J Biol Chem, 1983 Jul 25, 258(14), 8543 - 6 Mössbauer study of beef heart cytochrome oxidase . Comparative study of the bovine enzyme and cytochrome c1aa3 from Thermus thermophilus; Kent TA et al.; We have studied beef heart cytochrome c oxidase at 4.2 K with Mossbauer spectroscopy using the 57Fe present in natural abundance . The spectra observed are very similar to those of the a- and a3-sites of cytochrome c1aa3 from Thermus thermophilus . Thus, many conclusions derived from studies of the bacterial oxidase (available with enriched 57Fe) also apply to the mammalian enzyme . In the resting (as isolated) state, cytochrome a3 of the mammalian enzyme exhibits a doublet with quadrupole splitting, delta EQ = 1.0 mm/s and isomer shift, delta = 0.48 mm/s . These parameters suggest a high spin ferric heme and rule out an Fe(IV) assignment . The absence of magnetic features in the 4.2 K spectrum is consistent with earlier proposals that cytochrome a3 is spin-coupled to a cupric ion . The absorption lines are rather broad, suggesting that the a3-site is heterogeneous in the resting enzyme . Reduced cytochrome a3 has delta EQ = 1.85 mm/s and delta = 0.93 mm/s, demonstrating that the heme iron is high spin ferrous . The observed value for delta EQ is smaller than those of hemoglobin (2.4 mm/s), myoglobin (2.2 mm/s), and cytochrome a3 from T . thermophilus (2.06 mm/s) . The Mossbauer spectra of oxidized cytochrome a3-CN show that the heme iron is low spin ferric and that the ground state has integer spin S greater than or equal to 1, which plausibly results from ferromagnetic coupling of the S = 1/2 heme to an S = 1/2 cupric ion . Reduced cytochrome a is low spin ferrous, with parameters similar to those of cytochrome b5 and cytochrome c. Anal Biochem, 1983 Jul 15, 132(2), 413 - 7 The application of triazine dye affinity chromatography to the large-scale purification of glycerokinase from Bacillus stearothermophilus; Scawen MD et al.; Gram quantities of homogeneous glycerokinase have been prepared from the thermophilic bacterium, Bacillus stearothermophilus, using three major steps: precipitation of debris at pH 5.1, ion-exchange chromatography on DEAE-Sephadex, and affinity chromatography on Procion Blue MX-3G-Sepharose . This method is a considerable improvement over conventional techniques; the purified enzyme was obtained with a 40% recovery and a specific activity of 120 units (mumol/min)/mg protein . A modified culture medium enabled yields of 3.4 X 10(6) units of enzyme to be obtained from 400-liter production cultures. J Bacteriol, 1983 Jul, 155(1), 90 - 6 Temperature dependence of growth and membrane-bound activities of Chloroflexus aurantiacus energy metabolism; Oelze J et al.; The temperature dependence of various activities related to the energy metabolism of isolated membranes and whole cells of the thermophilic bacterium Chloroflexus aurantiacus was determined after phototrophic growth at either 40, 50, or 60 degrees C . The data obtained were expressed by use of Arrhenius plots . Maximum activities were determined at about 65 degrees C for succinate 2,4-dichlorophenol-indophenol reductase as well as NADH oxidase and at about 70 degrees C for Mg-ATPase and for light-induced proton extrusion by cells . Activation energies for Mg-ATPase and light-induced proton extrusion were about 40 kJ mol-1 from 30 degrees C to about 50 degrees C and they increased significantly at higher temperatures . Essentially the same dependency was detectable with NADH oxidase, except for an increase in activation energy below 41 degrees C . All of these responses were independent of growth temperature . Succinate-2,4-dichlorophenol-indophenol reductase showed a change in activation energy around 41 degrees C only with cells grown at 60 degrees C . Differences in the responses of cells grown at different temperatures were identified on the basis of changes from sigmoidal to hyperbolic kinetics for light saturation of proton extrusion . Moreover, the thermostability of proton extrusion was maximal when assayed at the corresponding growth temperatures . In any case, thermostability was lowest at the 65 and 68 degrees C assay temperatures . Differential scanning calorimetry with membranes revealed irreversible heat uptake from about 60 to 72 degrees C . The results are discussed in light of the activation energy for the specific growth rate, which is lowest at temperatures from 40 degrees C to the optimum at 60 degrees C. Dev Biol, 1983 Jul, 98(1), 173 - 81 An analysis of protein synthesis, membrane proteins, and concanavalin A-binding proteins during conjugation in Tetrahymena thermophila; Van Bell CT; Conjugation in the ciliate Tetrahymena thermophila has been used as a system in which to analyze biochemical events associated with the execution of a complex cell-cell interaction . Two-dimensional electrophoretic analysis of {35S}methionine-labeled whole-cell proteins revealed major changes in protein synthesis correlated with costimulation and the onset of pairing; specifically, the major induced polypeptide was one of 80 kDa . A second change in the pattern of protein synthesis was associated with the onset of meiosis; the major induced product was another, perhaps related, 80-kDa polypeptide . An effort was made to detect changes in the patterns of membrane proteins and Con A-binding proteins during conjugation; no changes were found . These results are discussed in the context of earlier hypotheses regarding the distribution of Con A receptors on the surfaces of conjugating cells. Mikrobiologiia, 1983 Jul-Aug, 52(4), 605 - 8 {Thermophilic Myceliophthora thermophila decomposes cellulose}; Loginova LG et al.; A thermophilic microscopic fungus was isolated from cattle rumen and identified as Myceliophthora thermophila (Apinis) van Oorschot . The culture synthesized cellulolytic enzymes and xylanase when it was grown in media containing cellulase at 50 degrees C under the conditions of submerged cultivation . The morphological and physiological characteristics of the culture are described and its taxonomic position is discussed. Res Commun Chem Pathol Pharmacol, 1983 Jul, 41(1), 149 - 55 Effect of various compounds on enkephalin hydrolysis by an aminopeptidase from the thermophiles Thermomonospora fusca ATCC 27730 and Thermus thermophilus ATCC 27634; Weiss B et al.; The microbial peptides amastatin and bestatin as well as several dipeptide analogues of the latter exerted little or no inhibitory effect on enkephalin hydrolysis by an aminopeptidase purified from the thermophiles Thermomonospora fusca, ATCC 27730 (Tf) and Thermus thermophilus, ATCC 27634 (Tt) . The enzyme catalyzes the cleavage of the tyrosyl-glycyl bond of leucine- and methionine-enkephalin . Intermediate compounds having the same amino acid sequence as the parent substrate disclosed that the residual tetrapeptide can be further degraded to its constituent parts . Each preparation also hydrolyzes to varying extents neutral dipeptides, tripeptides, tetrapeptides, can be further degraded to its constituent parts . Each preparation also hydrolyzes to varying extents neutral dipeptides, tripeptides, tetrapeptides, and larger molecules containing the Met-enkephalin sequence . The Tf enzyme has a pH optimum of 7.5, Km of 667 microM and Vmax of 92 nmol/min/mg of protein; the Tt enzyme, with a pH optimum of 7.2 has a Km of 400 microM and Vmax of 33 nmol/min/mg of protein . Activated by dithiothreitol (DTT) and inactivated by p-chloro- and p-hydroxymercuribenzoate, both are sulfhydryl enzymes . The activity lost by hydrolysis against EDTA can be restored, wholly or in part, by Co+2, Mg+2, and Mn+2; ions with an inhibitory effect were A1+3, Cd+2, Cu+2, Hg+2, and Zn+2 . The enzymes are not glycoproteins since they pass unretained through a Con A-Sepharose column. Hoppe Seylers Z Physiol Chem, 1983 Jul, 364(7), 879 - 92 Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria . II) The primary structure of thermophilic lactate dehydrogenase from Bacillus stearothermophilus . Cyanogen bromide fragments and partial sequence; Tratschin JD et al.; The polypeptide chain of thermophilic lactate dehydrogenase from Bacillus stearothermophilus was split with cyanogen bromide . The 6 cyanogen bromide fragments were then separated and isolated by gel filtration (Bio-Gel P 10, Sephadex G-75) and ionic exchange chromatography (Biorex 70), respectively . Peptide fractionation was performed in 50% formic acid . Fragment yield varied between 30 and 75% . About 75% of the amino-acid sequence was determined by the automatic N-terminal sequence analysis (amino-acid sequenator) of the cyanogen bromide fragments (41-57 cycles degraded) and N-terminal region of lactate dehydrogenase (74 cycles degraded) . Typical structure differences between thermophilic and mesophilic lactate dehydrogenases are already indicated by the comparison of the amino-acid composition of the thermophilic enzyme from B . stearothermophilus with the mesophilic from bacilli and higher organisms . Comparison of the N-terminal sequence reveals that sequence homology is higher (83-98%) between the thermophilic lactate dehydrogenases from B . stearothermophilus, B . caldotenax and B . caldolyticus than between the mesophilic lactate dehydrogenases of bacilli among each other or between thermophilic and mesophilic lactate dehydrogenases (about 60%) . High temperature would appear to limit variation in structure. Radiobiologiia, 1983 Jul-Aug, 23(4), 462 - 6 {Effect of MEA on the accumulation of DNA breaks in Bac . stearothermophilus irradiated with gamma and ultraviolet rays and treated with nitrosomethylurea}; Kuznetsova EA et al.; beta-Mercaptoethylamine (MEA) decreased the accumulation of enzymatic single- and double-strand breaks in DNA of thermophilic bacteria exposed to gamma- and UV-radiation and treated with N-nitroso-N-methylurea . The protective effect of MEA, as registered according to accumulation of single-strand and double-strand breaks in DNA of Bac . stearothermophilus immediately after irradiation and after 30 min postirradiation incubation, was similar. Equine Vet J, 1983 Jul, 15(3), 207 - 10 Chronic obstructive pulmonary disease in the horse . 2: Therapy; Thomson JR et al.; The therapy of equine chronic obstructive pulmonary disease (COPD) essentially entails minimising the horse's exposure to the aetiological antigens which are predominantly thermophilic actinomycetes and moulds occurring in hay and straw . This can be achieved, for example, by keeping affected horses permanently out of doors, or when stabled, using shredded paper, wood shavings or peat moss as bedding and feeding a complete cubed diet . There should be no supplementary hay feeding apart from dust-free vacuum-packed hay . Applying such measures generally allows horses to become asymptomatic in seven to 14 days . Bronchodilators and corticosteroids bring about a marked, but temporary, improvement and can be of value in the treatment of acute attacks . The use of oral bronchodilators in combination with environmental control measures may hasten the remission of clinical signs in affected horse . Inhaled sodium cromoglycate can be used prophylactically in asymptomatic horses to prevent the onset of COPD when unavoidable antigen exposure is anticipated. J Clin Pathol, 1983 Jul, 36(7), 829 - 34 A study of the oxygen and carbon dioxide requirements of thermophilic campylobacters; Bolton FJ et al.; The oxygen and carbon dioxide requirements of different biotypes of thermophilic campylobacters were investigated by means of (a) quantitative studies, and (b) total growth studies . Oxygen tolerance of the five test organisms differed markedly and varied with the carbon dioxide concentration . At most carbon dioxide concentrations tested, Campylobacter jejuni strains NCTC 11168 and NCTC 11392 tolerated 21% oxygen (growth reduced), C coli NCTC 11353 tolerated 15% oxygen (growth reduced), and C jejuni ATCC 3036 and (nalidixic acid resistant thermophilic campylobacter) NCTC 11352 tolerated 10% oxygen (growth not reduced) . Total growth studies indicated that 10% oxygen was the optimal concentration for growth of the five test organisms . All exhibited a requirement for carbon dioxide, and only C jejuni strains NCTC 11168 and NCTC 11392 tolerated its absence (growth reduced), when the oxygen concentration was low . The studies indicated that atmospheres containing 5% to 10% oxygen and 1.0% to 10% carbon dioxide are suitable for growth of the various biotypes of thermophilic campylobacters . The oxygen and carbon dioxide concentrations produced in anaerobic jars by variations of the evacuation-replacement technique were determined and suitable practices identified. Hoppe Seylers Z Physiol Chem, 1983 Jul, 364(7), 893 - 909 Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria . III) The primary structure of thermophilic lactate dehydrogenase from Bacillus stearothermophilus . Hydroxylamine-, o-iodosobenzoic acid- and tryptic-fragments . The complete amino-acid sequence; Wirz B et al.; Based on the partial sequence of the cyanogen bromide fragments {Tratschin, J.D., Wirz, B., Frank, G . and Zuber, H . (1983) Hoppe-Seyler's Z . Physiol . Chem . 