2,000-10,000), and F-IV (molwt, 700-2,000), F-IV specifically suppressed lymphocyte responses to E . coli LPS at concentrations of 50-200 microgram/ml . The other three fractions also showed immunosuppressive activities in both Con-A-induced and E . coli LPS-induced lymphocyte responses . The four fractions as well as crude culture fluids of mastocytoma cells expressed as immunosuppressive effect when injected into the peritoneal cavities of DBA/2 mice at doses of 24-180 microgram/day for 5 consecutive days . The fractions with a molecular weight equivalent to F-IV of mastocytoma culture supernatant were detected little, if any, in the supernatant from a nonmalignant cell culture . No suppressive effects of the fraction with a molecular weight greater than 2,000 (equivalent to F-I + F-II + F-III) from nonmalignant cell culture were found on lymphocyte responses to mitogens or SRBC at the concentrations used (100-200 microgram/ml). J Bacteriol, 1980 Oct, 144(1), 291 - 9 In vitro formation of beta-galactosidase with a template containing the lac genes fused to gene ilvD; Noti JD et al.; An in vitro coupled transcription-translation system was used to synthesize transaminase B and beta-galactosidase in the presence of a deoxyribonucleic acid template containing lac deoxyribonucleic acid under normal lac-specific control and in the presence of several deoxyribonucleic acid templates containing lac deoxyribonucleic acid fused to the ilvD gene . Time course experiments revealed that transcription of the lacZ gene from the fusion template required a longer time than did that initiated at the lac promoter . With a phage template containing an intact ilvE gene but lacking the normal ilv-specific promoter, synthesis of ilvE message was completed before synthesis of lacZ message . A phage template that contained the normal ilv-specific promoter but from which part of ilvE had been deleted also allowed formation of beta-galactosidase . Three plasmids containing the ilv-lac fusion were also used as templates . Two plasmids that contained both an intact ilvE gene and the normal ilv-specific promoter required longer times for lacZ transcription but were more efficient templates than was a plasmid in which the ilv-lac fusion, the ilvE gene, and the contiguous non-specific ilvE promoter were inverted with respect to the normal ilv-specific promoter . beta-Galactosidase synthesis was stimulated by guanosine 3'-pyrophosphate-5'-pyrophosphate with all templates tested except that in which the ilv-lac fusion had been inverted . Presumptive evidence was obtained for the generation of a limiting isoleucine signal by incorporating inhibitors of isoleucyl transfer ribonucleic acid synthetase into the coupled transcription-translation system. Biochim Biophys Acta, 1980 Oct 1, 632(2), 326 - 35 The interaction of the K88 antigen with porcine intestinal epithelial cell brush borders; Sellwood R; The interaction of 125I-labelled K88 antigen with brush borders of the epithelial cells of the pig small intestine has been studied . The iodinated antigen bound avidly to the brush borders prepared from adhesive (receptor-positive) pigs even after pretreatment of the brush borders with formaldehyde, whereas the brush borders from non-adhesive (receptor-negative) pigs failed to bind the antigen under these conditions . Treatment with glutaraldehyde rapidly destroyed the ability of both types of brush border to bind the K88 antigen . Studies on the binding of antigen to brush borders revealed the presence of high affinity receptors, but the non-linearity of the Scatchard plot could be explained by cooperative-like interactions, which view was supported by dissociation experiments . Rapid dissociation only in the presence of unlabelled K88 antigen suggested the existence of receptor site interactions of the negatively cooperative type . Attempts to inhibit the binding of 125I-labelled K88 with simple monosaccharides and oligosaccharides suggested that the binding of antigen to brush borders involves complex interactions and that galactosyl residues may be important. Clin Exp Immunol, 1980 Oct, 42(1), 77 - 85 Immune complex glomerulonephritis in mice infected with Escherichia coli; Fournie GJ et al.; C57Bl/6 mice were injected intraperitoneally with 10(8) to 2 x 10(8) living K 38 Escherichia coli (E . coli) and serological changes and kidney involvement were studied . E . coli were found in the blood 45 min to 24 hr after injection . In serum, large amounts of deoxyribonucleic acid (DNA) were present 24 hr after E . coli injection, and thereafter disappeared . Seven days after infection, antibodies directed against E . coli, anti-DNA antibodies and C1q-binding substances were found in serum and the kinetics of the variations of these parameters were studied until day 35 . Kidney lesions were evaluated immunochemically and by optical and electron microscopy . In the glomeruli, heavy granular deposits of IgG and IgM were constantly found in mesangium and along capillary walls . In most kidneys slight granular deposits of IgG and IgM were also found in the tubules . Histological studies revealed in the glomeruli mild endocapillary cell proliferation, focal thickening of glomerular basement membrane and dense deposits in mesangial and subendothelial areas and inside the glomerular basement membrane; in the tubules dense deposits were focally observed inside the tubular basement membrane. Gene, 1980 Oct, 11(1-2), 173 - 5 An easy method for the selection of restriction- and modification-deficient mutants of Escherichia coli K-12; Piechaczyk M et al.; An easy and rapid method for selecting restriction- and modification-defective mutants of Escherichia coli K-12 is described . This method employs selection of tetracycline resistant lysogens after infection with lambda::Tn10 phage and results in a high yield of spontaneous rk-mk- and rk-mk+ mutants. Cell, 1980 Oct, 21(3), 709 - 15 Molecular cloning and selection of genes regulated in Aspergillus development; Zimmermann CR et al.; Over 350 clones homologous to poly(A)+ RNAs that are significantly more prevalent in conidiating cultures of Aspergillus nidulans than in somatic cells have been selected from a recombinant DNA library formed between nuclear DNA and lambda Charon 4A . The procedure used for this selection involved in situ hybridization to a cDNA probe which had been selectively depleted of sequences represented in somatic cells by complement hybridization . Five of these clones have been characterized further . All but one encoded poly(A)+ RNAs that were at least ten times more prevalent in conidiating cultures than in somatic cells . One clone hybridized to a single, developmentally regulated RNA . The three others were complementary to several RNAs having different molecular weights, each of which was more prevalent in condiating cultures than in vegetative cells . These results and quantitative aspects of the selection procedure suggest that developmentally controlled poly(A)+ RNA coding regions may not be distributed randomly in the Aspergillus genome. J Clin Invest, 1980 Oct, 66(4), 621 - 8 Suppressor cell regulation of cell-mediated immune responses in renal infection in vitro modulation of suppressor cell activity; Miller T et al.; Infection-induced anergy is a frequent complication of bacterial, viral, and parsitic infection . A marked suppression of the thymus-derived (T) lymphocyte response to concanavalin A has been demonstrated in vitro during renal infection and the mechanisms by which suppression occurs have been investigated . In particular we have considered the possibility that suppression might result from the inhibitory effect of prostaglandins, secreted by activated macrophages with immunoregulatory potential . The experiments have shown that the T-lymphocyte effector status in experimentally-induced renal infection is determined by two suppressor cells, one infection-induced and the other naturally occurring . The inability to respond to mitogenic stimulation was reversible and restoration of immune responsiveness to splenic lymphocytes from infected animals could be achieved in two stepwise manipulations; differential centrifugation removed the infection-induced suppressor cells, and the suppressor activity of the naturally occurring suppressor cells could then be inhibited by indomethacin . Thus the two suppressor cells were distinguishable on the basis of their physical characteristics and their response to indomethacin . The dominant factor determining the immune responsiveness of splenic lymphocytes from the pyelonephritic animals was, however, the infection-induced suppressor cell . This cell has been characterized as a sedimentable cell (30 g) with suppressor activity demonstrable in co-culture experiments . Plastic-adherent cells from the sedimentable fraction of pyelonephritic animals' splenic cells were shown to have suppressor activity that was not inhibited by indomethacin . The infection-induced and naturally occurring suppressor cells can be viewed as prototypes for the equivalent cells in man and may be useful models for studying the role of these cells as determinants in the pathogenesis of infectious disease. Biochim Biophys Acta, 1980 Oct, 615(2), 489 - 96 Murein transglycosylase from phage lambda lysate . Purification and properties; Bienkowska-Szewczyk K et al.; Lysates of induced E . coli (lambda) lysogens contain two enzymes acting on murein: endopeptidase and murein transglycosylase . The transglycosylase was separated from the endopeptidase and purified to homogeneity . Its bacteriolytic activity was 200-fold higher than of hen egg lysozyme . The bacteriolytic activity of the lysate depends on the presence of the enzyme . The endopeptidase alone not lyse the cells, but it enhances the extent of lysis . The properties of the transglycosylase (molecular weight 17 500, pH optimum at 6.6, inactivation by Zn2+), show that it is entirely different from the bacterial enzyme of the same specificity described by others . Data are presented, which suggest that this enzyme is the phage lambda R-gene product. Biokhimiia, 1980 Oct, 45(10), 1909 - 12 {Localization and restriction mapping of the origin of replication of rat liver mitochondrial DNA}; Tomarev SI et al.; Based on plasmid pCV II, a recombinant comprising the origin of replication of rat liver mitochondrial DNA was obtained . A detailed restriction map of the region based on restrictases Hae III, Hpa II and Hind II was developed . The origin of mitochondrial DNA replication is localized in one of Hind II fragments. J Gen Microbiol, 