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With the help of an automatic protein sequenator, we have verified a prediction made by us in a recent paper, namely that residue 16 in Candida krusei cytochrome c is a glutamine and not a glutamic acid. Neurospora crassa cytochrome c now remains the only mitochondrial cytochrome c which apparently does not have a glutamine at this position. In the course of this investigation, from two strains of Candida krusei we isolated two cytochromes which differ in amino acid composition. One of them seems to correspond to that described in the literature. Two of the differences have been located. [ABSTRACT FROM AUTHOR]

Amino acid analyses of L-lactate dehydrogenase from baker's show that the minimum molecular weight (53 000 daltons) of the protein is much lower than found in the literature (80 000). This result, combined with those reported in the following papers, leads to a revision of the dimeric model generally accepted for cytochrome b2 [ABSTRACT FROM AUTHOR]

The neurohypophysial hormones of a chondrostean, the sturgeon (Acipenser sp.), have been purified by adsorption onto neurophysin, dissociation of the complex hormone-protein by trichloroacetic acid precipitation and isolation of active peptides, from the supernatant solution, by paper chromatoelectrophoresis. Arginine vasotocin has been characterized by amino acid composition, chromatographic and electrophoretic migrations and pharmacological properties as well. The amount of arginine vasotocin (about 50 nmol per g pituitary powder) is intermediary between those found for bony fishes about 1000 nmo]/g) and cartilaginous fishes (about 5 nmol/g). A second hormone, which can be classified in the oxytocin-likc type by its electrophoretic nigration and its pharmacological properties, has been disclosed. The very weak amount did not allow chemical identification. However the chromatographic behaviour and the pharmacological ‘profile’ indicate that this hormone differs from the six known oxytocin-like peptides. [ABSTRACT FROM AUTHOR]

Amino acid analyses of L-lactate dehydrogenase from baker's show that the minimum molecular weight (53 000 daltons) of the protein is much lower than found in the literature (80 000). This result, combined with those reported in the following papers, leads to a revision of the dimeric model generally accepted for cytochrome b2 [ABSTRACT FROM AUTHOR]

The species of methionine tRNA which places methionine into internal positions of growing polypeptide chains, methionine tRNAM, was purified from Escherichia coli strain CA265 labelled with 32>P by column chromatography on DEAE-Sephadex and benzolylated DEAE-cellulose. Sequence analysis of the products of complete and partial digestion of tRNAM by ribonucleic T1 and by pancreatic ribonuclease permitted the derivation of the total primary structure of this molecule. The sequence of methionine tRNAM is PGGCUACGU*AGCUCAGUD2'OMeGGDDAGAGCACAUCAACUCAUA*AΨGAUGGG7MeGXCACAGGtΨCGAAAUCCCGUCGUACCACCAOH, where U* is probable 4-thio-uridine, and C, A* X are unknown nucleotides. [ABSTRACT FROM AUTHOR]

A method is described for the preparation of DNA polymerase purified about 800-fold from rat liver nuclei. The yield of enzyme is about 140-200 µg from 200 g liver. Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the enzyme in the final step, shows a main band corresponding to a polypeptide of molecular weight of 29 000 ± 3%. Sephadex G-100 column chromatography indicates the enzyme to have an apparent molecular weight of approximately 60 000 ± 2% at an ionic strength of 0.15, suggesting that the enzyme is a dimer as isolated. In 2 M NaCl, the apparent molecular weight is 42 000. The enzyme prefers double-stranded DNA templates but utilises most efficiently those activated by deoxyribonuclease I. It has the ability to carry out limited synthesis using only one deoxynucleoside-5'-triphosphate in the assay. The final preparation of DNA polymerase has nucleoside diphosphate kinase associated with it. [ABSTRACT FROM AUTHOR]

A method is presented to measure the alkylation of phosphates in DNA after a treatment with an alkylating agent. Using this method, we have shown that phosphate alkylation represents 15% of total alkylation when DNA is alkylated with ethyl methanesulfonate and only 1% of total alkylation when DNA is alkylated with methyl methanesulfonate. Experiments are also presented which show that phosphate triesters resulting from the alkylation of DNA by ethyl methanesulfonate are very stable, most of them remaining intact after heating at 100 °C for 90 min at pH 7.0. [ABSTRACT FROM AUTHOR]

1. The action of insulin on [14C]glucosamine uptake and metabolism is analyzed in the isolated rat diaphragm. Various metabolites accumulating in the course of incubation are extracted, characterized and estimated by chromatographic, electrophoretic and colorimetric procedures. 2. Insulin greatly stimulates (up to three-fold) the uptake and time-dependent accumulation of metabolites derived from glucosamine. It is demonstrated that glycogen accounts but for a small part (less than 20%) of the accumulated material; the major part consists of glucosamine 6-phosphate, the level of which is increased up to six times by insulin in the cell. Hence the hormone affects glucosamine metabolism already at the level of its first steps: transport and phosphorylation. 3. The use of D-glucose and 3-O-methyl-D-glucose as competitive inhibitors of glucosamine metabolism shows that the mechanisms by which all three substrates are transported and by which two of them (glucose and glucosamine) are phosphorylated are essentially identical, both in the absence and in the presence of insulin. 4. The action of phlorizin as an inhibitor of sugar transport confirms this interpretation. 5. The results obtained are consistent with the hypothesis of an insulin-stimulated, facilitated diffusion step of glucosamine and of a bottle-neck reaction, which limits the deamination of glucosamine 6-phosphate and leads to its accumulation in the tissue. [ABSTRACT FROM AUTHOR]