364, 879-892}, the amino-acid sequence of thermophilic lactate dehydrogenase from B . stearothermophilus was completed by the preparation and sequencing (sequenator, carboxypeptidase A and Y) of further overlapping fragments . Suitable peptide fragments were obtained by lactate dehydrogenase cleavage with hydroxylamine, o-iodosobenzoic acid and trypsin . The polypeptide chain of thermophilic lactate dehydrogenase from B . stearothermophilus consists of 317 amino-acid residues . While sequence homology with mesophilic lactate dehydrogenase of higher organisms reaches 35%, it is substantially higher with this mesophilic enzyme of bacillae (greater than 60%, B . megaterium, B . subtilis) . The secondary structure elements and amino-acid residues of the active site of thermophilic lactate dehydrogenase deducted from primary structure data were compared with those from the mesophilic enzyme, the same was done for the internal sequence homology at the nucleotide-binding units . A comparative structure analysis (matrix system) based on the primary structure data of thermophilic enzyme should provide insight into the characteristic structure differences between thermophilic and mesophilic lactate dehydrogenase. FEBS Lett, 1983 Jun 27, 157(1), 95 - 9 NMR analyses on the molecular mechanism of the conformational rigidity of 2-thioribothymidine, a modified nucleoside in extreme thermophile tRNAs; Yamamoto Y et al.; 1H-NMR analyses have been made on the conformations of 2-thioribothymidine (s2T), 2-thiodeoxyribothymidine (s2dT), as well as ribothymidine (T) and deoxyribothymidine (dT) . s2T and s2dT exclusively take the anti form rather than the syn form . The C3'-endo-gg form of the sugar moiety is remarkably stabilized on modification of T to s2T, but not on modification of dT to s2dT . The steric effects of the 2-thiocarbonyl group and the 2'-hydroxyl group cause the rigidity of the C3'-endo-gg form of s2T . Such rigidity of s2T probably contributes to the thermostability of 2-thiopyrimidine polyribonucleotides and extreme thermophile tRNAs. Biochem Biophys Res Commun, 1983 Jun 15, 113(2), 575 - 80 Proton pumping and oxidase activity of thermophilic cytochrome oxidase remain after its extensive proteolysis; Yanagita Y et al.; A proton-pumping heme aa3-type cytochrome oxidase purified from the thermophilic bacterium PS3 was treated with trypsin, thermolysin, chymotrypsin, subtilisin, or pronase . The cleavage of the oxidase subunits and the effects of their cleavage on the oxidase activity and proton-pumping in reconstituted vesicles were studied . Trypsin and thermolysin cleaved some of the oxidase subunits without affecting the proton-pumping, but subtilisin and pronase cleaved all the subunits resulting in partial decrease in both activities . Chymotrypsin had an intermediate effect . Subunit II of this enzyme contains heme c which is also cleaved by proteases. Nucleic Acids Res, 1983 Jun 11, 11(11), 3487 - 502 Complex endonucleolytic cleavage pattern during early events in the processing of pre-rRNA in the lower eukaryote, Tetrahymena thermophila; Kister KP et al.; We have analysed nuclear RNA from T . thermophila by RNA transfer hybridization using cloned rDNA fragments . A very high number of in vivo intermediates and by-products of rRNA processing were identified . These include putative intermediates of the splicing process and alternative products resulting from temporal variability in various endonucleolytic cleavages . In addition, four small RNA species including only transcribed spacer sequences were detected . These are (1) the IVS RNA (approximately 400 bases), the by-product of the splicing process, (2) a fragment from the internal transcribed spacer (approximately 360 bases), possibly resulting from 3'-end processing of pre-17S rRNA, (3) a fragment comprising most or all of the external transcribed spacer (approximately 600 bases) obviously representing the major by-product of 5'-end processing, and, in addition, (4) a small fragment from the initiation region (approximately 230 bases) which might be a product of premature transcription termination. J Biol Chem, 1983 Jun 10, 258(11), 6899 - 905 Regulation of protein synthesis in Tetrahymena . RNA sequence sets of growing and starved cells; Calzone FJ et al.; The complexity of messenger RNA in growing or starved Tetrahymena thermophila is similar and unusually high (approximately 4.5 X 10(7) nucleotides) . The complexity of nuclear RNA in growing cells (approximately 7.8 X 10(7) nucleotides) is only about 1.7 times that of mRNA . The concentration of complex class (rare) messages (approximately 53 copies/growing cell and approximately 11 copies/starved cell) is low in comparison to the size of the cell . The concentration of complex nuclear transcripts is also very low (approximately 0.7 copies/growing cell nucleus and approximately 2.6 copies/starved cell nucleus) considering that the macronucleus contains 45 to 90 copies of each single copy sequence . The complex sequence sets found on polysomes of growing and starved cells overlap about 80% and about 60% of the complex nuclear transcripts appear to be held in common . About 60% of macronuclear single copy DNA is transcribed in one or both physiological states . Although growing and starved cells have extremely different fractions of their messages loaded onto polysomes, within each cell type the complex messages in polysomal and nonpolysomal cytoplasmic fractions are indistinguishable, suggesting that exchange may occur between loaded and unloaded messages . Although T . thermophila DNA has an unusually low G + C content (23%), sequences coding for complex RNAs have base ratios similar to those of total DNA . Therefore, codon usage in Tetrahymena must be extremely biased towards adenine- and uridine-rich codons. Arch Microbiol, 1983 Jun, 134(3), 247 - 50 Changes in enzyme stability and fatty acid composition of Streptomyces sp., a facultative thermophilic actinomycete; Heinen W et al.; The thermostability of several enzymes from the facultative thermophilic actinomycete Streptomyces sp., derived from cells grown in the temperature range from 37 degrees C to 60 degrees C, has been examined . A correlation between the growth temperature of the cultures and the heat stability of the enzymes could be demonstrated for alanine dehydrogenase, isocitrate dehydrogenase, myokinase and pyruvate kinase . Except for the isocitrate dehydrogenase, which showed a linear increase of its stability throughout the entire temperature range, all enzymes exhibited a steep increase of the heat stability up to about 50 degrees C, but no further increase in the higher growth range, suggesting, that from 50 degrees C upward a shift to the exclusive production of thermostable protein occurs . Furthermore, the stability of alanine dehydrogenase and pyruvate kinase was found to increase substantially in presence of their substrates . In contrast, substrate-mediated stabilization was very weak with glucose-6-phosphate dehydrogenase and totally absent with acetate kinase, isocitrate dehydrogenase and myokinase . A comparison with previous observations with the same enzymes from Bacillus flavothermus showed, that enzymes from different organisms can have different thermal properties . Determination of the fatty acid composition of Streptomyces sp . cells, grown at different temperatures, showed relatively small alterations, with the main changes occurring between 37 degrees C and 40 degrees C. Arch Microbiol, 1983 Jun, 134(3), 175 - 81 Activation and germination characteristics observed in endospores of thermophilic strains of Bacillus; Foerster HF; The properties of endospores of some thermophilic strains of Bacillus were examined . Included were strains isolated from thermal pools and springs in Yellowstone National Park, a strain of B . thermodenitrificans and two strains of B . stearothermophilus, ATCC 7953 (smooth) and T-10 . The spores of thermophilic strains of Bacillus contained relatively high levels of dipicolinic acid ranging from 11-14.8% of the spore dry weight, while the calcium levels were similar to those observed in other bacterial endospores including mesophilic bacilli and thermophilic actinomycetes . Spore populations of thermophilic bacilli could not be effectively germinated in solutions of sodium phosphate alone but germinated well in solutions supplemented with one of a variety of organic compounds . Solutions containing L-valine or L-leucine were particularly effective . A wide range of pH permitted the germination of fractions of spore populations, however, optimum germination was observed only at pH values of 6.0 and above . A range in incubation temperatures of less than 25 degrees C permitted 50% or more of the spores of each of the organisms to germinate . Freshly prepared spore did not germinate, but these spores germinated rapidly and completely if they were heated for 30 min at 100 degrees C just prior to germination testing, i.e., the spores were heat activatable . However, spores of thermophilic bacilli could also be activated by shifting them to and holding them at temperatures below their optimum growth temperature of 65 degrees C . Of the ten temperatures tested, ranging from 4 degrees C through 50 degrees C, the optimum reduced temperature for spore activation was 30 degrees C. Hoppe Seylers Z Physiol Chem, 1983 Jun, 364(6), 691 - 712 The complete amino-acid sequence of both subunits of phycoerythrocyanin from the thermophilic cyanobacterium Mastigocladus laminosus; Fuglistaller P et al.; The amino-acid sequences of both subunits of phycoerythrocyanin from the thermophilic cyanobacterium Mastigocladus laminosus have been determined . The alpha-subunit consists of 162 amino-acid residues and has a molecular mass of 18200 Da . The beta-subunit is 171 residues long and has a molecular mass of 19600 Da . The tetrapyrrole chromophores are bound at position 84 in the alpha- and beta-subunits and at position 155 in the beta-subunit . The homology between the two subunits is 21% . The homologies between the phycoerythrocyanin subunits and the corresponding subunits of C-phycocyanin and allophycocyanin are 63% and 26% for the alpha-subunits and 67% and 36% for the beta-subunits, respectively . Secondary structure predictions were calculated for all six subunits of the phycobiliproteins from M . laminosus . The most conservative regions of the biliproteins were found in segments C-terminal to the chromophore-binding sites. Proc Natl Acad Sci U S A, 1983 Jun, 80(11), 3406 - 10 Mitotic and meiotic stability of linear plasmids in yeast; Dani GM et al.; Circular recombinant DNA plasmids that contain autonomously replicating sequences (ARSs) are maintained in extrachromosomal form in transformed yeast cells . However, these plasmids are unstable, being rapidly lost from cells growing without selection . Although the stability of such a plasmid can be increased by the presence of yeast centromere DNA (CEN), even CEN plasmids are lost at a high rate compared to a bona fide yeast chromosome . Natural yeast chromosomes are linear molecules; therefore, we have asked if linearization can improve the stability of recombinant DNA plasmids . Linear plasmids with and without yeast CENs were constructed in vitro by using termini from the extrachromosomal ribosomal DNA (rDNA) of the ciliated protozoan Tetrahymena thermophila as "telomeres." These linear plasmids transformed yeast at high frequency and were maintained as linear extrachromosomal molecules during mitotic growth . Moreover, linear plasmids containing CENs were also transmitted through meiosis: these plasmids segregate predominantly 2+:2- at the first meiotic division, indicating that Tetrahymena rDNA termini can provide telomere function during yeast meiosis . Linear plasmids without CENs were about as stable in mitosis as the comparable circular plasmid . Thus, the Tetrahymena rDNA termini have no marked positive or negative effect on the mitotic stability of ARS plasmids . However, linear plasmids containing CENs are three to four times less stable in mitotic cells than circular CEN plasmids . This decrease in stability is not due to a functional change in the centromere itself; rather, linearization of a CEN plasmid has a direct detrimental effect on its mitotic stability . These results may reflect the existence of spatial constraints on the positions of centromeres and telomeres, constraints which must be satisfied to achieve stable segregation of chromosomes during mitosis. Antonie Van Leeuwenhoek, 1983 Jun, 49(2), 191 - 201 Thermal properties of enzymes from Bacillus flavothermus, grown between 34 and 70 degrees C; Lauwers AM et al.; The activity and stability of several enzymes from the facultative thermophile Bacillus flavothermus, grown within the mesophilic and thermophilic region at 34 degrees C, 43 degrees C, 52 degrees C and 70 degrees C, have been examined . While the temperature optima and maxima of all enzymes tested were found to remain unchanged at all growth temperatures, it was demonstrated that the heat stability of the proteins increased with ten perature, however, not uniformly for all enzymes . One exception was acetate kinase and the intrinsic stability of pyruvate kinase was found to increase only slightly . With all other proteins tested (alanine dehydrogenase, isocitric dehydrogenase and glucose-6-phosphate dehydrogenase, glutamate-oxalacetate and glutamate-pyruvate transaminase and myokinase) the intrinsic stability was found to increase to about 55 degrees C, but stayed unaltered at higher growth temperatures . Except for acetate kinase and myokinase, the enzymes could be stabilized by their respective substrates and the heat stability of the ES-complexes was found also to depend on the growth temperature of the cells . These data lead to the conclusion that the enzymes undergo a transition from heat-labile to thermostable within the growth temperature range between 44 degrees C and 51 degrees C while the thermal characteristics are not changed below and beyond this crucial region. J Bacteriol, 1983 Jun, 154(3), 1451 - 4 Base excision repair in the thermophile Thermus sp . strain X-1; Warner HR; The thermophile Thermus sp . strain X-1, grown at 70 degrees C, contains uracil-DNA glycosylase and apurinic endonuclease activities, both of which are known to have roles in the repair of DNA damaged by heat . Both of these activities have temperature optima of about 70 degrees C . However, neither of these activities is present in quantities significantly greater than that found in Escherichia coli grown at 37 degrees C . Therefore, it appears that thermophilic organisms may not contain greatly elevated levels of the enzymes thought to be involved in the