1980 Oct, 120(2), 475 - 83 Biochemical and genetic characterization of nirB mutants of Escherichia coli K 12 pleiotropically defective in nitrite and sulphite reduction; Cole JA et al.; Mutants of Escherichia coli K12 defective in the nirB gene lack NADH-dependent nitrite reductase activity and reduce nitrite slowly during anaerobic growth . With one exception these mutants require cysteine for growth . Cytochrome C552 synthesis and the assimilation of ammonia are unaffected by the nirB mutation . The defective gene is located between the crp and aroB genes at minute 73 on the E . coli chromosome . Mapping and reversion studies indicate the nirB is identical to the previously described cysG gene . It is suggested that the product of the cysG+ (nirB+) inverted question markgene is an enzyme required for the synthesis of sirohaem, a prosthetic group of enzymes which catalyse the six-electron reduction of nitrite to ammonia and sulphite to sulphide. Eur J Biochem, 1980 Oct, 111(2), 419 - 23 Molecular cloning of bovine thyroglobulin complementary DNA . Characterization of 2500-base-pair and 1900-base-pair fragments; Christophe D et al.; Double-stranded thyroglobulin complementary DNA (cDNA) was synthesized from purified 33-S bovine thyroglobulin mRNA . This synthetic structural gene has previously been shown to contain three sites for the restriction endonuclease HindIII, yielding two internal fragments of 1900 and 2500 base pairs respectively . Recombinant molecules were prepared by ligating the HindIII-restricted cDNA to the plasmid pBR322 which had been linearized by the same enzyme . When Escherichia coli was transformed with this mixture, it yielded two kinds of colonies each harboring recombinant plasmids containing one of the two cDNA fragments . Both recombinant molecules hybridized specifically to translatable thyroglobulin mRNA . Sequence homology between the two cloned DNAs could not be detected by cross-hybridization experiments; this argues against the existence of internal structural repetition in thyroglobulin subunits . Together, the two cloned DNA fragments represent 55% of the 8000-base-pair double-stranded thyroglobulin DNA. Proc Natl Acad Sci U S A, 1980 Oct, 77(10), 5799 - 801 Catabolite repression in Escherichia coli mutants lacking cyclic AMP receptor protein; Guidi-Rontani C et al.; Pleiotropic carbohydrate-positive pseudorevertants have been isolated from a specific class of rho-crp double mutants of Escherichia coli carrying both defective transcription termination protein, rho, and cyclic AMP receptor protein . The modulation of catabolite repression of beta-galactosidase, amylomaltase, and tryptophanase has been studied in the pseudorevertants . It has been found that these mutants exhibit catabolite repression . Because catabolite-sensitive operons can be expressed in the absence of functional cyclic AMP receptor protein, this would suggest on the one hand that the cyclic AMP-receptor protein complex is not the exclusive mediator of catabolite repression and on the other hand that rho might be involved in the regulation of catabolite-sensitive operons. J Virol, 1980 Oct, 36(1), 125 - 32 Expression of the gene for the polyoma small T antigen in Escherichia coli; Horwich A et al.; A cloned segment of the polyoma virus genome encoding the small T antigen has been fused, in the correct phase for translation, to the 5' end of the beta-galactosidase gene . The hybrid gene, cloned in Escherichia coli, produces a protein resembling the small T antigen. Gene, 1980 Oct, 11(1-2), 169 - 71 A cautionary note on the use of certain restriction endonucleases with methylated substrates; Backman K; Methylation of adenine and cytosine residues in DNA isolated from common strains of Escherichia coli K-12 can render that DNA resistant to cleavage by certain restriction endonucleases at those sites at which the recognition sequence for such an endonuclease overlaps (but does not include) a sequence recognized by methylases specified by the dam or dcm gene. J Bacteriol, 1980 Oct, 144(1), 346 - 55 Identification of a second tetracycline-inducible polypeptide encoded by Tn10; Zupancic TJ et al.; Three Tn10 polypeptides were detected by analyzing the proteins synthesized in ultraviolet light-irradiated Escherichia coli cells after infection with lambda::Tn10 . One of these polypeptides was the previously identified 36,000-dalton TET polypeptide . The other two had approximate sizes of 25,000 and 13,000 daltons . The syntheses of both the TET polypeptide and the 25,000-dalton polypeptide were inducible by tetracycline in lambda-immune hosts . Similarly, the synthesis of the TET polypeptide was inducible in nonimmune hosts . However, the synthesis of the 25,000-dalton polypeptide was constitutive in nonimmune hosts . An amber mutation in a gene required for tetracycline resistance on lambda::Tn10 was isolated that eliminated the synthesis of the TET polypeptide in sup+ hosts but not the synthesis of the 25,000-dalton or the 13,000-dalton polypeptides . The expression of tetracycline resistance from wild-type Tn10 was found to be anomalous in E . coli strains carrying the amber suppressors supD, supE, and supF . In general, strains containing these nonsense suppressors were less resistant to tetracycline. J Bacteriol, 1980 Oct, 144(1), 279 - 90 Regulation of ilvEDA expression occurs upstream of ilvG in Escherichia coli: additional evidence for an ilvGEDA operon; Subrahmanyam CS et al.; A low-copy-number plasmid was prepared that contained the entire ilv gene cluster of Escherichia coli . The introduction of an ilvO mutation allowed the ilvG gene of the plasmid to be expressed and imparted valine resistance to strains carrying it . Insertion of Tn10 into the ilvG gene of the plasmid resulted in a strong polar effect on ilv genes E, D, and A . Replacement of a region of ilv deoxyribonucleic acid between two KpnI sites on the high-copy-number plasmid carrying the entire ilv gene cluster with a KpnI fragment carrying an ilv-lac fusion but not extending into the ilv-specific control region resulted in a plasmid expressing the lacZ gene under ilv control when the fusion had been inserted in its normal orientation but not when it had been inserted in the opposite orientation . These experiments indicate that ilv-specific control over ilvE, ilvD, and ilvA expression is dependent on these genes being continguous with deoxyribonucleic acid that lies upstream of ilvG . The results also add further support to the concept of an ilvGEDA operon in E . coli. J Bacteriol, 1980 Oct, 144(1), 222 - 31 Physical and functional mapping of RP4-TOL plasmid recombinants: analysis of insertion and deletion mutants; Nakazawa T et al.; Cleavage sites for the restriction endonucleases XhoI, BamHI, HindIII, and EcoRI were mapped on the pTN2 plasmid, a recombinant of TOL and RP4, which specifies the toluene-degrading enzymes in the same way as the wild-type TOL plasmid . The pTN2 plasmid, purified from a strain of Escherichia coli, contained the entire length of the RP4 plasmid (about 54 kilobase pairs {kb}) and the TOL segment (about 56 kb) . The TOL segment is inserted at about 12 and 5 kb away from the EcoRI and BamHI cleavage sites of RP4, respectively . Cleavage sites for XhoI, BamHI, HindIII, and EcoRI were also mapped on an insertion mutant, pTN1, and two deletion mutants, pTN81 and pTN9 . Analysis of pTN81 and pTN9 allowed estimation of the region of the gene cluster for benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, as well as the region required for toluate oxygenase activity . Induction of TOL enzymes directed by pTN1 suggested the location and orientation of transcription of the gene cluster for catechol 2,3-oxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, and 2-hydroxymuconic semialdehyde hydrolase . Analysis of strains carrying both pTN9 and a xylR mutant of the TOL plasmid demonstrated that xylR+ is trans dominant over xylR. J Bacteriol, 1980 Oct, 144(1), 205 - 9 Suppression of defects in cyclic adenosine 3',5'-monophosphate metabolism in Escherichia coli; Alexander JK; Strain MM6-13 (ptsI suc lacI sup) of Escherichia coli contains a suppressor of the succinate-negative phenotype . In MM6-13, sup caused enhanced growth in glycerol, maltose, melibiose, and succinate media and increased activity of beta-galactosidase and tryptophanase relative to an isogenic strain without sup . In strain A61 (cya sup), sup partially suppressed cya . Cyclic guanosine monophosphate increased beta-galactosidase activity sevenfold in A61 and enabled this strain to grow on maltose, galactose, succinate, and arabinose . Strain A61 responded to much lower concentrations of cyclic adenosine monophosphate than cyclic guanosine monophosphate . It appears that sup is located in the crp locus . These results suggest that sup mutants have an altered cyclic adenosine monophosphate receptor protein which is activated by cyclic guanosine monophosphate and has an increased affinity for cyclic adenosine monophosphate. J Bacteriol, 1980 Oct, 144(1), 131 - 40 Insertion element IS102 resides in plasmid pSC101; Ohtsubo H et al.; In vivo recombination was found to occur between plasmid pHS1, a temperature-sensitive replication mutant of pSC101 carrying tetracycline resistance, and plasmid ColE1 after selection for tetracycline resistance at the restrictive temperature, 42 degrees C . Extensive analysis of the physical structures of three of these recombinant plasmids, using restriction endonucleases and the electron microscope heteroduplex method, revealed that the plasmid pHS1 was integrated into different sites on ColE1 . The recombinant plasmids contained a duplication of a unique 1-kilobase (kb) sequence of pHS1 in a direct orientation at the junctions between the two parental plasmid sequences . This was confirmed by comparing the nucleotide sequence of the recombinants and their parental plasmids . Nucleotide sequence analysis further revealed that nine nucleotides at the site of recombination of ColE1 were duplicated at the junction of each of the 1-kb sequences . The formation of recombinants was independent of RecA function . Based on our previous finding that a plasmid containing a deoxyribonucleic acid insertion (IS) element can recombine with a second plasmid to generate a duplication of the IS element, we conclude that the 1-kb sequence is an insertion sequence, which we named IS102 . For convenience, we have also denoted the IS102 sequence as eta theta to assign the orientation of the sequence . Eighteen nucleotides at one end (eta end) were found to be repeated in an inverted orientation at the other end (theta end) of IS102 . The nucleotide sequence of the eta end of the sequence was found to be identical to the sequence at the ends of the transposon Tn903, which is responsible for transposition of the kanamycin resistance gene. J Bacteriol, 1980 Oct, 144(1), 468 - 72 Macromolecular syntheses in an Escherichia coli adk mutant; Suit JL et al.; Preferential inhibition by high temperatures of synthesis of newly induced enzymes in Escherichia coli K-12 CR341T28 adk is only apparent; syntheses of all macromolecules cease simultaneously. J Membr Biol, 1980 Sep 30, 56(2), 169 - 75 Membrane transport of p-nitrophenyl-alpha-galactoside by the melibiose carrier of Escherichia coli; Ottina K et al.; p-Nitrophenyl-alpha-galactoside (alpha-pNPG) was found to be a substrate for the melibiose transport system of Escherichia coli . This sugar enters induced cells via the carrier and is split by alpha-galactosidase to galactose and p-nitrophenol . In mutant cells lacking the alpha-galactosidase {3H}-alpha-pNPG accumulated to concentrations 15 times higher than the external medium . The transport of alpha-pNPG is inhibited by both Na+ and Li+ . Na+ (10 mM) reduced the Km for alpha-pNPG from 0.45 to 0.18 mM and reduced the Vmax from 6.7 nmoles/min/mg dry wt to a value of 3.0. Biochemistry, 1980 Sep 30, 19(20), 4627 - 32 Methylation of chemotaxis-specific proteins in Escherichia coli cells permeable to S-adenosylmethionine; Rollins CM et al.; Using a modification of the EGTA treatment of Oishi and Smith {Oishi, M., & Smith, C . L . (1978) Proc . Natl . Acad . Sci . U.S.A . 75, 3569}, Escherichia coli cells have been made permeable to S-adenosylmethionine and other related molecules in order to facilitate the study of methylation in chemotaxis . The permeable cells are nonmotile but respond to chemotactic stimuli by reversible methylation of their methyl-accepting chemotactic proteins (MCP I and MCP II) in a manner similar to that of untreated, motile cells . Addition of S-adenosyl-L-{methyl-3H}methionine to the permeable cells specifically labels two proteins, MCP I and MCP II . Methylation of these MCP's is dependent on the presence of wild-type gene products of flaI, flaA, cheB, cheX, tsr, and tar . The extent of methylation of the MCP's is affected by the presence of attractants or repellents: addition of attractant increases the steady-state level of methylation; addition of repellent causes rapid demethylation to a new steady-state level . Methylation is inhibited by the addition of the transmethylase inhibitors A9145C and Sinefungin, which are S-adenosylmethionine analogues, and by S-adenosylhomocysteine. Biochemistry, 1980 Sep 30, 19(20), 4633 - 9 Chemotaxis in Escherichia coli: associations of protein components; Chelsky D et al.; Interactions between protein components of the chemotaxis mechanism in Escherichia coli were investigated by using the cleavable cross-linking reagent, dithiobis(succinimidyl propionate) . Two methods were used to allow detection of chemotaxis-specific proteins in intact cells . The first method was to program their synthesis in the presence of {35S}methionine using lambda E . coli hybrid phages which carry the chemotaxis genes . The second method was to label endogenous methyl-accepting chemotaxis proteins (MCP's), with the methyl donor S-adenosyl-L-{methyl-3H}methionine, after permeabilizing the cells with EGTA . Physical associations between proteins were analyzed, after cross-linking, by two dimensional NaDodSO4-polyacrylamide gel electrophoresis . Both labeling methods demonstrate that MCP I and MCP II exist as functional tetramers . Other proteins involved with chemotaxis were found to form dimers and higher polymers . Phage-directed products of cheW, cheX, motA, and cheA formed dimers . CheB and hag products formed multimers . A number of apparent interactions between different gene products were detected as well . Products of cheB, cheW, cheZ, motA, and motB were found to form complexes with other gene products . Included are results consistent with interactions between the products of cheB and cheZ. Proc R Soc Lond B Biol Sci, 1980 Sep 26, 209(1176), 431 - 9 The early pathogenesis of bovine mastitis due to Escherichia coli; Frost AJ et al.; The pathogenesis of coliform mastitis was studied after infusing each of ten lactating quarters of three dairy cows with a large dose (ca . 1 x 10(9) colony-forming units) of virulent Escherichia coli strain B117 . This approach was adopted first to maximize the chance of observing microscopic lesions in the tissues of a gland and secondly to overwhelm the differences that might be shown between animals in their response to the infection . The infected glands were examined at intervals of up to 4 h after infection by scanning and transmission electron microscopy and by light microscopy . The earliest lesions were seen after 1 h and consisted of necrosis and sloughing of the epithelial cells of the teat and lactiferous sinuses . After 2 h this was more severe, and was followed by an intense neutrophil response . Neutrophils migrated through the epithelial lesions and at first remained attached to the epithelial surface, forming large mounds . This resulted in gross underestimation of the number of cells in the lumen of the gland when neutrophils in the secretion were counted . At no stage was there evidence of attachment of organisms to the epithelial cells . Tissue damage did not extent beyond the basement membrane, which helps to explain the rapid clinical resolution seen in most field cases of the disease . There was considerable variation in the degree of response shown by the three cows, and also within the infected glands, where the damage was most severe in the lactiferous and teat sinuses . It seems unlikely that all aspects of the disease could be attributed to endotoxin. Nucleic Acids Res, 1980 Sep 25, 8(18), 4201 - 19 In vitro transcription of chromatin containing histones hyperacetylated in vivo; Dobson ME et al.; The culture of cells in the presence of sodium n-butyrate causes an accumulation of histones that are highly acetylated . When chromatin containing these histones was transcribed with E . coli RNA polymerase, an increase in the template activity compared to control chromatin was observed . Titration of chromatin with polymerase under both reinitiating and non-reinitiating conditions showed there was no increase in the number of regions available for transcription . Comparison of the kinetics for single and multiple rounds of transcription indicated that the rate of elongation was increased and probably the rate of reinitiation as well . Comparison of the size of transcripts from control and acetylated chromatin showed a small increase in the average size of transcripts from acetylated chromatin . When transcription was compared using partially purified HeLa polymerase, an increase was also seen . Studies under various ionic conditions showed that control chromatin required a higher salt concentration for optimum activity than did acetylated chromatin . In addition, at the optimum salt concentration for each chromatin, there was very little difference in the transcriptional activity using exogenous HeLa RNA polymerase. J Biol Chem, 1980 Sep 25, 255(18), 8431 - 6 Transient kinetic analysis of the catalytic cycle of alkaline phosphatase; Bale JR et al.; Transient kinetic studies were carried out to elucidate the catalytic cycle of Escherichia coli alkaline phosphatase at ph 8.3, 10 degrees C, and to evaluate the rate constants for the individual steps . Using a rapid mixing cell, we were able to detect the burst phase of the reaction, which could not be obtained at alkaline ph values with conventional mixing devices . Analysis of the burst phase revealed that the equilibrium of the initial binding of the substrate, 4-methylumbelliferyl phosphate, to the enzyme is attained rapidly, and that the production of the alcohol, 4-methylumbelliferone, is fast . Analysis of the steady state phase of the reaction yielded a phosphate release rate constant which agrees very well with the kcat determined by initial rate studies . Dephosphorylation of the phosphoryl enzyme prepared at pH 5.7 was studied by the pH-jump technique, using a three-syringe stopped flow apparatus . The results showed that the dephosphorylation step is not rate-limiting in the catalytic cycle and that the presence of substrates or inhibitor has no effect on this step . The lack of effect of substrates on the rate of dephosphorylation and on the rate of phosphate dissociation indicates that the flip-flop mechanism, in which the product release is supposedly facilitated by the binding of a 2nd molecule of substrate, is not valid for alkaline phosphatase . Kinetic constants for various steps in the catalytic cycle of alkaline phosphatase at pH 8.3, 10 degrees C, are reported and a reaction scheme which is in harmony with previous observations from other laboratories is described. J Biol Chem, 1980 Sep 25, 255(18), 8366 - 9 Identification of a cytoplasmic membrane-associated component of the maltose transport system of Escherichia coli; Bavoil P et al.; The maltose transport system of Escherichia coli contains at least five components, three of which, i.e . the products of lamB, malE, and malF genes, have so far been identified as constituents of the outer membrane, periplasmic space, and cytoplasmic membrane, respectively . We identified another component, a cytoplasmic membrane protein of an apparent molecular weight of 43,000, as the product of the malK gene on the basis of polyacrylamide gel electrophoretic analysis of various mutants and suppressed strains and by the incorporation of extra tyrosine residue into this proten in malK amber mutants containing the suppressor Su3+ allele . The transport of maltose thus appears to require at least two proteins associated with the cytoplasmic membrane. J Biol Chem, 1980 Sep 25, 255(18), 8706 - 10 Methylation of newly synthesized ribosomal protein L11 in a DNA-directed in vitro system; Jerez C et al.; The methylation of newly synthesized ribosomal protein L11 has been obtained in an in vitro system using lambda rifd 18 DNA as template and S-adenosyl{3H}methionine as methyl donor . About four methyl groups are incorporated per mol of L11 synthesized and the bulk of the methyl groups are present in the protein as trimethyl-lysine . lambda rifd 18 DNA also contains the genes for rRNA and newly synthesized 16 S and 23 S RNA are also methylated in this in vitro system. Nucleic Acids Res, 1980 Sep 25, 