repair of DNA damaged by heat. Biochem Biophys Res Commun, 1983 May 16, 112(3), 822 - 6 Resistance of thermophilic ATPase (TF1) to specific F1-atpase inhibitors including local anesthetics; Saishu T et al.; F1-ATPase obtained from mesophilic organisms is inhibited by specific inhibitors, such as aurovertin, efrapeptin, quercetin and several local anesthetics . This property has been explained by the common structure at the catalytic center of F1 . However thermophilic F1 (TF1), which has the same primary structure at the center as other F1's, was shown to be resistant to these F1-specific inhibitors . Thus, the inhibitory mechanism may be explained not by the common structure at the catalytic site, but by some conformational changes of the flexible mesophilic F1 molecules or the absence of an inhibitor binding site in thermophilic F1. Biochemistry, 1983 May 10, 22(10), 2314 - 9 Redox-linked hydrogen bond strength changes in cytochrome a: implications for a cytochrome oxidase proton pump; Babcock GT et al.; The heme a formyl group of cytochrome a in cytochrome oxidase appears to be involved in a hydrogen-bond interaction with a proton donor associated with the polypeptide backbone {Callahan, P.M., & Babcock, G.T . (1983) Biochemistry 22, 452-461} . Resonance Raman and optical absorption spectroscopies have been applied to the beef heart and Thermus thermophilus proteins and to heme a and copper porphyrin a models in order to assess the spectroscopic manifestations and the energetics of the hydrogen-bond interaction . We find a linear relationship between optical absorption red shift and carbonyl vibrational frequency decrease for a series of hydrogen-bonded model complexes; the magnitude of both changes increases as the hydrogen-bond strength increases . Comparison of the model compound data with analogous data for the proteins indicates that the strength of the formyl hydrogen bond in situ increases by 2-2.5 kcal/mol upon reduction of ferric cytochrome a . The selective stabilization of reduced cytochrome a by the stronger hydrogen bond is expected to increase the redox potential of this center; the energy made available as the hydrogen bond strengthens during reduction may be used to drive redox-coupled events in the protein . Thus, the linkage between cytochrome a redox state and chromophore/protein interaction energy provides a mechanism by which electron-transfer events and protein structure are coupled . Two models, which incorporate this linkage into a redox-driven proton pump centered at cytochrome a in cytochrome oxidase, are presented. FEBS Lett, 1983 May 2, 155(1), 173 - 7 Ribosome specificity of archaebacterial elongation factor 2 . Studies with hybrid polyphenylalanine synthesis systems; Klink F et al.; Polyphenylalanine synthesis with ribosomes and two separated, partially purified elongation factors (EF) was measured in cell-free systems from the archaebacteria Thermoplasma acidophilum and Methanococcus vannielii, in an eukaryotic system from rat liver and an eubacterial one with Escherichia coli ribosomes and factors from Thermus thermophilus . By substitution of heterologous EF-2 or EF-G, respectively, for the homologous factors, ribosome specificity was shown to be restricted to factors from the same kingdom . In contrast, EF-1 from T . thermophilus significantly cooperated with ribosomes from T . acidophilum. J Biochem (Tokyo), 1983 May, 93(5), 1455 - 6 Homocaldopentamine: a new naturally occurring pentaamine; Oshima T et al.; A new pentaamine was extracted from an extreme thermophile, Thermus thermophilus strain HB8, and its chemical structure was determined to be 1,16-diamino-4,8,12-triazahexadecane (see structure 1) . A trivial name, homocaldopentamine, was proposed for the new naturally occurring polyamine. J Biochem (Tokyo), 1983 May, 93(5), 1329 - 36 Carbon monoxide-binding cytochromes in the respiratory chain of the thermophilic bacterium PS3 grown with sufficient or limited aeration; Sone N et al.; The effects of aeration on the growth and cytochrome patterns of thermophilic bacterium PS3 were studied; bacteria grown with strong aeration synthesized cytochromes c, b, and aa3, while those grown with low aeration, showing non-exponential growth, synthesized higher amounts of cytochromes c and b including o, and a lower amount of cytochrome a (a3) . The CO-difference spectra indicated that the terminal oxidase was cytochrome aa3 for high aeration conditions and the cytochrome o for low aeration conditions . Cytochrome o can be solubilized by Triton X-100 from the membrane fraction of bacteria grown under oxygen-limited conditions . The carbon monoxide complex of cytochrome o, obtained by exposing this extract to CO, was photolyzed and the subsequent rebinding of CO was analyzed; it followed first order kinetics with a rate constant of around 8 s-1 at 25 degrees C . At liquid nitrogen temperature, CO-rebinding did not occur . The CO-difference spectrum of purified cytochrome oxidase sample from the bacteria grown with strong aeration (Sone, N., et al . (1979) FEBS Lett . 106, 39-42) revealed the presence of a small amount of a cytochrome o-like pigment besides cytochrome aa3 . Analysis of the CO complexes of these chromophores showed rate constants of 29-30 s-1 for cytochrome aa3 and 35-42 s-1 for the o-like pigment, indicating that the cytochrome o-like pigment contaminating the purified cytochrome oxidase preparation was not typical cytochrome o. Arch Biochem Biophys, 1983 May, 223(1), 193 - 201 Effect of modification of membrane phospholipid composition on the activity of phosphatidylethanolamine N-methyltransferase of Tetrahymena; Smith JD; The activity of phosphatidylethanolamine N-methyltransferase is less than 10% of control levels in microsomes prepared from the ciliate protozoan Tetrahymena thermophila whose phospholipid composition had been altered by being cultured on media containing phosphonic acids . The primary modification obtained is decreased levels of phosphatidylethanolamine (J.D . Smith and D.A . Giegel, Arch . Biochem . Biophys., 206, 420-423 (1981) and 213, 595-601 (1982)) . The enzyme protein is present in these cells at normal levels since addition of the substrate phosphatidylethanolamine to the assay system restores enzyme activity of the lipid-modified microsomes to control levels, while the enzyme from control microsomes is not affected by added phosphatidylethanolamine . The microsomal enzyme is inhibited by the anionic phospholipids cardiolipin, phosphatidylglycerol, and phosphatidylinositol and by lysophosphatidylethanolamine while it is activated only by phosphatidylserine in addition to the substrates phosphatidylethanolamine and phosphatidyldimethylethanolamine . The added phosphatidylethanolamine acts directly as a substrate for the methyltransferase rather than acting by merely stimulating utilization of endogenous lipid since added phosphatidyl{14C}ethanolamine is directly converted to phosphatidylcholine . The results suggest that the technique of phosphonic acid-induced modification of lipid composition will be useful for the study of other membrane-bound enzymes. J Protozool, 1983 May, 30(2), 403 - 9 The pathogenic amoeboflagellate Naegleria fowleri: environmental isolations, competitors, ecologic interactions, and the flagellate-empty habitat hypothesis; Griffin JL; From several surveys of environmental sites, the virulent human pathogen, Naegleria fowleri, was isolated from a pond in Georgia, a sewage treatment plant in Missouri, and from the Potomac and Anacostia rivers near and in Washington, D.C . Widely scattered, sparse populations seemed only a potential threat to human health at the time of sampling . The data support an estimate that the sites sampled contain 10,000 typical, low temperature, bactivorous amoebae for each heat tolerant amoeba able to grow at 45 degrees C . Heat tolerant competitors were much more common than N . fowleri . Naegleria lovaniensis, which is heat tolerant but nonpathogenic, was isolated from and downstream from an open air thermal pollution temperature gradient . Hot piles of composting sewage sludge yielded no amoeboflagellates, many heat tolerant (45-49 degrees C) amoebae, and one thermophilic (52 degrees C) Acanthamoeba . Features of the methods used include two-stage incubation to increase isolation of sparse organisms and distinction of N . fowleri from almost all other amoebae on agar plates . The flagellate-empty habitat hypothesis postulates a general model in which human intervention and/or natural events remove usual competitors and the ability to transform to a motile flagellate confers an advantage in recolonizing. Infect Immun, 1983 May, 40(2), 553 - 62 Isolation and possible relevance of Thermoactinomyces candidus proteinases in farmer's lung disease; Roberts RC et al.; The thermophilic actinomycetes are the most common etiological agents causing hypersensitivity pneumonitis . Antigen preparations of these organisms contain proteolytic activity . Further investigation of the proteinases of the thermophilic actinomycetes was undertaken to determine whether this activity may contribute directly to the pathogenesis of hypersensitivity pneumonitis and pulmonary mycotoxicosis . The presence of proteolytic activity in aerosolized dust from moldy silage was demonstrated, and antibodies to two proteolytic enzymes from Thermoactinomyces candidus were found in the blood of farmer's lung patients who had been sensitized to this organism . These two enzymes were isolated from culture filtrate antigen preparations that had been partially characterized with respect to the proteolytic activities and their interaction with human serum proteinase inhibitors . Both proteinases belonged to the serine class of endopeptidases . Neither proteinase was inhibited by alpha 1-proteinase inhibitor or alpha 1-antichymotrypsin . Both proteinases were inhibited by alpha 2-macroglobulin . One of the proteinases had elastase activity . Inhalation of these proteinases apparently does occur, and they may induce an inflammatory response in the lungs since they are not inhibited by the main proteinase inhibitors protecting the lung. Mycopathologia, 1983 Apr 22, 82(1), 61 - 4 Allergenic fungi and actinomycetes in smoking materials and their health implications; Kurup VP et al.; Street marijuana, commercial cigarettes and pipe tobaccos were studied for the presence of fungi and actinomycetes associated with hypersensitivity pneumonitis . Aspergillus species and thermophilic actinomycetes were isolated from the smoking materials . In addition, Aspergillus fumigatus spores were isolated from marijuana smoke, indicating the potential hazard involved in developing serious disease . Precipitin antibodies against fungi, particularly Aspergillus, showed a higher prevalence in marijuana smokers, whereas only very few cigarette smokers and nonsmokers demonstrated antibodies to fungi . Cigarette smokers and nonsmokers showed more or less similar prevelance of antibodies against thermophilic actinomycetes. Nucleic Acids Res, 1983 Apr 11, 11(7), 2093 - 109 Different nucleosome spacing in transcribed and non-transcribed regions of the ribosomal RNA gene in Tetrahymena thermophila; Gottschling DE et al.; The chromatin structure of the palindromic macronuclear ribosomal RNA genes of Tetrahymena thermophila was probed with micrococcal nuclease . Independent of the state of transcriptional activity, the transcribed region had a shorter nucleosome repeat (184 +/- 3 base pairs) than the non-transcribed central spacer or bulk chromatin (both 200 base pairs) . The transcribed region displayed an increased sensitivity to micrococcal nuclease in rapidly growing cells, which suggested an altered chromatin structure during transcription . At early stages of nuclease digestion, the central spacer appeared to be in a highly structured nucleosomal array . Based on the differences in nucleosome repeat distance and sensitivity to nuclease, we conclude that quite different chromatin structures are maintained in two adjacent regions of the Tetrahymena ribosomal RNA gene . The DNA of the non-transcribed terminal spacer was found to contain sequences which are highly susceptible to micrococcal nuclease, precluding any conclusions about nucleosome structure in this region. Appl Environ Microbiol, 1983 Apr, 45(4), 1271 - 6 Adaptation of mesophilic anaerobic sewage fermentor populations to thermophilic temperatures; Chen M; Thermophilic (50 degrees C) and obligately thermophilic (60 degrees C) anaerobic carbohydrate- and protein-digesting and methanogenic bacterial populations were enumerated in a mesophilic (35 degrees C) fermentor anaerobically digesting municipal primary sludge . Of the total bacterial population in the mesophilic fementor, 9% were thermophiles (36 x 10(6)/ml) and 1% were obligate thermophiles (4.5 x 10(6)/ml) . Of these 10%, the percentages of bacteria (thermophiles and obligate thermophiles, respectively) able to use specific substrates were further enumerated as follows: bacteria able to digest albumin, casein, starch, and mono- and disaccharides, 30 and 10%; pectin degraders, 10 and 0.2%; cellulose degraders, 2 and 0.06%; methanogens that grow with H2 and CO2, methanol, and dimethylamine, 9 and 1%; methanogens that grow with formate, 8 and 5%; and methanogens that grow with acetate, 25 and less than 0.8% . Shortly after the temperature was elevated from 35 to 50 or 60 degrees C, the digestion of albumin, casein, starch, and mono- and disaccharides was detected, and methane was produced from H2 and CO2 . Methane produced from acetate was not delayed at 50 degrees C, but was delayed by 29 days at 60 degrees C . Methane produced from formate was delayed by 3 days, from methanol by 7 days, and from dimethylamine by 5 days at 50 and 60 degrees C . A 10- and 20-day acclimation period was required for hydrolysis of pectin and cellulose, respectively, at 50 degrees C . Digestion of pectin required 20 days