8(18), 4235 - 46 Involvement of DNA gyrase in the transcription of ribosomal RNA; Oostra BA et al.; The DNA gyrase inhibitor novobiocin specifically inhibits the transcription of ribosomal RNA in vivo while protein synthesis and the mRNA transcription are only partly affected . In vitro the novobiocin inhibition is only observed when protein fraction, which stimulates ribosomal RNA synthesis, is present . These results indicate that DNA gyrase is involved in the transcription of ribosomal RNA, probably at an initiation step. Nucleic Acids Res, 1980 Sep 25, 8(18), 4165 - 84 Equilibrium melting of plasmid ColE1 DNA: electron-microscopic visualization; Borovik AS et al.; The fine structure of the melting curve for the linear colE1 DNA has been obtained . To find the ColE1 DNA regions corresponding to peaks in the melting curve's fine structure, we fixed the melted DNA regions with glyoxal /12/ . Electron-microscopic denaturation maps were obtained for nine temperature points within the melting range . Thereby the whole process of colE1 DNA melting was reconstructed in detail . Spectrophotometric and electron microscopic data were used for mapping the distribution of Gc-pairs over the DNA molecule . The most AT-rich DNA regions (28 and 37% of GC-pairs), 380 and 660 bp long resp., are located on both sides of the site of ColE1 DNA's cleavage by EcoR1 endonuclease . The equilibrium denaturation maps are compared with maps obtained by the method of Inman /20/ for eight points of the kinetic curve of ColE1 DNA unwinding by formaldehyde. J Biol Chem, 1980 Sep 25, 255(18), 8928 - 35 Cloning of fragments of lambda dapB2 DNA and identification of the dapB gene product; Mackie GA; DNA of the specialized transducing phage lambda dapB2 has been digested with the restriction endonucleases Bam I, HindIII, or both together to generate fragments originating from the bacterial substitution on the phage . Seven such fragments ranging in size from 0.8 to 7.1 kilobases and encompassing the entire bacterial substitution of 18 kilobases of DNA have been covalently ligated to the vector pBR322 . The recombinant plasmids so constructed have been tested for their ability to complement the dapB17 allele in Escherichia coli strain AT999 . Only pGM4, which contains a 7.1 kilboase fragment generated by Bam I cleavage of lambdadapB2 inserted into pBR322, relieves this strain's requirement for DL-diaminopimelic acid and restores dihyrodipicolinic acid reductase activity to wild type levels . Deletions were obtained in pGM4 by two methods . None of the resultant shortened plasmids were proficient in complementation of the dapB17 allele . The proteins encoded by the parental plasmid and by those of seven deletions derived from it have been identified by coupled transcription and translation of plasmid DNA templates in vitro or by the stimulation of protein synthesis promoted by these plasmids in an ultraviolet irradiated host . The parent encodes four proteins unique to the 7.1 kilobase insert whose apparent molecular weights are 48,000, 36,000, 32,000, and 17,000 . Of these, the protein of 32,000 is consistently missing when noncomplementing pasmids harboring deletions are used as templates . This protein is tentatively identified as the product of the dapB gene . The role of the other three proteins whose genes are closey linked to the dapB gene is unknown . There appear to be at least two transcriptional units within this cluster of genes, however, suggesting independent regulation and, possibly, function. J Biol Chem, 1980 Sep 25, 255(18), 8655 - 62 Structure of the rat prolactin gene; Gubbins EJ et al.; The organization and sequence of the rat preprolactin gene has been investigated . Analysis of two different plasmids containing pituitary cDNA inserts has provided the complete 681-nucleotide coding sequence of preprolactin as well as 17 nucleotides preceding the initiation codon and 90 nucleotides following the termination codon . Digestion of rat chromosomal DNA with the restriction endonuclease Eco RI followed by size fractionation and hybridization to a labeled prolactin cDNA probe has demonstrated that prolactin genomic sequences are located on 6.0-, 3.9-, and 2.9-kilobase fragments . The 6.0- and 3.9-kilobase fragments were isolated from a library of cloned rat DNA fragments . The sequence of more than 1800 nucleotides of the cloned DNA has been determined . The sequenced region contains coding regions of 180 and 189 nucleotides which specify the COOH-terminal 123 amino acids of the 227-amino-acid sequence of rat preprolactin . These coding regions are separated by an intervening sequence of 597 nucleotides . At least one other large intervening sequence separates this region from the region coding for the NH2-terminal portion of preprolactin . Hybridization experiments suggested that the intervening sequences of the rat prolactin gene contain DNA sequences which are repeated elsewhere in the rat genome. J Biol Chem, 1980 Sep 25, 255(18), 8623 - 8 Characterization of yeast iso-1-cytochrome c mRNA; Boss JM et al.; The iso-1-cytochrome c mRNA has been identified by hybridization of a 32P probe prepared from a plasmid containing the iso-1-cytochrome c gene to RNA size-fractionated on agarose gels and transferred to paper . A hybridization band was visible with RNA prepared from wild type cells, but not with RNA prepared from an iso-1-cytochrome c deletion mutant . RNA prepared from cells containing a nonsense mutation in the iso-1-cytochrome c gene showed reduced levels of hybridization . The RNA that hybridized to the probe was 700 +/- 50 nucleotides in length and was polyadenylated . The cellular levels of this RNA were repressed by glucose, and this repression was achieved within 5 min after glucose addition to a derepressed culture . No precursors of this RNA were detected in wild type cells or in an RNA1 mutant, temperature-sensitive for RNA metabolism . The length of the 3' noncoding region of this RNA was determined to be 200 +/- 25 nucleotides (excluding the poly(A) tail) and the 5' noncoding region was estimated to be about 120 nucleotides in length. Nucleic Acids Res, 1980 Sep 25, 8(18), 4131 - 42 The role of the basic N-terminal region of protein L18 in 5S RNA-23S RNA complex formation; Newberry V et al.; Of the three proteins, L5, L18 and L25, which bind to 5S RNA, the former two effect the interaction of 5S RNA with 23S RNA . We have used trypsin as a probe to investigate the roles of the proteins in this RNA-RNA assembly, with the following results: (1) In complexes with 5S RNA, the highly basic N-terminal region of L18 is accessible to trypsin . This accessibility is unaffected by L25 . However, its presence is essential for stimulating L5 binding . (2) In 5S RNA-protein-23S RNA complexes proteins L5 and L18 are both strongly resistant to proteolysis . (3) No 5S RNA-23S RNA complex formation occurs in the presence of L5 and the C-terminal L18 fragment . Two possible models for the mechanism of RNA-RNA assembly are proposed. Biochim Biophys Acta, 1980 Sep 19, 609(2), 313 - 20 A circular dichroism study of Escherichia coli Initiation Factor-1 binding to polynucleotides; Schleich T et al.; Binding of Escherichia coli Initiation Factor-1 protein to the nucleic acid lattice induces alterations in the secondary structures of a variety of purine and pyrimidine containing polynucleotides in both the single and double stranded conformations, as assessed by circular dichroism spectroscopy . The helical hairpin form of poly(U), the single-stranded stacked form of poly(C), and the duplex poly(A) x poly(U) (in the presence of Mg2+) are stoichiometrically converted by Initiation Factor-1 (IF-1) to structures spectrally indistinguishable from their partially or completely thermally denatured forms . By contrast, the binding of IF-1 to double stranded poly(C), single- and double-stranded poly(A) elicited spectral responses which were interpreted in terms of diminished base-base interaction, not equivalent to that induced by thermal means . Stoichiometric endpoints of 3-5 nucleotide residues/IF-1 were determined for polynucleotide structures in those cases where light scattering artifacts at low nucleotide residue to protein ratios were absent . In the absence of Mg2+ IF-1 was unable to elicit a conformation alteration effect in poly(A) x poly(U), while for poly(U) much less of an effect was observed than in the presence of this divalent ion . The functional significance of these results is briefly considered. Biochim Biophys Acta, 1980 Sep 19, 609(2), 264 - 71 Specificity of the spermidine requirement for the replication of phi X174 DNA be cell-free extracts of Escherichia coli; Geiger LE et al.; A new experimental approach for assessing the biological significance of spermidine interactions in isolated systems is applied to the stimulation by spermidine of the conversion of phi X174 virion DNA to its replicative form by cell-free extracts of Escherichia coli . At 2.5 mM Mg2+, spermidine activated the reaction 20-fold . Varying the spermidine concentration affected both the rate and extent of this DNA synthetic reaction without altering the nature of the reaction products . We evaluated the biological significance of the spermidine requirement by measuring reaction rates in the presence of a homologous series of spermidine analogs of known activity in vivo . There was a lack of specificity, in that all of these analogs were capable of efficiently substituting for spermidine in stimulating the reaction rate . The relevance of this in vitro spermidine stimulation to Escherichia coli chromosome replication in vivo is discussed in light of the results obtained with the spermidine analogs. Science, 1980 Sep 19, 209(4463), 1343 - 7 At least three human type alpha interferons: structure of alpha 2; Streuli M et al.; The sequence of a human leukocyte-derived complementary DNA (cDNA), Hif-2h, which directs the formation in Escherichia coli of a polypeptide, IFN-alpha 1, with interferon (IFN) activity has been described . A second IFN cDNA, Hif-SN206, which also elicits synthesis of a biologically active IFN, IFN-alpha 2, is described in this article . Whereas IFN-alpha 2 is twice as active on human as on bovine cells, IFN-alpha 1 is 10 to 20 times more active on bovine than on human cells . As deduced from the cDNA's, the messenger RNA's for the two IFN's differ in length and in 20 percent of the nucleotides; the mature IFN polypeptides differ in 17 percent of the amino acids . Both IFN-alpha 1 and IFN-alpha 2 differ from the lymphoblastoid IFN described by others . Therefore, at least three different IFN-alpha genes are expressed in man; studies on genomic DNA reveal the presence of at least eight IFN-related genes. Nature, 1980 Sep 18, 287(5779), 203 - 8 DNA N-glycosylases and UV repair; Demple B et al.; Repair of some DNA photoproducts can be mediated by glycosylic bond hydrolysis . Thus, Escherichia coli endonuclease III releases 5,6-hydrated thymines as free bases, while T4 UV endonuclease releases one of two glycosylic bonds holding pyrimidine dimers in DNA . In contrast, uninfected E . coli apparently does not excise pyrimidine dimers via a DNA glycosylase. Biochim Biophys Acta, 1980 Sep 17, 632(1), 35 - 46 Mode of action of heat-stable Escherichia coli enterotoxin . Tissue and subcellular specificities and role of cyclic GMP; Rao MC et al.; Some enteric strains of Escherichia coli release a heat-stable enterotoxin which, in contrast to cholera and heat-labile E . coli enterotoxins, stimulates guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) . We have examined the tissue spcificity of its action and the relation of its action to those of the 8-bromo analogues of cyclic GMP and cyclic AMP . Heat-stable enterotoxin stimulated guanylate cyclase activity and increased cyclic GMP concentration throughout the small and large intestine . It increased transepithelial electric potential difference and short-circuit current in the jejunum, ileum and caecum but not in the duodenum or distal colon . This pattern of electrical responses was mimicked by 8-bromo-cyclic GMP . However, 8-bromo-cyclic AMP produced an electrical response in all intestinal segments . The enterotoxin failed to stimulate guanylate cyclase inliver, lung, pancreas or gastric antral mucosa . In the intestines, it stimulated only the particulate and not the soluble form of the enzyme . Preincubation of the toxin with intestinal membranes did not render it capable of stimulating pancreatic guanylate cyclase . Cytosol factors did not enhance the toxin's stimulation of intestinal guanylate cyclase . This study supports the role of cyclic GMP as intracellular mediator for heat-stable enterotoxin and suggests that the toxin affects a membrane-mediated mechanism for guanylate cyclase activation that is unique to the intestines. Biochemistry, 1980 Sep 16, 19(19), 4527 - 33 Mechanism of reconsitution of the apo beta 2 subunit and the alpha 2 apo beta 2 complex of tryptophan synthase with pyridoxal 5'-Phosphate: kinetic studies; Bartholmes P et al.; The mechanism of pryidoxal 5'-phosphate (PLP) binding to both the alpha apo beta 2 complex and the apo beta 2 subunit of tryptophan synthase was investigated by rapid mixing experiments . Absorption and fluorescence changes were used to monitor the binding reaction directly . Reduction with sodium borohydride provided the rate of formation of the internal aldimine with the lysine amino group of the enzyme, and substrate turnover monitored the rate of formation of acive enzyme . The alpha 2 apo beta 2 complex binds PLP in a sequence of three steps of decreasing rate: formation of a noncovalent complex, which isomerizes to an enzymically inactive internal aldimine, followed by formation of an active alpha 2 holo beta 2 complex . The two binding sites appear to bind PLP independently . The apo beta 2 subunit binds PLP cooperatively in a sequence of three steps of decreasing rate: formation of a nonconvalent complex, which isomerizes to an enzymically inactive internal aldimine, followed by the formation of the enzymically active holo beta 2 subunit . Taken together with kinetic studies of pyridoxine phosphate binding {Tschopp, J., & Kirschner, K . (1980) Biochemistry (second paper of three in this issue)}, the rate data of the apo beta 2 subunit are shown to be consistent with the concerted mechanism . The difference between the values of the isomerization rate constants of bound PLP and bound PNP appear to result from the convalent internal aldimine, which is formed with PLP but not with PNP. Biochemistry, 1980 Sep 16, 19(19), 4514 - 21 Subunit interactions of tryptophan synthase from Escherichia coli as revealed by binding studies with pyridoxal phosphate analogues; Tschopp J et al.; An improved purification procedure for the alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli has been developed . It consists of DEAE-Sephacel chromatography, followed by hydrophobic chromatography on Sepharose CL 4B, and leads to material with a higher specific activity than reported previously . Inhibition studies, equilibrium dialysis, and spectrophotometric titration were used to study the binding both of pyridoxal phosphate analogues and of bisubstrate analogues . Pyridoxine 5'-phospate and N-phosphopyridoxyl-L-serine bind to the enzyme, but pyridoxamine 5'-phoshate and N-phosphopridoxyl-L-alanine do not . N-Phosphopyridoxyl-L-tryptophan is bound only weakly, although L-tyrptophan binds strongly to the alpha 2 holo beta 2 complex . It is likely that either differences is protonation or in geometry are responsible for the low affinity of the bisubstrate analogues in comparison to that of the external aldimines of either L-serine or L-tyrptophan with pyridoxal 5'-phosphate . As previously found with pyridoxal 5'-phosphate, pyridoxine 5'-phosphate, and N-phosphopryidoxyl-L-serine bind noncooperatively to two identical binding sites in the alpha 2 apo beta 2 complex . The same ligands bind with positive cooperatively to two binding sites in the apo beta 2 subunit . Because the analogues mimic the binding behavior of pyridoxal 5'-phosphate to both proteins, the internal aldimine of pyridoxal 5'-phosphate to the lysine amino group contributes only to the strength of that binding . The nickel apo beta 2 subunit, which is produced by limited proteolysis with trypsin, binds pyroxine 5'-phosphate noncooperatively to two identical sites . Therefore, the loop of polypeptide chain connecting the two autonomous domains of folding must be intact for enzyme activity, for the binding of the alpha subunit, and for cooperative binding of pyridoxine 5'-phosphate. Biochemistry, 1980 Sep 16, 19(19), 4486 - 92 Circular dichroism study of Escherichia coli initiation factor 3 binding to nucleic acids; Schleich T et al.; The circular dichroic spectral features of (A)10-20, (C)10-20, A8UGU6, poly(A), and poly(C), at both neutral and acidic pH values and in the presence and absence of Mg2+, are significantly altered by Escherichia coli initiation factor 3 (IF3), implying the occurrence of protein-induced changes in nucleic acid secondary structure . Similarly, the circular dichroic spectral characteristics of helical poly(U), poly-(A)-poly(U), and poly(I)-poly(C) are modified by IF3 . However, no structural perturbation of poly(A)-poly(U) occurs in the absence of Mg2+ by IF3 . The oligonucleotides (A)10-20 and (C)10-20 at both pH 7.5 and 5.5 titrate to end point of 26 +/- 4 nucleotide residues per IF3 {except (C) 10-20 at pH 5.5 which titrates to 17 +/- 1 nucleotide residues per IF3}, whereas the hairpin A8UGU6 under similar conditions at neutral pH and in the presence of Mg2+ titrates to an end point of 56 +/- 3 nucleotide residues per IF3, thereby suggesting the presence of multiple binding sites on the protein . By contrast, poly(A) and poly(C) at neutral pH and in the absence of Mg2+ titrate to an end point of 13 +/- 1 nucleotide residues per IF3 . The occurrence of significant light-scattering artifacts precluded a determination of the end point stoichiometry in most other cases . The circular dichroic spectra of E . coli tRNA, MS2 RNA, phiX174 DNA, and sonicated calf thymus DNA were unaffected by IF3 at physiological concentrations . Addition of an equimolar mixture of IF3 and ribosomal protein S1 titrates the circular dichroism of poly(C) at acid pH as did S1 alone . However, addition of IF3 to mixture of poly(A) and S1 at neutral pH did not result in significant titration of the optical activity until IF3 was in excess over S1, even though filter binding assays indicate normal IF3 binding to the polynucleotide . The possible relation of these observations to the biological function of IF3 is briefly considered. Biochemistry, 1980 Sep 16, 19(19), 4521 - 7 Kinetics of cooperative ligand binding to the apo beta 2 subunit of tryptophan synthase and its modulation by the alp ha subunit; Tschopp J et al.; The different binding mechanisms of pyridoxine 5'-phosphate and N-phophopridoxyl-L-serine have been investigated by kinetic studies with rapid reaction techniques . Pyridoxine 5'-phosphate binds in a single rapid step to the alpha 2 apo beta 2 complex and in a single slow step to the nicked apo beta 2 subunit that is obtained by limited proteolysis with trypsin . Both pyridoxine 5'-phosphate and N-phosphopyridoxyl-L-serine bind to the apo beta 2 subunit with a comparatively slow binding step, followed by an event slower isomerization reaction . These findings are consistent with nonexclusive concerted mechanism of cooperative binding but cannot be explained by the simple sequential mechanism . A quantitative fit of the rate and equilibrium data to the concerted mechanism generally yielded the pertinent rate and equilibrium constants . In particular, the same value of L0 = {T0}/{R0} = 200 +/- 50 simultaneously satisfies the data obtained with three different ligands . The comparison of the mechanisms of ligand binding to the three states of the apo beta 2 subunit suggests that the alpha 2 apo beta 2 complex is similar to the high-affinity R state and the nicked apo beta 2 subunit is similar to the low-affinity T state of the apo beta 2 subunit . The slow isonerization involved in the cooperative binding of the ligands to the intact apo beta 2 subunit is discussed in terms of local and concerted conformational changes involving the two autonomously folding domains of the beta protomer. C R Seances Acad Sci D, 1980 Sep 15, 291(2), 203 - 6 {Inhibition of the 3' to 5' exonuclease activity of the DNA polymerase I of Escherichia coli by deoxyribonucleotides}; Granger M et al.; The 3' a 5' exonuclease activity of E . coli DNA-polymerase I is inhibited by nucleotides and deoxynucleotides at concentrations (< 1 mM) where polymerase activity is not affected . This inhibitory effect depends on the nature of the excised deoxynucleotide, excision of purines being much less inhibited than that of pyrimidines . It does not depend on the purine or pyrimidine nature of the inhibitor. Nucleic Acids Res, 1980 Sep 11, 8(17), 3851 - 64 Use of RNA polymerase as an enzymatic probe of nucleosomal structure; Hodo HG 3rd et al.; Nucleosomes