and cellulose longer than 85 days when the temperature was elevated abruptly from 35 to 60 degrees C . The acclimation period for the digestion of pectin and cellulose at 60 degrees C was shortened to 3 and 15 days, respectively, by seeding with a small amount of a culture acclimated to 50 degrees C . The data suggest that enrichment of cellulolytic, pectinolytic, and acetate-utilizing bacteria is crucial for the digestion of sewage sludge at 60 degrees C. Mol Cell Biol, 1983 Apr, 3(4), 503 - 10 Characterization of a cycloheximide-resistant Tetrahymena thermophila mutant which also displays altered growth properties; Hallberg RL et al.; A cycloheximide-resistant strain of Tetrahymena thermophila, expressing a mutant chx-B gene (Ares and Bruns, Genetics 90:463-474, 1978), displayed very different temperature-dependent growth characteristics than either wild-type cells or another cycloheximide-resistant strain expressing a different mutant gene . Whereas wild-type cells showed an immediate decline in ribosome translocation rates when shifted from 30 to 38 or 40 degrees C, this mutant strain (X-8) showed no such decline . These results directly correlated with the growth rate differences we found for these cells at these temperatures . By genetic analysis, we showed that the phenotype of cycloheximide resistance cosegregated with the ability to grow rapidly at 40 degrees C . Analyses, both direct and indirect, suggested that a number of functional and structural characteristics of the ribosomes from strain X-8 cells are most likely conformationally different from those of wild-type ribosomes. J Allergy Clin Immunol, 1983 Apr, 71(4), 389 - 93 Marijuana smoking and fungal sensitization; Kagen SL et al.; The possible role of marijuana (MJ) in inducing sensitization to Aspergillus organisms was studied in 28 MJ smokers by evaluating their clinical status and immune responses to microorganisms isolated from MJ . The spectrum of illnesses included one patient with systemic aspergillosis and seven patients with a history of bronchospasm after the smoking of MJ . Twenty-one smokers were asymptomatic . Fungi were identified in 13 of 14 MJ samples and included Aspergillus fumigatus, A . flavus, A . niger, Mucor, Penicillium, and thermophilic actinomycetes . Precipitins to Aspergillus antigens were found in 13 of 23 smokers and in one of 10 controls, while significant blastogenesis to Aspergillus was demonstrated in only three of 23 MJ smokers . When samples were smoked into an Andersen air sampler, A . fumigatus passed easily through contaminated MJ cigarettes . Thus the use of MJ assumes the risks of both fungal exposure and infection, as well as the possible induction of a variety of immunologic lung disorders. J Dairy Sci, 1983 Mar, 66(3), 444 - 9 Contribution of Streptococcus thermophilus to growth-stimulating effect of yogurt on rats; Wong NP et al.; The origin of the growth-stimulating factor in yogurt was studied in rats fed liquid or freeze-dried diets of milk, yogurt, milks fermented individually by Streptococcus thermophilus and Lactobacillus bulgaricus, milks to which cells of Streptococcus thermophilus and Lactobacillus bulgaricus were added . Diets containing sonicated cells, cell supernatant, and cell fractions also were fed . Milk fermented by Streptococcus thermophilus and milk plus Streptococcus thermophilus cells stimulated growth as effectively as did yogurt . That finding and the absence of stimulation in rats fed Lactobacillus bulgaricus showed that Streptococcus thermophilus is responsible for stimulation of growth by yogurt . Growth was stimulated by an intracellular factor and not by fermentative changes in the milk. J Bacteriol, 1983 Mar, 153(3), 1266 - 71 Bacterial elongation factor Ts: isolation and reactivity with elongation factor Tu; Wittinghofer A et al.; An improved method for the purification of bacterial polypeptide elongation factor Ts (EF-Ts) from one mesophile (Escherichia coli) and two thermophiles (Bacillus stearothermophilus and PS3) is described . The improvements are both in the facility of isolation and in increased yields . The purified factors were used for cross-reactivity studies with elongation factor Tu (EF-Tu) obtained from the same bacterial strains . In all combinations studied, the efficiency of EF-Ts in catalyzing the exchange of EF-Tu-bound GDP was proportional to the strength of the protein-protein complex . Whereas the factors from the two thermophiles were interchangeable, the mesophilic EF-Ts formed a very weak complex with thermophilic EF-Tu; however, thermophilic EF-Ts formed very strong complexes with mesophilic EF-Tu . Thus, e.g., EF-Tu from E . coli formed a complex with EF-Ts from B . stearothermophilus which was 10 times more stable than the corresponding homologous complex. Nature, 1983 Feb 10, 301(5900), 511 - 3 Chemolithoautotrophic metabolism of anaerobic extremely thermophilic archaebacteria; Fischer F et al.; Several types of extremely thermophilic archaebacteria have recently been isolated from solfataric water holes, hot springs and hot sea floors . It has been shown that some of them can live using sulphur respiration of reduced carbon substrates as a source of energy, a type of metabolism previously described for the eubacterium Desulfuromonas . We report here that several extremely thermophilic archaebacteria can live with carbon dioxide as their sole carbon source, obtaining energy from the oxidation of hydrogen by sulphur, producing hydrogen sulphide . They are thus capable of a new type of anaerobic, purely chemolithoautotrophic metabolism, a possible primaeval mode of life. J Appl Bacteriol, 1983 Feb, 54(1), 115 - 25 Development of a blood-free Campylobacter medium: screening tests on basal media and supplements, and the ability of selected supplements to facilitate aerotolerance; Bolton FJ et al.; The capacity of six basal media to support the growth of thermophilic campylobacters was tested . The most successful was Nutrient Broth No . 2 (Oxoid) solidified with New Zealand agar but it gave at best only a 9% recovery rate . Various blood products, iron compounds, detoxifying agents, reducing agents, growth stimulants and an antimetabolite were added to the selected basal medium and counts of inoculated organisms were compared with counts on basal medium containing 5% lysed horse blood . Of 22 supplements tried only blood, Fildes' peptic digest of blood, heamatin, iron salts, charcoal, sodium metabisulphite and sodium pyruvate greatly improved the basal medium . The ability of these supplements used singly and in combinations to facilitate aerotolerance of campylobacters was investigated . Two aspects of aerotolerance were tested; (a) the ability of the supplements to sustain the viability of campylobacters seeded onto culture plates left on the bench for up to 6 h before microaerobic incubation; and (b) the ability of the supplements to facilitate the growth of campylobacters at increasing oxygen tension (6, 10 and 17% oxygen) . A combination of 0.4% charcoal, 0.025% ferrous sulphate and 0.025% sodium pyruvate was found to be as effective as blood in both tests. J Biochem (Tokyo), 1983 Feb, 93(2), 503 - 11 Purification and properties of adenosine 5'-triphosphate-dependent deoxyribonuclease from Thermus thermophilus HB8; Watanabe N et al.; An ATP-dependent DNase has been purified from Thermus thermophilus HB8 by a procedure involving streptomycin precipitation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and heparin-agarose affinity chromatography . ATP-dependent DNase activity was separated into two distinct peaks, Peak A and Peak B, by heparin-agarose affinity chromatography . Each peak fraction was further purified by ATP-agarose affinity chromatography . Peak A and Peak B were eluted from an ATP-agarose column at 0.14 M and 0.28 M KCl, respectively, each as a single peak . Both enzyme activities require ATP and Mg2+ for the degradation of double- and single-stranded DNAs, and degrade denatured DNA about 1.5 times faster than native DNA . The two peaks are optimally active at 69 degrees C and have similar optimal pH ranges from 8.2 to 9.2 . The two purified peaks were unstable on storage at -20 degrees C, but were remarkably stabilized by addition of 0.4 mg/ml bovine serum albumin . Ammonium sulfate strongly inhibits the activities of both peaks . The molecular weights of Peak A and Peak B are about 170,000 as estimated by glycerol gradient sedimentation . The average chain lengths of denatured DNA produced by Peak A and Peak B were 4.2 and 3.6, respectively, and the products were terminated by 5'-phosphoryl and 3'-hydroxyl groups . The limit-digested products of denatured DNA produced by Peak B consist of mono-, di-, tri-, tetra-, and pentanucleotides along with some larger fragments . The mode of action of both activities is processive and Peak A does not attack double-stranded circular DNA. J Biochem (Tokyo), 1983 Feb, 93(2), 461 - 8 Investigation of actin in Tetrahymena cells . A comparison with skeletal muscle actin by a devised two-dimensional gel electrophoresis method; Hirabayashi T et al.; Total protein constituents of Tetrahymena thermophila strain B1868 III were studied by two-dimensional agarose-polyacrylamide gel electrophoresis to detect actin among the constituents . In the attempts to prepare a whole-cell extract of Tetrahymena, it was found that protease activity in the extract was so high that high molecular components were quickly digested with the endogenous protease into small peptides unless the homogenization and heat-treatment in a sodium dodecylsulfate solution were performed within 5 s . It was eventually found that employment of 8 M guanidine hydrochloride (HCl) in the homogenization of cells perfectly prevented the degradation of protein components, even through a long preparation procedure . A devised two-dimensional agarose-polyacrylamide gel electrophoresis of the guanidine HCl extract gave a 'protein map' on which most proteins were located in their respective positions, including proteins with more than 200,000 mol . wt . Addition of rabbit skeletal muscle actin on the protein map revealed that no protein with isoelectric point and molecular weight identical with those of the actin was contained in the whole Tetrahymena extract, suggesting that Tetrahymena actin may have characteristics far different from those of skeletal muscle actin. Lab Anim Sci, 1983 Feb, 33(1), 56 - 9 Preliminary findings on the use of protozoa (Tetrahymena thermophila) as models for ocular irritation testing in rabbits; Silverman J; The ciliated protozoan Tetrahymena thermophila (ATCC 30008) was used as an indicator organism in the development of an in vitro test to replace in vivo testing of chemicals in the eyes of rabbits . Fifty microliters of Tetrahymena suspension were mixed with fifty microliters of varying dilutions of test chemicals . After 2 minutes, a bacteriological loopful of the mixture was examined microscopically to evaluate the motility of the organism . The minimum dilution allowing nearly 100% typical cell motility was recorded as was the maximum dilution causing nearly 100% cell death . The reciprocals of these two dilutions were added together, and the higher this number, the more toxic the compound . The results were compared to published reports of rabbit eye irritancy studies . The preliminary in vitro testing indicated a close correlation with in vivo testing with many, but not all chemicals tested . The incidence of false negatives was minimal. Appl Environ Microbiol, 1983 Feb, 45(2), 381 - 3 House flies (Musca domestica) as possible vectors of Campylobacter fetus subsp . jejuni; Rosef O et al.; A total of 161 strains of Campylobacter fetus subsp . jejuni were isolated from house flies (Musca domestica) . The carrier rates detected were 50.7% in flies captured on a chicken farm and 43.2% in flies from a piggery . The relative prevalences of Campylobacter coli, C . jejuni, and nalidixic acid-resistant thermophilic campylobacters were 90.1, 6.2, and 3.7%, respectively . The results indicate that flies may play a linking role in the epidemiology of Campylobacter infection in humans by transmitting campylobacters from animals to human food. Biochimie, 1983 Feb, 65(2), 149 - 56 Purification and characterization of an endoglucanase from a newly isolated thermophilic anaerobic bacterium; Creuzet N et al.; An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) from a new cellulolytic thermophilic bacterium was purified to apparent homogeneity after being separated from a xylanase . Little carbohydrate was associated with the endoglucanase . A molecular weight of 91,000 and 99,000 was determined by SDS-polyacrylamide gel electrophoresis and by gel filtration of the native enzyme on Ultrogel ACA 34 . The optimal pH was approximately 6.4 and the enzyme was isoelectric at pH 3.85 . The enzyme was found highly thermostable: it retained 50 per cent of its activity after 1 hour at 85 degrees C . Hydrolysis of CMC took place with a rapid decrease in viscosity but a slow liberation of reducing sugars, indicating endo-enzyme activity . It showed little capacity to hydrolyze highly ordered cellulose . Cellobiose inhibited the activity of the endoglucanase . None of the metal ions tested stimulated the activity . The enzyme was completely inactivated by 1 mM Hg2+ and was inhibited by thiol reagents. Exp Cell Res, 1983 Feb, 143(2), 461 - 7 An amicronucleate mutant of Tetrahymena thermophila; Kaney AR et al.; A stable amicronucleate strain of Tetrahymena thermophila was isolated following nitrosoguanidine mutagenesis . The mutant has the same growth rate and viability as the micronucleate parent strain, and has no micronucleus detectable by chromatin-specific staining in vegetative growth or during conjugation . The mutant pairs with normal efficiency with cells of complementary mating type . Matings of the mutant with aneuploid strains which lose their micronucleus during