prepared from human placental nuclei and Escherichia coli DNA-dependent RNA polymerase (nucleoside triphosphate: RNA nucleotidyl transferase EC.2.7.7.6) form stable initiation complexes . This property is utilized as a probe of nucleosome structure . RNA polymerase initiation has been studied on purified nucleosomes, nucleosome cores, and nucleosomal DNA . The affinity of E . coli RNA polymerase for both nucleosome cores and monomers was 5-6 fold less than found for nucleosomal DNA . No difference in apparent initiation Km was found between cores and mononucleosomes . This suggests that initiation does not preferentially occur on the DNA tails of nucleosomes . Once initiated and allowed to form nascent RNA, these complexes are very stable to ionic strength changes . Under conditions in which free enzyme is inactivated with rifampicin, the enzyme in the complex retains activity as demonstrated by its ability to transcribe and reinitiate on both nucleosomes and free DNA . These complexes can be well resolved from free nucleosomes on preparative polyacrylamide gels and both can be eluted from gels for analysis of proteins and DNA sequence complexity . Studies using (125I) labelled nucleosomes show that histones are retained in the initiation complex, and are not dissociated by the enzyme during initiation. Nucleic Acids Res, 1980 Sep 11, 8(17), 3865 - 74 DNA gyrase subunit stoichiometry and the covalent attachment of subunit A to DNA during DNA cleavage; Sugino A et al.; Escherichia coli DNA gyrase contains a 1:1 ratio of protomers coded by the genes gyrA and gyrB . This along with previous results shows that the enzyme has two copies of each protomer and thus a molecular weight of 400,000 . Abortion of the gyrase reaction results in double-strand breakage of the DNA and covalent attachment of both gyrA protomers to the 5'-cut ends . We conclude that the gyrA protomer contains a critical part of the active site for the concerted breakage and reunion reaction of gyrase, the topoisomerase activity of the enzyme. Nucleic Acids Res, 1980 Sep 11, 8(17), 3809 - 27 Sequence of the distal tRNA1Asp gene and the transcription termination signal in the Escherichia coli ribosomal RNA operon rrnF(or G); Sekiya T et al.; Several DNA fragments carrying tRNA genes have been cloned from EcoRI endonuclease digests of Escherichia coli DNA . Using cloned DNA, the sequence of the region around the distal gene for tRNA1Asp (F(or G)) in the E . coli ribosomal RNA operon {rrnF(or G)} has been determined . In the distal portion of rrnF(or G), the genes for 23S, 5S rRNA and tRNA1Asp (F(or G)) are located in that order and separated by intergenic spacers of 93 and 52 base pairs, respectively . A possible hairpin structure, with its center between the 22nd and 23rd base pair downstream from the 3'-end of the tRNA1Asp(F(or G)) gene, followed by a sequence of eight thymidine residues was identified as the transcription termination signal for rrnF(or G) . The termination is rho-independent, at least in vitro, and occurs within the region of the contiguous thymidine residues . A possible promoter for a protein gene is present about 50 base pairs downstream from the rrnF(or G) terminator. J Biol Chem, 1980 Sep 10, 255(17), 8109 - 15 Subunit interaction during catalysis . ATP modulation of catalytic steps in the succinyl-CoA synthetase reaction; Bild GS et al.; A new approach for assessing of catalytic cooperativity may occur between subunits has been applied to succinyl-CoA synthetase . This is based on the extent of oxygen exchange between medium {18O}Pi and succinate per molecule of ATP cleaved during steady state succinyl-CoA synthesis . Suitable traps are used to remove succinyl-CoA and ADP as soon as they are released to the medium . With the Escherichia coli enzyme, which has an alpha 2 beta 2 structure, a pronounced increase in oxygen exchange per ATP cleaved occurs as ATP concentration is lowered . In contrast, when the CoA concentration is varied, the oxygen exchange per molecule of product formed remains constant . Also, with the pig heart enzyme, which is shown to retain its alpha beta structure during catalysis and thus has only one catalytic site, no modulation of oxygen exchange by ATP concentration is observed . These experimental findings show that the binding of an ATP either promotes the dissociation of bound succinyl-CoA or decreases its participation in exchange . Measurement of the distribution of {18O}Pi species found as exchange occurs shows that only one catalytic sequence is involved in exchange at various ATP concentrations . These observations along with other controls and results eliminate most other explanations of the ATP modulation of the exchange and suggest that binding of ATP at one catalytic site promotes catalytic site promotes catalytic events at an alternate catalytic site. J Biol Chem, 1980 Sep 10, 255(17), 8027 - 30 On the mechanism of ribonucleoside diphosphate reductase from Escherichia coli . Evidence for 3'-C--H bond cleavage; Stubbe J et al.; The 3'-carbon--hydrogen bond of {3'-3H}uridine 5'-diphosphate is cleaved during its conversion to 2'-deoxyuridine 5'-diphosphate catalyzed by Esherichia coli ribonucleoside diphosphate reductase . A selection against 3H of approximately 3.3 is observed on this reduction reaction . During the course of this reaction, a small but significant amount of 3H is released to the solvent. J Biol Chem, 1980 Sep 10, 255(17), 8116 - 20 Interaction of 70 S ribosomes from Escherichia coli with spin-labeled N-Cbz-Phe-tRNAPhe . An electron paramagnetic resonance study; Rodriguez A et al.; Two selectively spin-labeled Cbz-Phe-tRNAsPhe, one at position s4U8 and the other at position U33, have been used to study the dynamics of tRNA-ribosome interaction in the presence of poly(U) and factors washable from ribosomes . Upon binding to the ribosome, the correlation time of the spin label at position s4U8 decreases markedly while the same parameter for the label in the anticodon increases . The presence of poly(U) is not a prerequisite condition for the EPR spectral changes observed but larger variation occurs in the presence of factors washable from ribosomes . No variation in the correlation time is observed if uncharged spin-labeled tRNAPhe (on the s4U8 residue) is used in these experiments . Most of the ribosome-bound spin-labeled Cbz-Phe-tRNAPhe are puromycin-reactive, and consequently, the observed effect is manifested mainly at the ribosomal P site . These observations seem to suggest that the interaction between the N-blocked aminoacyl residue on the tRNA and the ribosome results in a conformational change on the tRNA, possibly involving tertiary interactions in a region close to s4U8 . The role that the amino acid at the 3'-end can possibly play on this structural change is discussed. J Biol Chem, 1980 Sep 10, 255(17), 8069 - 73 Enzyme-catalyzed DNA unwinding . The role of ATP in helicase III activity; Das RH et al.; The enzyme helicase III catalyzes ATP-dependent unwinding of double-stranded DNA (Yarranto, G . T., Das, R . H., and Gefter, M . L . (1979) J . Biol . Chem . 254, 11997-12001) . The free enzyme is able to bind to double- and single-stranded DNA . In the presence of ATP the enzyme can bind single- but not double-stranded DNA . The enzyme catalyzes an ADP-ATP exchange reaction in the absence of DNA . It is suggested that there is an enzyme.phosphate complex that discriminates between the two forms of DNA . These results are discussed in relation to a model that accounts for catalytic unwinding of DNA coupled to ATP hydrolysis. Biochim Biophys Acta, 1980 Sep 9, 615(1), 59 - 69 Incorporation of amino acid analogs during the biosynthesis of Escherichia coli aspartate transcarbamylase; Gueguen P et al.; Amino acid-requiring mutants capable of producing derepressed levels of aspartate transcarbamylase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) were obtained and used for the incorporation in this enzyme of eight different amino acid analogs . These amino acid replacements enabled the biosynthesis of a series of modified aspartate transcarbamylases altered in their catalytic or regulatory properties . The enzyme in which phenylalanine was rereplaced by 2-fluorophenylalanine was purified to homogeneity and appeared to have the same specific activity as normal asparate transcarbamylase but lacking both homotropic and heterotropic interactions. Biochim Biophys Acta, 1980 Sep 9, 615(1), 10 - 8 The binding of a fluorescent activator 2-(N-decyl)aminonaphthalene-6-sulfonic acid to pyruvate oxidase; Schrock HL et al.; E . coli pyruvate oxidase (pyruvate:ferricytochrome b1 oxidoreductase, EC 1.2.2.2) is a peripheral membrane flavoenzyme which has been purified to homogeneity . In vivo the oxidase resides on the inner surface of the cytoplasmic membrane and is coupled to the bacterial electron transport chain . In vitro, the purified oxidase requires lipids for full enzymatic activity . Previous studies have characterized the conformational and energetic coupling between the lipid-binding site(s) and the catalytic active site . The affinity of the enzyme for phospholipids and detergents is significantly enhanced when the flavoprotein is in the reduced form, i.e., in the presence of pyruvate and the required cofactor, thiamin pyrophosphate . The lipid-binding studies were hindered due to the complicating factor of the self-association of the substrate-reduced flavoprotein . In this paper, fluorescence techniques are employed to measure the binding of a detergent-like activator to the oxidase . The experiments are performed at much lower protein concentrations than previously employed, so that protein aggregation is not a problem . The chromophore on the activator, 2-(N-decyl)aminonaphthalene-6-sulfonic acid is effective at quenching the pyruvate oxidase intrinsic tryptophan fluorescence . Quenching titrations are used to obtain the binding isotherm . AT DNS concentrations less than 10(-5) M, the results show a larger amount of DNS binding to the reduced flavoprotein than to the oxidized form of the enzyme . This is the concentration range where DNS is an effective activator of the enzyme . This represents a class of binding sites specifically found on pyruvate oxidase and not apparent in other proteins such as lysozyme or aldolase . At the DNS concentration which is optimum for activation approx . 