meiosis produced cell pairs yielding one viable and one inviable cell . The mutant receives a micronucleus from a normal mating partner, but this micronucleus is lost by the mutant cells within two hundred generations. Tsitologiia, 1983 Feb, 25(2), 210 - 3 {Comparison of the heat resistance of ATP-hydrolyzing enzymes in 2 species of frogs}; Pisareva LN et al.; A comparison was made between the heat resistance levels of two enzymatic preparations: the actomyosinic and the transport ATPases of two species of frogs (grass and lake frogs) differing in their thermophilia . The interspecies differences in the heat resistance were found to be 6 degrees C for the actomyosinic and 3 degrees C for the transport ATPase in favour of the more thermophilic lake frog . In both species the myosinic ATPase is more sensitive to heat than the transport one . The high heat resistance of the transport ATPase is due to a higher threshold of its sensitivity to temperature. Philos Trans R Soc Lond B Biol Sci, 1983 Jan 26, 300(1100), 249 - 61 Large-scale purification of enzymes; Bruton CJ; Many standard procedures for the purification of proteins in the laboratory do not readily lend themselves to scaling up, whereas, on the other hand, some techniques relatively unsatisfactory in the laboratory are much more effective on a large scale . When producing gram or kilogram quantities of enzymes for use over an extended period, the storage properties and general tractability of the purified products become increasingly important . Hence enzymes from thermophilic sources frequently have advantages over those from mesophiles . The possible economic advantages of simultaneous large-scale multi-enzyme isolation over separate individual enzyme purifications are evaluated . Batchwise adsorption and elution from ion-exchange celluloses frequently replace traditional precipitation techniques in the early stages of a large-scale purification . Dialysis is replaced by concentration, dilution and reconcentration with the use of hollow-fibre ultrafiltration equipment . Antiphonally direct scaling-up of column chromatographic procedures is usually possible . Modifications to column geometry to maximize flow rates are often desirable but purification factors and recoveries comparable with those obtained on the laboratory scale can be achieved relatively easily . Classical affinity chromatographic techniques have not proved so amenable to large-scale work, mainly because of the enormous expense and rather short life of the matrices . However, the quasi-affinity chromatography afforded by the triazine dye conjugates has proved of great benefit . The materials are cheap to prepare . The coupling procedures are both simple and rapid and do not involve the use of noxious chemicals such as cyanogen bromide . Moreover the triazine linkage is more stable under a variety of conditions than the isourea formed in cyanogen bromide coupling . Considerable further exploitation of these versatile matrices is expected. Biochemistry, 1983 Jan 18, 22(2), 484 - 9 Glycosylation, ADP-ribosylation, and methylation of Tetrahymena histones; Levy-Wilson B; We have examined some of the postsynthetic modifications that occur in macronuclear histones from Tetrahymena thermophila . When purified macronuclei are incubated with {32P}NAD+, histones H1, H2A, H2B, and H3 are ADP-ribosylated . Furthermore, histones H1, H2A, H2B, and H3 contain fucose and mannose residues as evidenced by the incorporation of {3H}fucose and by the specific binding to these proteins of gorse seed lectin and concanavalin A . Finally, our studies on incorporation of methyl groups into histones show that histone H2A, together with the related nonhistone protein A24, is methylated in Tetrahymena. Biochim Biophys Acta, 1983 Jan 12, 742(1), 197 - 205 Ornithine decarboxylase activity from an extremely thermophilic bacterium, Clostridium thermohydrosulfuricum . Effect of GTP analogues on enzyme activity; Paulin L et al.; The activity of ornithine decarboxylase has been detected for the first time in extracts of a thermophilic bacterium, Clostridium thermohydrosulfuricum . The temperature optimum of the thermoresistant ornithine decarboxylase was 55 degrees C and the pH optimum was 7.5 . It required pyridoxal phosphate and a thiol (dithiothreitol) for activity . The activity of the enzyme was closely connected to the growth of the thermophilic bacteria, since the activity was highest during the logarithmic growth . The enzyme was not inhibited (in contrast to the enzyme from Escherichia coli) by putrescine, spermidine or other naturally occurring polyamines . When the effect of GTP and a number of GTP analogues was tested on the activity of the enzyme, it was observed that GTP or dGTP was necessary for the full activity . The modification of either the purine base or 5'-phosphate chain of GTP leads to a stimulation smaller than that caused by GTP . Modification of the 3'-carbon of the ribose part of GTP (magic spot I and II of Cashel and Gallant, Nature 221 (1969) 838-841) caused a distinct inhibition of the enzyme activity, indicating that ornithine decarboxylase contains at least two domains for binding of GTP . The enzyme was inhibited irreversibly by high concentrations (50 mM) of difluoromethylornithine . Extracts of the bacterium contained also arginine decarboxylase, but its activity was always very much lower than that of ornithine decarboxylase . The activity of arginine decarboxylase was inhibited irreversibly by difluoromethylarginine, which is an irreversible suicide inhibitor of bacterial arginine decarboxylase (Kallio, A., McCann, P.P . and Bey, P . (1981) Biochemistry 20, 3163-3166). Biochemistry, 1983 Jan 4, 22(1), 98 - 102 Separation and comparison of 2-thioribothymidine-containing transfer ribonucleic acid and the ribothymidine-containing counterpart from cells of Thermus thermophilus HB 8; Watanabe K et al.; For the extreme thermophile Thermus thermophilus HB 8, a positive correlation was observed among the growth temperatures of the cells, the melting temperature, and the 2-thioribothymidine (s2T) content of tRNA extracted from cells grown at various temperatures {Watanabe, K., Shinma, M., Oshima, T., & Nishimura, S . (1976) Biochem . Biophys . Res . Commun . 72, 1137-1144} . On the basis of these observations, studies were carried out from which the following results were obtained . (1) Both RNase T1 and U2 digestions of tRNA gave only four fragments containing s2T or T: s2T psi CGp, s2T psi CAp, T psi CGp, and T psi CAp . For the different growth temperatures, the ratio of the content of s2T psi CGp plus s2T psi CAp to that of T psi CGp plus T psi CAp was almost the same as that of the s2Tp to Tp content reported previously . (2) The midpoint of the s2T-specific circular dichroism spectral change induced by heat was constant for all tRNAs extracted from cells grown at various temperatures, suggesting that the s2T-containing tRNAs melt at about the same temperature, which is independent of the growth temperature of cells . (3) s2T-containing tRNA was separated from the T-containing counterpart quantitatively by a specific modification of s2T with bromoaceto-2,4-dinitroanilide followed by BD-cellulose column chromatography . The molar ratio of the s2T- and T-containing tRNAs was also similar to that of s2Tp to Tp as mentioned above . These results demonstrate that T . thermophilus cells have both s2T- and T-containing tRNAs, whose relative content is controlled by the growth temperature . This phenomenon may be necessary to enable the thermophile to adapt to higher temperatures. Biochemistry, 1983 Jan 4, 22(1), 85 - 93 Localization of the elongation factor Tu binding site on Escherichia coli ribosomes; Rychlik W et al.; Fluorescent techniques were used to study binding of peptide elongation factor Tu (EF-Tu) to Escherichia coli ribosomes and to determine the distances of the bound factor to points on the ribosome . Thermus thermophilus EF-Tu was labeled with 3-(4-maleimidylphenyl)-4-methyl-7-(diethyl-amino)coumarin (CPM) without loss of activity . In the presence of Phe-tRNA and a nonhydrolyzable analogue of GTP, 70S ribosomes bind the CPM-EF-Tu {Kb = (3 +/- 1.2) X 10(6) M-1} causing a decrease of CPM fluorescence . Binding of CPM-EF-Tu to 50S subunits was at least 1 order of magnitude lower than with 70S ribosomes, and binding to 30S subunits could not be detected . Reconstituted 70S ribosomes containing either S1 labeled with fluoresceinmaleimide or ribosomal RNAs labeled at their 3' ends with fluorescein thiosemicarbazide were used for energy transfer from CPM-EF-Tu . The distances between CPM-EF-Tu bound to the ribosomes and the 3' ends of 16S RNA, 5S RNA, 23S RNA, and the closest sulfhydryl group of S1 were calculated to be 82, 70, 73, and 62-68 A, respectively. Nutr Cancer, 1983, 5(3-4), 159 - 64 Effect of feeding fermented milk on the incidence of chemically induced colon tumors in rats; Shackelford LA et al.; The effect of feeding skim milk fermented by Streptococcus thermophilus or Lactobacillus bulgaricus on the incidence of chemically induced colon tumors was studied in rats . Weanling Fisher-344 rats were fed chow plus skim milk (SM), chow plus SM fermented by S . thermophilus, chow plus SM fermented by L . bulgaricus, or chow plus water until sacrifice at 36 weeks, or before if moribund . Colon tumors were induced by s.c . injections of 1,2-dimethylhydrazine hydrochloride during weeks 3 through 22 . The control (chow + water) group received saline injections . The survival rate of the rats fed fermented milks was significantly higher than that of the rats fed nonfermented milk . The latter had a significantly higher incidence of ear-duct tumors than the rats receiving fermented milk . The percentage of rats showing colon tumors was similar among all three experimental groups . The control group did not have any tumors . The rats receiving fermented milk had a significantly higher incidence of small-intestine tumors than those receiving nonfermented milk . The rats on S . thermophilus milk had the lowest percentage of malignant colon tumors of the three experimental groups . Results indicated that the feeding of fermented milks altered the metabolism of 1,2-dimethylhydrazine and shifted the target organ from the ear duct to the small intestine . In addition, the colon tumor distribution for the fermented-milk groups appeared to shift toward the anus. Nucleic Acids Symp Ser, 1983, (12), 217 - 20 An algorithm for the bonding-probability map of nucleic acid secondary structure; Suyama A et al.; We report a more efficient and well-defined algorithm for predicting a secondary structure of single-stranded nucleic acid from a primary nucleotide sequence . Using this algorithm, one- and two-dimensional bonding-probability maps of 5S rRNA of thermus thermophilus HB8 were calculated . These maps well express the stability of the secondary structure. Nucleic Acids Symp Ser, 1983, (12), 173 - 6 Synthesis of model compounds for the interaction between modified nucleic acid bases; Higashii T et al.; In order to study the stacking interaction of modified nucleic acid bases, which is assumed to be responsible for the anomalous thermostability of thermophile tRNA, several model compounds 1-6 are synthesized . On the basis of their hypochromicities, the interaction between bases are discussed. Nucleic Acids Symp Ser, 1983, (12), 153 - 4 Aminoacyl-tRNA synthetases from an extreme thermophile, Thermus thermophilus HB8; Kohda D et al.; Thermostable aminoacyl-tRNA synthetases specific to Val, Ile, Met and Glu were purified from an extreme thermophile, Thermus thermophilus HB8 . As for the subunit compositions and molecular weights, these four aminoacyl-tRNA synthetases are similar to the corresponding enzymes from E . coli and B . stearothermophilus . Val-tRNA, Ile-tRNA and Met-tRNA synthetases from T . thermophilus have two tightly bound zinc ions, whereas Glu-tRNA synthetase does not . The amino acid compositions and secondary structures of Val-tRNA, Ile-tRNA and Met-tRNA synthetases are quite similar to one another . The conformational transition involving the anticodon of E . coli tRNAGlu as complexed with Glu-tRNA synthetase from T . thermophilus is necessary for the aminoacylation activity. Int J Vitam Nutr Res, 1983, 53(4), 394 - 7 {Microbiological assay of pantothenic acid in rat liver using Tetrahymena}; Lhuissier M; Microbiological Assay of Pantothenic Acid in Rat Liver using Tetrahymena . The quantitative determination of total pantothenic acid in rat liver using Tetrahymena was carried out in the same way as a microbiological assay . The strain selected was Tetrahymena thermophila grown on rat liver . Inoculum was obtained from a commercial medium . The assay medium consisted of a dry powder to be rehydrated and heated to boiling, and then adjusted to pH 7 . Liver pantothenic acid was extracted by autolysis . Tetrahymena use made one accustomed to a biological reagent easy to manipulate but with complex nutritional requirements . Consequently the organism should be more extensively used for nutritional or toxicological studies. Biochem Soc Symp, 1983, 48, 133 - 46 Industrial prospects for thermophiles and thermophilic enzymes; Hartley BS et al.; Reasons for enzyme instability are discussed . Thermophiles are a promising source of more stable intracellular enzymes . This aids their purification as well as providing desirable industrial properties . The organisms themselves have advantages for high temperature fermentations, e.g . for ethanol production . Systems for cloning genes into them are under development . An example is given of genetic and physiological manipulation of a fast-fermenting Bacillus stearothermophilus to increase ethanol yields at 70 degrees C similar to those obtained with yeasts. J Biochem (Tokyo), 1983 Jan, 93(1), 7 - 13 Heat-stable extracellular proteolytic enzyme produced by Thermus caldophilus strain GK24, an extremely thermophilic bacterium; Taguchi H et al.; Proteolytic activity was detected in the culture supernatant of a newly isolated, extremely thermophilic bacterium belonging to the genus Thermus, and tentatively named T . caldophilus sp . n . strain GK24 . The enzyme activity continued to increase for at least three days after cells reached the stationary phase of growth . Purification of the proteolytic enzyme was tried with ammonium sulfate fractionation, gel filtration, and ion exchange chromatography . The most purified enzyme fraction thus obtained appeared to be homogeneous in a chromatographic analysis, but still had seven bands of proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis . Treatment of the protease with denaturing reagents or organic solvents did not alter the chromatographic profile and the purified enzyme sample showed a large sedimentation coefficient of about 11S . The optimal pH of the hydrolytic activity of the enzyme was observed at around 7.8 for casein and 7.2 for N-carbobenzoxy-L-leucyl-L-tyrosinamide (Z-Leu-Tyr-NH2) . The enzyme was stable in the pH range of 5 to 11 for 1 day at 4 degrees C or for 1 h at 70 degrees C . The enzyme sample showed a maximal activity at 90 degrees C and had an extreme stability toward treatment by heat and denaturing reagents . The enzyme sample was inactivated almost completely by diisopropyl fluorophosphate (DFP), but not by ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA) . From these results, the enzyme seems to be a serine protease, and not to be a metallo-enzyme such as thermolysin . The enzyme also was hydrolytic active toward an ester compound, N-benzoyl-L-tyrosine ethyl ester (BTEE), but not toward N-benzoyl-L-arginine ethyl ester (BAEE). J Membr Biol, 1983, 75(1), 45 - 56 Monolayer black membranes from bipolar lipids of archaebacteria and their temperature-induced structural changes; Gliozzi A et al.; The membrane of Caldariella acidophila, an extreme thermophilic archaebacterium, is characterized by unusual bipolar complex lipids . They consist of two nonequivalent polar heads, linked by a C40 alkylic component . The molecular organization of these lipids in the plasma membrane is still a matter of study . In this paper, we present current-voltage measurements on artificial bipolar lipid membranes, indicating that molecules are indeed organized as a covalently bound bilayer, in which each molecule is completely stretched and spans its entire thickness . Furthermore, conformational transitions of these artificial membranes (which could be formed only above 70 degrees C from a lipid/squalene dispersion) are analyzed in the 80 to 15 degrees C temperature range . Abrupt variations in capacitance and valinomycin-induced conductance seem to indicate the occurrence of at least two structural changes . Measurements are also extended to different solvent systems . Results are consistent with the picture of a monolayer bipolar lipid membrane in which few solvent molecules align themselves parallel to the lipophilic chains . The amount of solvent as well as the temperature at which conformational transitions occur, depend on the solvent system in which the lipid is dispersed. J Biochem (Tokyo), 1983 Jan, 93(1), 225 - 34 Outer and inner membrane preparations of extreme thermophile and their physico-chemical properties; Oshima M et al.; Outer and inner (cytoplasmic) membranes were partially purified from the gram negative extremely thermophilic bacteria, Thermus thermophilus HB-8 by sucrose density gradient centrifugation . In spite of our efforts to separate them, the inner membrane fraction contained some outer membrane components as determined by enzyme assay and electrophoresis . When studied by 5DS spin labeling, the outer membranes showed a larger 2T11 value (lower fluidity) than the inner membranes, although the fatty acid compositions were similar . The inner membranes of the cells cultured at higher temperature showed a larger 2T11 value than the cells cultured at lower temperature . A similar phenomenon was observed with the TEMPO parameter of liposomal membranes . The upper break point (Th) of the inner membranes observed by spin labeling was slightly lower than the culture temperature of the cells, and the lower break point (T1) corresponded well to the lowest temperature limit of growth . The calorimetric heating curve of the inner membranes had a broader temperature range of transition than that of the liposomal membranes . The transition temperature observed by calorimetry seems to reflect the melting properties of the membrane lipids, while fatty acid spin probe probably reports the local environment of the membrane, which is more directly related to its biological function. Eur J Biochem, 1982 Dec 15, 129(2), 429 - 36 Purification and characterization of tRNA (adenine-1-)-methyltransferase from Thermus flavus strain 71; Morozov IA et al.; tRNA (adenine-1-)-methyltransferase was isolated from the extreme thermophile Thermus flavus, strain 71 . It was purified about 2000-fold by ammonium sulfate fractionation and affinity chromatography on tRNA bound to aminohydroxybutylcellulose via its oxidized 3' end . The purified protein preparation is free of nuclease and aminoacyl-tRNA synthetase activity and contains no more than 4% of tRNA (guanine-7-)methyltransferase activity . The only activity of the enzyme is to methylate A58 in the T psi loop of tRNA . Out of the eight purified tRNAs examined, only yeast tRNATrp was not utilized as a substrate . The enzyme is highly thermostable . It is most active at 75 degrees C . tRNA (adenine-1-)-methyltransferase has a Km of 0.4-0.5 microM for tRNA2Gln from Escherichia coli and a Km of 6 microM for S-adenosyl-L-methionine. J Anim Sci, 1982 Dec, 55(6), 1293 - 302 Nutritive value of methane fermentation residue in diets fed to feedlot steers; Harris JM et al.; Nutritive value of the methane fermentation residue (MFR) from the effluent of a large scale thermophilic methane generator was determined in diets fed to feedlot steers . The MFR contained 22.2% dry matter and 21.9% crude protein (dry basis) . Two diets containing 10.6% (dry basis) MFR were formulated using the Urea Fermentation Potential (UFP) system such that in one diet N was in excess (-1.6 UFP) while in the other diet energy was in excess (+2.6 UFP) . These two diets were compared in a California Net Energy trial with a feedlot diet (-.3 UFP) containing the same ingredients except the MFR . Six steers were fed in a replicated 3(2) Latin square metabolism trial and 70 steers were fed in a 118-d comparative-slaughter, feedlot trial . Digestibilities of dry matter, organic matter, crude protein, acid detergent fiber, ash, total digestible nutrients (TDN) and metabolizable energy were depressed (all P less than .05) in the MFR-containing diets . Steers fed the MFR-containing diets had lower (P less than .05) rates of gain and increased (P less than .05) feed requirements per unit gain . Net energies for maintenance and gain were slightly lower for the MFR-containing diets than the control diet . Crude protein digestibility for the MFR calculated by difference, for the -UFP and the +UFP diets were 37.8 and 50.7%, while corresponding values for TDN were 28.8 and 12.8%, respectively . Concentrations of potentially toxic elements in kidney, liver and muscle as well as flavor and tenderness of steaks were not affected by feeding MFR. J Lipid Res, 1982 Dec, 23(9), 1301 - 7 The effect of temperature on glyceryl ethers in Tetrahymena pyriformis W; Lund-Katz S et al.; The effect of temperature on the ether content of the glycerophospholipids of Tetrahymena pyriformis W was examined . The only ether detected was 1-O-hexadecyl glycerol (alpha-chimyl alcohol) . The data provide evidence that the class 1-O-alkyl-2-acyl-sn-glycero-3-(2-aminoethyl)-phosphate (1-alkyl PsE), in addition to the previously reported 1-O-alkyl-2-acyl-sn-glycero-3-(2-aminoethyl)-phosphonate (1-alkyl PnE) and 1-O-alkyl-2-acyl-sn-glycero-3-phosphorylcholine (1-alkyl PC), exists in this ciliate species . A comparison was made of the ether content of the glycerophospholipids from cells grown at 15 degrees and 28.5 degrees C . An elevation in the amount of ether was noted in all glycerophospholipids at the lower temperature with the largest proportional change in 1-alkyl PsE . Tetrahymena species have a high gamma-linolenic acid content in the sn-1 position of the glycerophospholipids in addition to the usual saturated acids and ether . The replacement at low temperature of gamma-linolenic acid by a saturated hydrocarbon at the sn-1 position of the glycerophospholipids of Tetrahymena pyriformis W should increase the microviscosity of the membranes; thus, it is difficult to envision this alteration in the glycerophospholipids as an adaptive change beneficial for growth . These findings are in direct contrast to the situation in Tetrahymena thermophila where the percentage of ether glycerophospholipids increases at the expense of gamma-linolenate as the temperature rises. J Clin Microbiol, 1982 Dec, 16(6), 995 - 9 Comparison of methods for isolation and enumeration of thermophilic actinomycetes from dust; Treuhaft MW et al.; Thermophilic actinomycetes are the primary sensitizing agents in farmer's lung disease . We compared dilution pour-plate and spread-plate methods for their usefulness in enumerating thermophilic actinomycetes in moldy silage dust and evaluated the ability of a nonquantitative gravity settling technique to recover thermophilic actinomycetes from moldy silage . Spread plates and pour plates yielded similar estimates of total thermophiles . Higher counts were observed on spread plates (P less than 0.05) for Thermoactinomyces candidus, Micropolyspora faeni, and Saccharomonospora viridis . M . faeni and S . viridis were less efficient than T . candidus in breaking through the agar of pour plates to form colonies which could be identified . Coefficients of variability were less than 10% for the two methods . The relative proportion of organisms recovered by the settling method correlated well with that recovered on spread plates for M . faeni (r = 0.79), S . viridis (r = 0.88), and Thermomonospora spp . (r = 0.79), but not well for T . candidus (r = 0.28) . When sophisticated air-sampling equipment is not available, dilution spread plates of dust washings provide a reproducible method for enumerating a broad range of thermophilic actinomycetes of interest . The gravity settling method is a simple, rapid alternative when isolation is all that is required. J Biochem (Tokyo), 1982 Dec, 92(6), 1823 - 32 A new aspect of a restriction endonuclease Tth111 I . It has a degenerated specificity (Tth111 I); Shinomiya T et al.; We previously reported that Thermus thermophilus 111 contained two restriction enzymes, Tth111 I and Tth111 II . The former does not cleave phi X174RFDNA and the latter does . We have now found another endonuclease activity able to cleave phi X174RFDNA in the cell extract of T . thermophilus 111 . The protein with this activity was purified in a homogeneous state by chromatography on cellulose phosphate, heparin-Sepharose 4B and hydroxylapatite, successively . However, this endonuclease activity was always accompanied with Tth111 I activity during the purification procedure and the purified protein also showed a strong Tth111 I activity, suggesting that the Tth111 I activity and the phi X174RFDNA-cleaving activity reside in a single molecule . The phi X174RFDNA-cleaving activity was enhanced more strongly with Mn2+ than with Mg2+ and seemed to be attributable to a relaxed specificity of Tth111 I activity as seen in the cases of EcoRI* and BamHI* Thus we designated the phi X174RFDNA-cleaving activity Tth111 I* . The molecular weight of the protein with both Tth111 I and Tth111 I* activities was determined to be about 76,000 by gel filtration on a Sephadex G-100 column and 39,000 by SDS-polyacrylamide gel electrophoresis, suggesting the enzyme to be a dimer consisting of identical polypeptide chains . The phi X174RFDNA sequences surrounding Tth111 I* cuts were determined by the chain terminator method of Sanger et al . The results confirmed that Tth111 I* recognized a degenerated form of the Tth111 I recognition sequence, i.e., a sequence such that one of the specified nucleotides in the Tth111 I recognition sequence, 5'GACNNNGTC3', was substituted with N (N stands for any of A, G, C, and T), such as 5'NACNNNGTC3', 5'GACNNNNTC3', 5'GACNNNGNC, and so on (arrows indicate cleavage sites). J Biochem (Tokyo), 1982 Dec, 92(6), 2043 - 6 Nanosecond fluorometric investigation of hydrodynamic properties of adenosine triphosphatase from thermophilic bacterium PS3; Kinosita K Jr et al.; The soluble portion (TF1) of proton-translocating ATPase from thermophilic bacterium PS3 was labeled with a fluorescent dye N-(1-pyrene)maleimide . The decay of fluorescence anisotropy of the adduct showed that TF1 in aqueous solution was characterized by a volume of equivalent sphere of 1,120 nm3 . This value is 2.4 times the volume calculated from the molecular weight and partial specific volume, indicating a non-spherical shape and/or extensive hydration . A prolate ellipsoid with an axial ratio of 2 to 3 is suggested as a first approximation of the shape of hydrated TF1 . The presence or absence of ATP, ADP, or Mg2+ did not alter the volume of the equivalent sphere appreciably; the probable conformational change of TF1 induced by these ligands does not lead to a gross alteration of its hydrodynamic properties. J Biol Chem, 1982 Nov 10, 257(21), 12600 - 4 Proton transport by cytochrome c oxidase from the thermophilic bacterium PS3 reconstituted in liposomes; Sone N et al.; Cytochrome oxidase from the thermophilic bacterium PS3 which contains three types of polypeptide subunits are reconstituted into liposomes by a freeze-thaw technique . The reconstituted enzyme caused acidification of the medium during cytochrome c oxidation with a