20 molecules of DNS are bound per enzyme tetramer in the absence of the substrate . The pyruvate-reduced form of the enzyme binds about 40--50 molecules of DNS per tetramer . Qualitatively, the results are similar to what was previously found for both sodium dodecyl sulfate and cetyl trimethylammonium bromide . However, in both these cases, the amount of bound detergent was nearly an order of magnitude less than the values obtained using DNS. Biochim Biophys Acta, 1980 Sep 9, 615(1), 103 - 12 Further kinetic characterization of the non-allosteric phosphofructokinase from Escherichia coli K-12; Ewings KN et al.; The labile non-allosteric form of phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) was purified to a specific activity of 107 U/mg (2078-fold) from aerobic cultures of Escherichia coli K-12 . The enzyme has an isoelectric point (pI) of 5.1, a native molecular weight of 67 000 +/- 3000 and a subunit weight of 34 000 +/- 400 . A number of divalent metal ions can substitute for Mg2+ in the enzyme reaction in decreasing order Mn2+ > Mg2+ > Co2+ > Ca2+ . In the presence of excess Mg2+, nucleotides do not affect the Km for fructose 6-phosphate with a value of 0.042 mM . The order of efficiency for nucleotides to act as phosphoryl donors is ATP > ITP > GTP > UTP > CTP . This remains unchanged in the presence of excess Mn2+, but V is increased 2.4-fold with ATP . A 2 : 1 ratio of Mn2+/nucleotide 5'-triphosphate produced an equivalent dissociation constant of 1.1 mM for all nucleotides, which was markedly decreased at a high Mn2+ level . The rate of enzyme catalysis was found to be dependent on the concentration of MnATP2- . Mn2+ at non-limiting values does affect the binding of fructose 6-phosphate to the enzyme. Biochim Biophys Acta, 1980 Sep 9, 615(1), 121 - 31 Inactivation of Escherichia coli acetate kinase by N-ethylmaleimide . Protection by substrates and products; Wong SS et al.; Acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) from Escherichia coli exhibited a time-dependent loss of activity when incubated with N-ethylmaleimide at micromolar concentrations . However, prolonged incubation did not eliminate all catalytic activity and generally about 15% of its initial activity remained . When incubated with 7.2 microM N-ethylmaleimide, acetate kinase was inactivated with a rate constant of 0.063 min-1 . Adenine nucleotides, ATP, ADP and AMP, protected the enzyme against such inactivation, but acetate up to 3.0 M and in the presence of 0.2 M MgCl2 and acetyl phosphate at 24 mM did not interfere with the rate of inactivation . While both acetate and acetyl phosphate did not affect the protection rendered by AMP, the presence of acetyl phosphate altered ADP protection . However, both substrates prevented ATP from protecting the enzyme . These data suggest that the binding sites for acetate and acetyl phosphate are different from that of the adenosine binding domain, but are in close vicinity to the phosphoryl binding regions of the nucleotides. Biochemistry, 1980 Sep 2, 19(18), 4237 - 43 Mechanism of the melibiose porter in membrane vesicles of Escherichia coli; Cohn DE et al.; The melibiose transport system of Escherichia coli catalyzes sodium--methyl 1-thio-beta-D-galactopyranoside (TMG) symport, and the cation is required not only for respiration-driven active transport but also for binding of substrate to the carrier in the absence of energy and for carrier-mediated TMG efflux . As opposed to the proton--beta-galactoside symport system {Kaczorowski, G . J., & Kaback, H . R . (1979) Biochemistry 18, 3691}, efflux and exchange of TMG occur at the same rate, implying that the rates of the two processes are limited by a common step, most likely the translocation of substrate across the membrane . Furthermore, the rate of exchange, as well as efflux, is influenced by imposition of a membrane potential (delta psi; interior negative), suggesting that the ternary complex between sodium, TMG, and the porter may bear a net positive charge . Consistently, energization of the vesicles leads to a large increase in the Vmax for TMG influx, with little or no change in the apparent Km of the process . It is proposed that the sodium gradient (Na+out < Na+in) and the delta psi (interior negative) may affect different steps in the overall mechanism of active TMG accumulation in the following manner: the sodium gradient causes an increased affinity for TMG on the outer surface of the membrane relative to the inside and the delta psi facilitates a reaction involved with the translocation of the positively charged ternary complex to the inner surface of the membrane. Biochemistry, 1980 Sep 2, 19(18), 4208 - 13 Elementary steps in the reaction mechanism of the pyruvate dehydrogenase multienzyme complex from Escherichia coli: kinetics of acetylation and deacetylation; Akiyama SK et al.; The kinetics of the acetylation of the pyruvate dehydrogenase complex from Escherichia coli by {3-14C}pyruvate and of the deacetylation of the complex by coenzyme A have been studied by using rapid mixing-quench techniques . The time course for acetylation in 4 mM thiamin pyrophosphate, 2 mM MgSO4, and 0.02 M potassium phosphate (pH 7.0) at 4 degrees C can be analyzed in terms of two kinetic processes . At long times 10 nmol of acetyl groups is incorporated per mg of enzyme complex (48 sites per complex of molecular weight 4.8 X 10(6)) . The slower process is much too slow to be of catalytic significance . The rate constant for the faster process is not dependent on enzyme concentration and reaches a limiting value of 40--65 s-1 at high pyruvate concentrations; the exact value is dependent on the detailed acetylation mechanism assumed . The minimum molar turnover number of the enzyme complex is 420 s-1 (17.5 s-1 per pyruvate decarboxylase) . The acetylated lipoic acids are deacetylated by coenzyme A at a rate much faster than that of acetylation . Complete deacetylation is obtained only if the deacetylation is carried out within seconds of the acetylation, apparently because dead-end intramolecular transfers of acetyl groups from the lipoic acids to other functional groups on the enzyme not essential for catalytic activity can occur . The results obtained suggest only about half of the acetylation reactions are on the main catalytic pathway. Biochemistry, 1980 Sep 2, 19(18), 4179 - 88 Precise localization of the site of cross-linking between protein L4 and 23S ribonucleic acid induced by mild ultraviolet irradiation of Escherichia coli 50S ribosomal subunits; Maly P et al.; Mild ultraviolet irradiation of Escherichia coli 50S ribosomal subunits causes a cross-linking reaction between protein and RNA, whose primary target is protein L4 {Moller, K., & Brimacombe, R . (1975) Mol . Gen . Genet . 141, 343} . Here we have determined the site of this cross-link both on L4 and on 23S RNA . For the site on the protein, a cross-linked protein-oligonucleotide complex was isolated and subjected to successive digestions with various proteases . In each case the peptide-oligonucleotide complexes formed were analyzed . It could clearly be shown that the cross-link site was contained within a characteristic peptide 16--20 amino acids long and that the amino acid concerned was the tyrosine residue at position 35 in the recently completed L4 sequence (M . Kimura and B . Wittmann-Liebold, personal communication) . For the site on the RNA, a cross-linked L4--23S RNA complex was subjected to mild nuclease digestion, producing a range of L4--RNA fragments which were isolated with the help of a new two-dimensional gel electrophoresis system . Oligonucleotide analyses of these fragments, combined with successive nuclease digestions of the residual oligonucleotide attached to protein L4, established that the site of cross-linking was homogeneous, involving a uridine residue at position 615 in the recently determined 23S RNA sequence {Brosius, J., Dull, T . J., & Noller, H . F . (1980) Proc . Natl . Acad . Sci . U.S.A . 77, 201}. Biochemistry, 1980 Sep 2, 19(18), 4201 - 8 Escherichia coli photoreactivating enzyme: purification and properties; Snapka RM et al.; We have purified large quantities of Escherichia coli photoreactivating enzyme (EC 4.1.99.3) to apparent homogeneity and have studied its physical and chemical properties . The enzyme has a molecular weight of 36 800 and a S020,W of 3.72 S . Amino acid analysis revealed an apparent absence of tryptophan, a low content of aromatic residues, and the presence of no unusual amino acids . The N terminus is arginine . The purified enzyme contained up to 13% carbohydrate by weight . The carbohydrate was composed of mannose, galactose, glucose, and N-acetylglucosamine . The enzyme is also associated with RNA (approximately 10 nucleotides/enzyme molecule) containing uracil, adenine, guanine, and cytosine with no unusual bases detected. Kidney Int, 1980 Sep, 18(3), 366 - 74 Immunologic studies in IgA nephropathy; Woodroffe AJ et al.; Circulating immune complexes (CIC) were detected in 43.6% of 78 patients with primary IgA nephropathy by the solid-phase Clq radioimmunoassay . The IC were intermediate (9 to 17S) in size and contained IgA, IgG, and less commonly IgM . CIC were often present intermittently, correlating with episodes of macroscopic hematuria . Elevated serum IgA concentrations (38.7%) did not correlate with the detection of CIC . Similar findings were observed in sera samples from patients with Henoch Schonlein purpura and in IgA glomerulonephritis associated with alcoholic cirrhosis and/or portal systemic shunts . The factors responsible for the mesangial localization of the IC are not clear, but elevations in serum antibody titers to respiratory pathogens (mycoplasma pneumoniae, herpes virus, influenza), gut flora (E . coli 07), and bovine serum albumin suggest that common exogenous antigens may be involved in the pathogenesis . Primary defects in either mucosal antigen exclusion or reticuloendothelial IC sequestration are proposed to account for these findings. Zentralbl Bakteriol Mikrobiol Hyg {B}, 1980 Sep, 171(4-5), 448 - 54 {The in vivo influence of gibberellic acid (GA3) on the phagocytic properties of peritoneal macrophages (author's transl)}; Schwartz E et al.; Different concentrations of gibberellin acid GA3 - 10.0 mg, 1.0 mg and 0.1 mg - were applied intraperitoneally to white mice of the SPF strain on four consecutive days . Following the last application we injected E . coli, P . aerug . and Pr . vulg . intraperitoneally into the peritoneum of these animals, in a quantity of 1.5-2.0 X 10(6)/0.5 ml of sterile phosphate buffer . The peritoneal macrophages obtained by puncturing the peritoneum of the animals killed under chloroform anaesthesia showed, as compared with those of the control animals