stoichiometry of up to 0.8 H+/e . Uptake of K+ ions in the presence of valinomycin occurred with a stoichiometry between 1.5 and 2 K+/e . Dicyclohexylcarbodiimide inhibited the acidification and decreased the stoichiometry of K+ ion uptake to about 1 K+/e . This bacterial oxidase thus appears to be a proton pump with properties similar to the mitochondrial enzyme. J Biol Chem, 1982 Nov 10, 257(21), 12489 - 92 Mössbauer study of a bacterial cytochrome oxidase: cytochrome c1aa3 from Thermus thermophilus; Kent TA et al.; Cytochrome c1aa3 from Thermus thermophilus has optical and EPR properties similar to bovine cytochrome c oxidase . We have studied 87Fe-enriched samples with Mossbauer spectroscopy in the fully oxidized and fully reduced states and in the oxidized state complexed with cyanide . The cytochromes a and c1 yielded spectra quite similar to those reported for the cytochromes c and b5; in the oxidized state the spectra reflect noninteracting, low spin ferric hemes, whereas the a- and c1-sites of the reduced enzyme are typical of low spin ferrous hemochromes . The spectra of the reduced enzyme show that reduced cytochrome a3 is high spin ferrous, with Mossbauer parameters quite similar to those of deoxymyoglobin . Upon addition of cyanide to the oxidized enzyme, the a3-site exhibits in the absence of an applied magnetic field and at temperatures down to 1.3 K a quadrupole doublet with parameters typical of low spin ferric heme-CN complexes . The low temperature spectra taken in applied magnetic fields show that the electronic ground state of the a3-CN complex has integer electronic spin, suggesting ferromagnetic coupling of the low spin ferric heme (S = 1/2) to Cu2+ (S = 1/2) to yield as S = 1 ground state . We have examined the oxidized enzyme from two different preparations . Both had good activity and identical optical and EPR spectra . The Mossbauer spectra, however, revealed that the a3-site had a substantially different electronic structure in the two preparations . Neither configuration had properties in accord with the widely accepted spin-coupling model proposed for the bovine enzyme. J Gen Microbiol, 1982 Nov, 128 (Pt 11), 2515 - 22 Deoxyribonucleic acid sequence relatedness between thermophilic members of the genus Campylobacter; Belland RJ et al.; The genomic relatedness among 28 catalase-positive Campylobacter strains was assessed by determination of DNA base composition, by DNA:DNA hybridization followed by S1 endonuclease digestion of single-stranded DNA, and by determining the thermal stability of homologous and heterologous double-stranded DNA . The catalase-positive Campylobacter strains were shown to comprise 3 species, C . coli, C fetus and C . jejuni; each included the type strain (respectively, CIP 7080, CIP 5396 and CIP 702). Biokhimiia, 1982 Nov, 47(11), 1785 - 91 {Isolation and properties of DNA polymerase from the extreme thermophilic bacterium Thermus ruber}; Kaledin AS et al.; A DNA-polymerase from the external thermophylic bacteria Thermus ruber has been isolated . A six-step purification procedure resulted in an electrophoretically homogeneous enzyme preparation with m . w . of 70 000 . The isolated enzyme is thermostable and has a temperature optimum on DNA templates at 70 degrees and that on RNA templates at 50 degrees . The enzyme does not contain contaminant endo- and exonuclease activities . The maximal activity of the enzyme requires the presence of template, four deoxyribonucleoside triphosphates, monovalent and divalent cations in the incubation mixture . The catalytic activity of the enzyme on various templates of DNA- and RNA-types has been investigated . A comparative physico-chemical study of this DNA-polymerase and an analogous enzyme isolated earlier from the external thermophylic bacteria Thermus aquaticus YT-1 has been carried out. Cell, 1982 Nov, 31(1), 147 - 57 Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena; Kruger K et al.; In the macronuclear rRNA genes of Tetrahymena thermophila, a 413 bp intervening sequence (IVS) interrupts the 26S rRNA-coding region . A restriction fragment of the rDNA containing the IVS and portions of the adjacent rRNA sequences (exons) was inserted downstream from the lac UV5 promoter in a recombinant plasmid . Transcription of this template by purified Escherichia coli RNA polymerase in vitro produced a shortened version of the pre-rRNA, which was then deproteinized . When incubated with monovalent and divalent cations and a guanosine factor, this RNA underwent splicing . The reactions that were characterized included the precise excision of the IVS, attachment of guanosine to the 5' end of the IVS, covalent cyclization of the IVS and ligation of the exons . We conclude that splicing activity is intrinsic to the structure of the RNA, and that enzymes, small nuclear RNAs and folding of the pre-rRNA into an RNP are unnecessary for these reactions . We propose that the IVS portion of the RNA has several enzyme-like properties that enable it to break and reform phosphodiester bonds . The finding of autocatalytic rearrangements of RNA molecules has implications for the mechanism and the evolution of other reactions that involve RNA. J Biol Chem, 1982 Oct 10, 257(19), 11395 - 404 Structural studies of 5 S ribosomal RNAs from a thermophilic fungus, Thermomyces lanuginosus . A comparison of generalized models for eukaryotic 5 S RNAs; Wildeman AG et al.; The cytoplasmic ribosomes of the thermophilic fungus Thermomyces lanuginosus contain two types of 5 S RNA . The nucleotide sequence for approximately 80% of the molecules is (pp)pA-C-A-U-G-C-G-A-C-C-A-U-A-G-G-G-U-G-U-G-G-A-A-A-A-C-A-G-G-G-C-U-U-C-C-C-G-U-C-C-G-C-U-C-A-G-C-C-G-U-A-C-U-U-A-A-G-C-C-A-C-A-C-G-C-C-G-G-C-U-G-G-U-U-A-G-U-A-G-U-U-G-G-G-U-G-G-G-U-G-A-C-C-A-C-C-A-G-C-G-A-A-U-C-C-C-A-G-C-U-G-U-U-G-C-A-U-G-UOH . The remainder contains two nucleotide substitutions, C19 and G60, which preserve base complementarity . The secondary structure was probed using partial T1, pancreatic, and S1 nuclease digestion under a variety of ionic and temperature conditions and fragments were analyzed by rapid gel sequencing techniques . The results support the Y-shaped secondary structure model originally proposed by Nishikawa, K., and Takemura, S . (1974) FEBS Lett . 40, 106-109, for eukaryotic 5 S RNAs . When the thermal denaturation profile was compared with that of the yeast 5 S RNA, the thermophilic RNA exhibited not only a higher Tm but also an unusual decline in absorbency at moderate temperatures . This suggests that a functionally important structure may be maintained only at higher temperatures. Mol Cell Biochem, 1982 Oct 1, 48(1), 48 - 58 Glucose regulation of specific gene expression is altered in a glucokinase-deficient mutant of Tetrahymena; Lavine JE et al.; Expression of the galactokinase gene in Tetrahymena thermophila can be repressed by glucose, glucose analogs, and epinephrine, each apparently acting through increased intracellular levels of adenosine 3':5'-cyclic monophosphate (cAMP) (1) . To characterize further the initial steps in the control of galactokinase gene expression by glucose, we have analyzed mutants which are defective in the metabolism of this sugar; these mutants were selected for their resistance to the glucose analog, 2-deoxyglucose (2) . In one such mutant that is deficient in glucokinase, the synthesis of galactokinase is totally resistant to repression by glucose or its analogs, while repression by exogenous catecholamines or dibutyryl cAMP is unaffected . Radiochromatographic analyses of extracts of wild-type cells incubated with {14C}-deoxyglucose reveal intracellular conversion to several deoxyglucose metabolites, principally deoxyglucose-6-P and smaller amounts of deoxyglucose-1-P and 2-deoxygluconate; extracts of glucokinase-deficient cells prepared in a similar manner contain only trace amounts of deoxyglucose-6-P . The glucose analog 3-O-methylglucose, which is transported but not phosphorylated in wild-type cells, also cannot maintain repression of galactokinase . These results establish that the transport and subsequent phosphorylation of glucose are required for glucose-initiated repression of galactokinase gene expression, possibly acting by modulation of catecholamine or cyclic AMP levels . Additionally, we show unequivocally that: (a) cells containing derepressed levels of galactokinase are repressed upon the addition of glucose by inhibition of the synthesis of new enzyme and dilution of preformed enzyme concomitant with cell division, rather than through selective inactivation or degradation of galactokinase; and (b) glycerol kinase, glucokinase and fructokinase activities also are repressed by glucose in wild-type Tetrahymena, indicating that the glucose repression phenomenon is pleiotropic . Because the glucose repression of the synthesis of each of these enzymes is abolished in cells deficient in glucokinase, the regulatory mechanisms elucidated for repression of galactokinase synthesis are likely to be of wide significance. J Hyg (Lond), 1982 Oct, 89(2), 191 - 4 The occurrence of Campylobacter jejuni in dog faeces from a public park; Wright EP; Dog faeces collected from a public park were cultured on selective media for Campylobacter spp . Campylobacter jejuni was isolated from 12 (4.6%) of 260 specimens . In contrast Salmonella spp . were found in only three (1.2%) . Six of the 12 isolates were nalidixic acid-resistant thermophilic campylobacters (NARTC), whereas during the same period of study none were found among human isolates . Most of the campylobacter positive faeces were found during June and July . Dog faeces deposited in public places constitute only a small potential source of infection by this organism. Can J Microbiol, 1982 Oct, 28(10), 1181 - 8 {Peptide hydrolases of lactobacilli of the Thermobacterium group . I . Demonstration of these activities in Lactobacillus helveticus, L . acidophilus, L . lactis and L . bulgaricus}; El Soda M et al.; The intracellular peptide hydrolase activities of Lactobacillus helveticus, L . acidophilus, L . lactis, and L . bulgaricus were determined using various aminopeptidase, dipeptidase, and carboxypeptidase substrates in addition to casein and whey protein fractions . The different activities were then separated using disc gel electrophoresis . Each bacterium had aminopeptidase activity towards various amino acid beta-naphthylamides and dipeptides . The four species also showed bands of true dipeptidase activities on a large number of dipeptides . Intracellular enzymes from thermophilic lactobacilli also hydrolysed the whey proteins (alpha-lactalbumin and beta-lactoglobulin) . From the results of electrophoresis on beta-casein and alpha s1-casein it was shown that beta-casein was totally hydrolysed by L . lactis while it was only partially hydrolysed by the intracellular enzymes of L . acidophilus and L . bulgaricus . On the other hand, alpha s1-casein was only partially hydrolysed by L . helveticus, L . lactis, and L . bulgaricus. J Biol Chem, 1982 Sep 10, 257(17), 9913 - 4 A pentaamine is present in an extreme thermophile; Oshima T; A pentaamine was found in cells of an extreme thermophile, Thermus thermophilus, grown at 80 degrees C . The chemical structure was determined to be 1,15-diamino-4,8,12-triazapentadecane, NH2(CH2)3NH(CH2)3NH(CH2)3NH(CH2)3NH2 . A trivial name "caldopentamine" is proposed for the new naturally occurring polyamine. Med J Aust, 1982 Sep 4, 2(5), 244 - 6 Hypersensitivity pneumonitis in a mouldy house; Saltos N et al.; Two cases of hypersensitivity pneumonitis, one confirmed by histopatholgy, are described . The patients were members of different families and developed the disease as occupants, at separate times, of the same inner-city dwelling . We believe the disorder resulted from exposure to thermophilic microorganisms prevalent in their domestic environment . Both patients recovered after moving from this place of residence . The need for greater awareness of this variety of hypersensitivity pneumonitis is stressed. Can J Biochem, 1982 Sep, 60(9), 847 - 53 Reorganization of unique and repetitive sequences during nuclear development in Tetrahymena thermophila; Brunk CF et al.; Genomic libraries of macro- and micro-nuclear DNA of the ciliate protozoan Tetrahymena thermophila were constructed in the bacteriophage vector lambda gt WES lambda B . Screening of these libraries with a probe for the repeated hexamucleotide sequence C4A2 showed many phage from the micronuclear library but few if any macronuclear sequences having homology to this probe . This is consistent with C4A2-repeating elements being present predominantly if not exclusively at or near the termini of macronuclear DNA . Sequences flanking C4A2-repeating elements were isolated from a number of purified phage and were used as hybridization probes to restriction endonuclease digested macro- and micro-nuclear DNA . These experiments revealed a repeated sequence family as well as unique sequences present only in micronuclear DNA . The repeated sequence element appears to be dispersed throughout the genome . Phage-containing individual members of this micronucleus limited sequence family were purified from the micronuclear library . Some of these phage contained sequences which hybidized to macronuclear DNA . These fragments therefore contain a "transition" region between micronucleus-limited sequences and sequences present in both nuclei. Appl Environ Microbiol, 1982 Sep, 44(3), 757 - 60 Thermophilic biotransformations of 2,4,6-trinitrotoluene under simulated composting conditions; Kaplan DL et al.; The biotransformations of 14C-labeled 2,4,6-trinitrotoluene by thermophilic microorganisms in a compost system were determined . The reduction products identified in solvent extracts were similar to those identified in mesophilic systems . A significant percentage of the 14C-labeled products were bound to humus fractions. Nature, 1982 Aug 26, 298(5877), 867 - 9 E . coli F1-ATPase interacts with a membrane protein component of a proton channel; Walker JE et al.; The ATP synthases of bacteria, mitochondria and chloroplasts, which use the energy of a transmembrane proton gradient to power the synthesis of ATP, consist of an integral membrane component F0--thought to contain a proton channel--and a catalytic component, F1 . To help investigate the way F0 and F1 are coupled, we have sequenced the b-subunit of the Escherichia coli F0, which seems to be the counterpart of a thermophilic bacteria F0 subunit thought to be essential for F1 binding . We report here that its sequence is remarkable, being hydrophobic around the N-terminus and highly charged in the remainder . We propose that the N-terminal segment lies in the membrane and the rest outside . The extramembranous section contains two adjacent stretches of 31 amino acids where the sequence is very similar: in the second of these stretches there is further internal homology . These duplicated stretches of the polypeptide probably fold into two alpha-helices which have many common features able to make contact with F1 subunits . Thus protein b occupies a central position in the enzyme, where it may be involved in proton translocation . It is possibly also important in biosynthetic assembly. J Biol Chem, 1982 Aug 10, 257(15), 9114 - 8 On the phylogeny of Phycomyces blakesleeanus . Nucleotide sequence of 5 S ribosomal RNA; Andersen J et al.; The nucleotide sequence of the major 5 S ribosomal RNA from the lower fungus Phycomyces blakesleeanus has been determined . The sequence is 5' AAUCUACGGCCAUACAGAUAGUAACACACCGGAUCCCGUCUGAUCUCCGCAGUUAAGUCUCUCCUGGUAGCGUCAGUAC UAUGGUGGGGGACCACAUGGGAAUACGCUAUGUCGUAGGUU3'OH . The Phycomyces 5 S RNA sequence has invariant nucleotide positions characteristic of other eukaryotic 5 S RNAs and fits currently proposed secondary structural models . The Phycomyces of 5 S RNA shows relatively low overall sequence homology to the higher fungal (Ascomycetes) 5 S RNAs (56-60%) but shows higher sequence homology to those 5 S RNAs from Tetrahymena thermophila (68%), human KB cells (67%), and Spinacia oleracea (62%) . A comparison of individual segments of the RNA also shows that the structure of Phycomyces 5 S RNA has several major differences from structures common to the higher fungi . Positions 2-14 are homologous with those of metazoan and some protozoan 5 S RNAs . At positions 30-45, the RNA sequence is closer to metazoan 5 S RNAs than to the Neurospora of Aspergillus 5 S RNAs . The Phycomyces 5 S RNA shares similar sequences with both Aspergillus and Tetrahymena 5 S RNAs at positions 79-99 . Several other important homologies in primary and proposed secondary structures also have been observed in comparing Phycomyces 5 S RNA with animal and plant 5 S RNAs . We conclude that Phycomyces may not be as closely related phylogenetically to the Ascomycetes as previously thought. Biochim Biophys Acta, 1982 Aug 10, 705(3), 293 - 305 Purification and some properties of an extracellular protease (caldolysin) from an extreme thermophile; Cowan DA et al.; An extracellular metal-chelator-sensitive lytic protease (assigned the trivial name caldolysin) was isolated from a Thermus-like organism, Thermus T-351 . Caldolysin was purified by affinity chromatography on Cbz-D-phenylalanine-TETA-Sepharose 4B and by gel filtration . It contained 13% carbohydrate, a single zinc atom, had a molecular weight of approx . 21,000, a pH optimum of 8 (azocasein substrate), and an isoelectric point of about 8.5 . It was capable of hydrolysing many soluble and insoluble protein substrates, including collagen and elastin . No esterase activity was detected, and small peptides (less than four amino acids) and low molecular weight chromogenic substrates were not hydrolysed . A specificity for small aliphatic amino acids on either side of the splitting point was indicated . Caldolysin lysed heat-killed Gram-negative bacterial cells, but had little effect on Gram-positive organisms . Caldolysin exhibited a very high degree of thermostability (t 1/2(80 degrees C) approximately 30 h, t 1/2(90 degrees C) = 1 h) . The stability (but not activity) was shown to be dependent on the presence of Ca2+ (t 1/2(75 degrees C, 10 mM calcium) greater than 193 h; t 1/2(75 degrees C, no calcium) = 4.8 min) . None of the other metal ions tested (Co, Zn, Sr, Mg, Ba and Cu) was as effective as calcium in conferring thermostability of EDTA-treated caldolysin . Caldolysin was stable at room temperature in moderately acid and alkaline (pH 5 to 11) buffers for periods of greater than 90 days . Little loss of enzyme activity was detected after the incubation of caldolysin at 18 degrees C in the presence of 8 M urea, 6 M guanidine hydrochloride or 1% sodium dodecyl sulphate for 24 h . At 75 degrees C, the activity half-life of caldolysin in these denaturing agents was reduced to approx . 1 h, 1 h and more than 5 h, respectively. J Allergy Clin Immunol, 1982 Aug, 70(2), 101 - 8 Evaluation of indoor plantings as allergen exposure sources; Burge HA et al.; The role of indoor plantings as allergen sources was assessed by direct sampling of interior air . Homes with 10 or more plants in one room and three University of Michigan greenhouses were studied by means of a dc-powered rotorod and separate Andersen viable sampler collections incubated at 23 degrees and 50 degrees C . Sequential 30 and 60 sec Andersen samples were obtained during 15 min rotorod collections before and during watering of plants as well as during disturbance of foliage by a small fan . Relative humidity averaged 51% in homes and 78% in greenhouses . Aspergillus fumigatus recoveries were rare . Thermophiles, primarily bacteria, were present at low-to-moderate levels in homes, did not increase with watering of fan in homes, and rose only slightly with disturbance at greenhouse sites . Cladosporium and Penicillium dominated Andersen collections . Watering and fan increased levels of these taxa as well as rotorod recoveries of Alternaria . Epicoccum, and Pithomyces slightly in homes and markedly at greenhouse sites . We conclude that modest numbers of undisturbed house plants contribute minimally to aeroallergen prevalence in homes . However, especially under greenhouse conditions, plantings can harbor abundant fungus growth that may become airborne, especially when agitated directly. Mol Cell Biol, 1982 Aug, 2(8), 930 - 8 Mutant strains of Tetrahymena thermophila defective in thymidine kinase activity: biochemical and genetic characterization; Cornish KV et al.; Three mutant strains, one conditional, of Tetrahymena thermophila were defective in thymidine phosphorylating activity in vivo and in thymidine kinase activity in vitro . Nucleoside phosphotransferase activity in mutant cell extracts approached wild-type levels, suggesting that thymidine kinase is responsible for most, if not all, thymidine phosphorylation in vivo . Thymidine kinase activity in extracts of the conditional mutant strain was deficient when the cells were grown or assayed or both at the permissive temperature, implying a structural enzyme defect . Analysis of the reaction products from in vitro assays with partially purified enzymes showed that phosphorylation by thymidine kinase and nucleoside phosphotransferase occurred at the 5' position . Genetic analyses showed that the mutant phenotype was recessive and that mutations in each of the three mutant strains did not complement, suggesting allelism. Nucleic Acids Res, 1982 Jul 24, 10(14), 4409 - 12 The nucleotide sequences of 5S rRNAs from three ciliated protozoa; Kumazaki T et al.; The nucleotide sequences of 5S rRNAs from three ciliated protozoa, Paramecium tetraurelia, Tetrahymena thermophila and Blepharisma japonicum have been determined . All of them are 120 nucleotides long and the sequence of probable tRNA binding site of position 41-44 is GAAC which is characteristic of the plant 5S rRNAs . The sequence similarity percents are 87% (Paramecium/Tetrahymena), 86% (Paramecium/Blepharisma) and 79% (Tetrahymena/Blepharisma), suggesting a close relationship of these three ciliates. Nucleic Acids Res, 1982 Jul 24, 10(14), 4279 - 91 Rearrangement of repeated DNA sequences during development of macronucleus in Tetrahymena thermophila; Iwamura Y et al.; Three clones of non-repetitive sequences and six clones containing repetitive sequences were obtained from micronuclear DNA of Tetrahymena thermophila . All the non-repetitive and three repetitive sequences had the same organization in micro- and macronuclear DNAs as revealed by blot hybridization . On the other hand, the remaining three clones with repetitive sequences had apparently different organization in the two nuclear DNAs . All these repetitive sequences showed a smear on the blot in addition to a number of discrete bands when micronuclear DNA was digested with EcoR I . In macronuclear DNAs, these sequences invariably became one or two bands and the smear disappeared . We conclude that, when a macronucleus develops from a micronucleus, the non-repetitive sequences amplify by more than 20 times with relatively few rearrangement, whereas some selected portions of repeated and/or repeat-contiguous sequences are amplified with rather extensive reorganization. Biochim Biophys Acta, 1982 Jul 16, 717(1), 76 - 85 Purification and properties of galactokinase from Tetrahymena thermophila; Lavine JE et al.; Galactokinase (EC 2.7.1.6; ATP: D-galactose-1-phosphototransferase) was purified 152-fold with an 11% yield from Tetrahymena thermophila maximally derepressed for enzyme synthesis in late stationary phase . The purification procedure utilized sequential acid precipitation, batch DEAE-Sephacel chromatography, differential ammonium sulfate precipitation and narrow range electrofocusing . The apparent molecular weight of the holoenzyme as determined by gel filtration on Sephadex G-200 is 50000-55000 . The holoenzyme consists of two subunits of approx . 28000 daltons each, as determined by SDS-polyacrylamide gel electrophoresis . The native enzyme appears to be a single species with an isoelectric point at pH 5.1 . Optimal activity was obtained at pH 7.8 and 41 degrees C, with no added monovalent salt . D-Galactose, 2-deoxygalactose and galactosamine all are suitable carbohydrate substrates for the stereospecific galactokinase; only substitution at the C-2 position of galactose retains enzyme recognition . The enzyme utilizes ATP, 2'-dATP and 3'-dATP as phosphate donors; ADP and adenosine-5'-{gamma-thio}triphosphate are inhibitory . The Km values for galactose and ATP were determined to be 0.60 mM and 0.15 mM, respectively . The enzyme requires a divalent cation for activity, with effectiveness being in the order: Mg2+ greater than Co2+ greater than Mn2+ greater than Fe2+ . Galactokinases from all eucaryotic sources studied thus far seem to be very similar . Based upon the results reported here, the galactokinases from Tetrahymena and yeast appear to be most similar in their biophysical and biochemical properties. J Biol Chem, 1982 Jul 10, 257(13), 7388 - 95 A thermostable tRNA (guanosine-2')-methyltransferase from Thermus thermophilus HB27 and the effect of ribose methylation on the conformational stability of tRNA; Kumagai I et al.; An S-adenosylmethionine-=dependent tRNA (guanosine-2'-)-methyltransferase (EC 2.1.1.34) was purified to the homogeneous state (2,400-fold) from a cell-free extract of an extreme thermophile, Thermus thermophilus HB27 . The enzyme was highly resistant to heat as reported for other enzymes from thermophilic organism . The enzyme is monomeric and its molecular weight was estimated to be about 20,000 . The Km values for S-adenosylmethionine and for Escherichia coli tRNAPhe were determined to be 0.47 microM and 10 nM, respectively, while the Ki for a competitive inhibitor S-adenosylhomocysteine, was 1.67 microM . When yeast tRNAPhe was methylated with the purified Gm-methyltransferase, a stoichiometric amount of methyl group was incorporated into the invariant guanosine at position 18 in the D-loop . Yeast tRNAPhe and E . coli tRNAMet, which were quantitatively methylated with the enzyme, were very similar to the native tRNAs with regard to amino acid acceptor activity and melting temperature, but were more resistant to RNase T1 and RNase A digestions than the corresponding native tRNAs. Biochemistry, 1982 Jul 6, 21(14), 3294 - 8 Complete amino acid sequence of the 4Fe-4S, thermostable ferredoxin from Clostridium thermoaceticum; Elliott JI et al.; The complete amino acid sequence of the 4Fe-4S ferredoxin from the thermophilic bacterium Clostridium thermoaceticum has been determined . The protein is extremely thermostable and is the only known clostridial ferredoxin to contain a single {4Fe-4S} cluster . The sequence totals 63 residues and includes the first tryptophan (Trp-26) reported for a clostridial ferredoxin, and other amino acids not commonly found in clostridial or clostridial-like ferredoxins: methionine (Met-1), histidine (His-33), arginine (Arg-49), and leucine (Leu-9, -19, and -31) . Sequence homology to clostridial and other 8Fe-8S ferredoxins is limited to eight to nine residues at the amino-terminal sulfhydryl grouping (Cys-10, -13, -16, and -20) and two to five residues in the carboxyterminal region . This ferredoxin is, thus, sequentially distinct from all known clostridial ferredoxins and from other bacterial ferredoxins in both the 8Fe-8S and 4Fe-4S classes. Mikrobiologiia, 1982 Jul-Aug, 51(4), 611 - 5 {Cytological characteristics of the thermophilic bacterium Thermus ruber}; Shadrina IA et al.; The obligate thermophilic bacterium Thermus ruber 12b was examined using optical and electron microscopy . Its cells were shown to be polymorphous . In a liquid medium, the cells associate yielding complex spherical bodies having a surface layer in common and complexes consisting of two or more cells but without a common envelope . A large nuc