statistically most significantly higher values (P < 0.001) of the phagocytic factor and phagocytic index as well as of the activity of the acid phosphatase . In the test animals which received gibberellin acid, the inhibition of the lysosomal activity of peritoneal macrophages as a result of the application of trypan blue did not reach the level which was found to exist in the control animals and those infected with B CG - Praha. Am J Trop Med Hyg, 1980 Sep, 29(5 Suppl), 1033 - 6 The genes for variant antigens in trypanosomes; Borst P et al.; We have studied the mechanism of antigenic variation by using DNA complementary to the messenger RNAs for four variant surface glycoproteins of Trypanosoma brucei . Pure complementary DNAs were obtained by cloning as recombinant DNA in Escherichia coli . Using these complementary DNAs as hybridization probes, we have analyzed the genes for these variant surface glycoproteins . The results provide new information on the origin and evolution of antigenic variation, and on the mechanism involved in switching from one antigenic type to another. Nippon Yakurigaku Zasshi, 1980 Sep, 76(5), 363 - 72 {Effect of prednisolone 17-valerate 21-acetate on immunological responses in mice (author's transl)}; Kobayashi F et al.; The in vivo effects of prednisolone 17-valerate 21-acetate (PVA), an anti-inflammatory glucocorticoid on several immunological responses in mice were investigated in comparison with hydrocortisone 17-butyrate (HB) and betamethasone 17-valerate (BV), when given subcutaneously . PVA reduced the spleen weight, the number of splenic nucleated cells, the formation of hemolytic plaque forming cells (PFC), delayed type footpad reaction and the responsiveness of splenic lymphocytes to concanavalin A . These suppressive effects were almost the same as those seen with HB and weaker than those of BV . However, the responsiveness of splenic cells to lipopolysaccharide and circulating IgM antibody response to sheep red blood cells were suppressed by a smaller dose of PVA than that of HB . PVA had no effect on the responsiveness to phytohemagglutinin-P, whereas HB and BV enhanced the phytohemagglutinin-P responsiveness . The suppressive effect of PVA on the host defense to experimental infection with Escherichia coli was weaker than those of HB and BV . From these results, PVA appears to be similar to other glucocorticoids in that it exerts complicated effects on several immunological responses in mice. Eur J Biochem, 1980 Sep, 110(2), 555 - 63 The role of eucaryotic factor Tu in protein synthesis . The measurement of the elongation factor Tu content of rabbit reticulocytes and other mammalian cells by a sensitive radioimmunoassay; Slobin LI; A sensitive radioimmunoassay for eucaryotic elongation factor Tu (eEF-TU) was developed using radioiodinated elongation factor T (eEF-T) and goat anti-(rabbit eEF-T) immunoglobulins coupled to a solid support . eEF-T was iodinated with 125I to a specific activity of 7 x 10(3) counts min-1 ng-1 using a system employing lactoperoxidase and glucose oxidase coupled to a solid support . The assay exhibits a limit of detection of about 1 ng eEF-TU and an intraassay variability of < 10% . By using the radioimmunoassay, it was found that eEF-Tu is a major non-hemoglobin protein of rabbit reticulocyte postribosomal supernatant proteins, comprising about 3% of the total hemoglobin and 10--13% of the non-hemoglobin proteins . Similar results were found for a number of different tissues and cells, including rabbit heart, brain, liver and kidneys, as well as both induced and non induced Friend erythroleukemia cells . Values of eEF-Tu ranged from 1% of supernatant proteins in heart to about 11% in noninduced erythroleukemic cells . The levels of eEF-Tu in these mammalian tissues were comparable to the level of the homologous factor EF-Tu in Escherichia coli . It has previously been found that EF-Tu constitutes about 6--8% of the supernatant proteins of E . coli {Furano, A . V . (1975) Proc . Natl Acad . Sci . USA, 72, 4780--4784} . The level of eEF-Tu in reticulocytes was compared to the abundance of other components of protein synthesis in reticulocytes, such as translocase (eEF-G), tRNA, ribosomes and eIF-2 . In all cases eEF-Tu was present in large excess over these other components. Pflugers Arch, 1980 Sep, 387(2), 175 - 81 Effects of potential blood substitutes (perfluorochemicals) on rat liver and spleen; Lutz J et al.; The effect of an emulsion of perfluorochemicals (PFC) (7 parts perfluorodecalin and 3 parts perfluorotripropylamine, 4.4 g PFC/kg body weight) on organ function was determined . Whereas maximal storage of PFC was reached in the spleen as early as 12 h after PFC administration, the liver attained a maximal PFC content only after 2 days . The increase in weight also differed: a maximum occurred in the spleen on the 4th day, in the liver on the 8th day . Indocyanine green (ICG) clearance showed a small decrease, statistically significant after 12 and 24 h . Colloidal carbon clearance, used as a measure of the function of the reticuloendothelial system (RES) decreased instantly after PFC to less than half the control value; after full recovery a second decrease was seen which lasted till the 4th day after PFC . Pretreatment with C 48/80 or with increasing doses of E . coli endotoxin could largely obviate the depressive effect of PFC-loading on carbon clearance . Serum transaminases increased to about twice the control levels but were normal by the 2nd day, and thereafter . Alkaline phosphatase showed a 2.5 fold increase but returned to control level after the 2nd day . It is concluded that while a severe disturbance of liver function did not occur, the reduction in the capacity of the RES can become a serious factor in the defence against a simultaneously appearing infection if not compensated by activating the RES. Biophys J, 1980 Sep, 31(3), 381 - 92 Sensitivity to ultraviolet radiation as a function of DNA content in Escherichia coli B/r; Bronk BV et al.; Populations of Escherichia coli B/r A were grown to log phase at various growth rates determined by the richness of the medium . The genome content, G, was calculated from log phase doubling times by means of the Cooper-Helmstetter formula . Cell volumes were measured and found to vary linearly with this genome content . Cells with various DNA contents were prepared for ultraviolet irradiation and plated for dark repair under similar conditions . The resulting logarithmic survival curves were all similar in shape: convex up, with straight line portions having approximately the same slope (D0 = 11.4 +/- 0.2 J/m2) . The shoulders however increase in width with calculated DNA content giving an extrapolation number which varies roughly as exp(G) or exp (0.6 Gmax). Int J Radiat Biol Relat Stud Phys Chem Med, 1980 Sep, 38(3), 323 - 34 Cerenkov ultraviolet radiation (137Cs gamma-rays) and direct excitation (137Cs gamma-rays and 50 kVp X-rays) produce photoreactivable damage in Escherichia coli; Moss SH et al.; The mechanism of formation of photoreactivable damage in deoxyribonucleic acid (DNA) by ionizing radiation in a dark repair deficient strain of Escherichia coli (uvrA recA) has been investigated . By altering the ratio of damage produced directly (by ionization) to that formed indirectly (by Cerenkov ultraviolet (U.V.) radiation by 137Cs gamma-rays, it has been demonstrated that the major portion of the photoreactivable damage is produced by Cerenkov U.V . radiation . The amount of photoreactivable damage produced by 50 kVp X-rays, which cannot generate Cerenkov radiation, is similar to that component of photoreactivable damage produced by 137Cs gamma-rays that is not attributed to Cerenkov radiation . It is suggested that the second mechanism of formation of photoreactivable damage in DNA by ionizing radiation is the direct excitation of DNA . The possible role of Cerenkov U.V . radiation in ionizing radiation mutagenesis is discussed. Biochem J, 1980 Sep 1, 189(3), 421 - 33 Analysis of progress curves . Rate law of pyruvate kinase type I from Escherichia coli; Markus M et al.; Progress curves of the reaction catalysed by pyruvate kinase from Escherichia coli K12, designed to cover the four-dimensional concentration space of phosphoenolpyruvate, ADP, Mg2+ and ATP in the regulatory region, were recorded with the pH-stat method (pH 7.0 and 25 degrees C) . Additional initial-rate measurement were performed to assess specific points . Two methods for the evaluation of progress curves were used: fitting the rate law to the rates obtained from the tangents of the progress curves and fitting the integrated rate law directly to the curves . Two models, both extensions of the concerted model given by Monod, Wyman & Changeux {(1965) J . Mol . Biol . 12, 88--118} with four protomers, could be fitted to the data within the experimental error . Model discrimination in favour of one of these models was possible by proper experimental design . In the selected model one conformational state of the enzyme forms the active complex . The active site of a second conformational state forms abortive complexes with Mg2+, causing strong inhibition at high Mg2+ concentrations . In the absence of ligands, most of the enzyme is in a third state that binds ATP at an allosteric site. J Appl Physiol, 1980 Sep, 49(3), 516 - 20 Diphenhydramine reduces endotoxin effects on lung vascular permeability in sheep; Brigham KL et al.; Because, in sheep, histamine-induced increased lung vascular permeability is prevented by diphenhydramine, we tested the effects of diphenhydramine on the sheep lung vascular response to endotoxin . We infused E . coli endotoxin (0.40-1.00 micrograms/kg) with and without diphenhydramine (3.0 mg/kg bolus + 1.5 mg . kg-1 . h-1) in the same unanesthetized sheep while measuring pulmonary arterial (Ppa) and left atrial (Pla) pressures, lung lymph flow (Qlym) and lymph (L) and plasma (P) protein concentrations . Endotoxin caused pulmonary hypertension soon after infusion (base-line Ppa = 22 +/- 3 (SE) cmH2O; after endotoxin Ppa = 40 +/- 2; P less than 0.05, n = 6) and after several hours an increase in permeability reflected in high flow of protein-rich lymph (base-line; Qlym = 7.5 +/- 1.4 (SE) ml/h, L/P protein concentration = 0.60 +/- 0.02: after endotoxin; Qlym = 21.4 +/- 3.1, P less than 0.05; L/P = 0.66 +/- 0.03, P less than 0.05) . In the presence of diphenhydramine, endotoxin caused identical pressure changes but Qlym was lower during the period of increased permeability (16.7 +/- 3.0 (SE) ml/h, P less than 0.05 comp