Gonads of the male Holothuria tubulosa contain two families of lysine-rich histones. One of these families resembles the lysine-rich histones found in somatic tissues of higher organisms (e.g. calf and rabbit). The other family, which may be restricted to the male gonad, is recognizably related to the first family and yet is quite distinct. About 35% of the tryptic peptides differ between these families. These findings support the notion that a broad spectrum of structural variation may exist in lysine-rich histones, perhaps even merging into structures of the slightly lysine-rich class. [ABSTRACT FROM AUTHOR]

Lipase from different tissues was extracted in potassium phosphate buffer containing sodium deoxycholate and purified by column chromatography on Sephadex and Sepharose gels. A 500fold purification was achieved by this procedure. This purified enzyme has an estimated molecular weight greater than 3 × 106, and it runs as a single protein band in polyacrylamide preparative disc gel electrophoresis. After acetone — ether treatment of the etude rat pancreactic lipase extract. the resulting enzyme has an estimated molecular weight less than 3 × 104, similar to those reported in the literature. The purified enzyme catalyzes the hydrolysis of long and short chain fatty acid glycerol esters. Phenoxybenzamine and phentolamine were found to inhibit the hydrolysis of the long chain fatty acid glycerol esters by the high molecular weight lipase, while that by the low molecular weight lipase was not inhibited. When administered to rats in vivo, phenoxybenzaamine and phentolamine inhibited the hydrolysis of tripalmitin by the epididymal fat lipase: but the pancreatic or intestinal lipase activity was not affected. [ABSTRACT FROM AUTHOR]

An X-ray diffraction study has been performed by the powder technique on two crystalline forms of yeast cytochrome b2. Type II (DNA-free) cytochrome b2 crystallizes in the trigonal system, the parameters of the unit cell being a = 168.2 Å and c = 112.0 Å, whereas type I (DNA-containing) cytochrome b2 crystallizes in the tetragonal system with a = 99.1 Å and c = 87.0 Å. Measurement of the crystal density and of the partial specific volume of the protein (found to be 0.754 cm³ g at 25°) enables a precise determination of the molecular weight of cytochrome b2 to be obtained. In this respect, the values 234 6000 - 8000 and 235000 - 10000 were found for type II and type I crystals respectively. These results, although in complete concordance, are quite different from the previously accepted value (approx. 180000 from sedimentation studies based on v = 0.71 cm³ /g). Space-groups and protein contents of the crystals show that this protein is composed of at least two, or even four, identical protomers. [ABSTRACT FROM AUTHOR]

1. Porcine proelastase was purified from frozen pancreases by extraction at pH 4.5, precipitation with (NH4)2SO4, isolation of the pH 5.7 insoluble euglobulin fraction, selective (pH dependent) adsorption on elastin and chromatography on CM-cellulose at pH 4.5. The active fraction was dialyzed and freeze dried. All the steps of purification except the last one were performed in presence of trypsin and chymotrypsin inhibitor AA from soybeans. All the process was done at 4°.2. The proelastase thus obtained was found to be pure by acrylamide gel electrophoresis and ultracentrifugation. The extinction coefficient of the freeze-dried material, E1 cm1% at 280 nm was 17.0. The pure preparation was free of α-N-benzoyl-L-benzoyl-L-arginine [ABSTRACT FROM AUTHOR]

A series of 62 mutants of Saccharomyces cerevisiae showing high levels of resistance to oligomycin and rutamycin have been isolated. These mutants can be subdivided into two classes on the basis of their cross resistance to a variety of inhibitors and uncouplers of mitochondrial energy-conservation reactions. Class I mutants show cross resistance to aurovertin, triethyltin, ‘1799’ (a hexafluorotriacetone derivative), uncoupling agents and inhibitors of mitochondrial protein synthesis such as chloramphenicol, mikamycin and spiramycin. Class II mutants are specifically resistant to oligomycin and rutamycin and show no cross resistance to aurovertin, triethyltin or any of the other agents tested. The majority of the class I mutants and one of the class II mutants exhibit cold sensitivity of the ohgomycin resistance, i.e. they lose resistance at the non-permissive temperature of 20 °C, and this provides a further basis for classification of the mutants. The growth rates of the majority of these resistant mutants from both classes were unaltered from the wild type and the growth yield of the class II mutants was also the same as that of the parental strains. The growth yield of the class I mutants, however, was 15–20% less than that of the wild type. No differences in mitochondrial morphology or of the number of mitochondria per cell were seen on examination by the electron microscope. The isolation and properties of specific mitochondrial oligomycin-resistant mutants supports the biochemical evidence for different sites of action of oligomycin, triethyltin and uncoupling agents on energy-conservation reactions. [ABSTRACT FROM AUTHOR]

Cytoplasmic messenger-like RNA not associated with ribosomes has been investigated in immature duck erythrocytes and compared with polyribosome-bound messenger RNA. It is shown that 9-S RNA of the same electrophoretic mobility as the polyribosomal 9-S globin messenger is present free in the cytoplasm. Its rate of synthesis and decay is consistent with a role as a precursor of polyribosomal 9-S globin messenger. mRNA species different from 9-S RNA, among them a predominant 12-S species, are found associated with polyribosomes as well as in the free cytoplasmic pool. The free cytoplasmic mlRNA pool is more heterogeneous compared to the polyribosomal mRNAs, suggesting a translational control qualitativeiy and quantitatively selecting among the free cytoplasmic mlRNAs for those to be translated. [ABSTRACT FROM AUTHOR]

The specificity of yeast hexokinase for ATP was examined with the aid of a number of synthetic and natural analogs. The main features observed are: 1. The presence of a 2-amino group either alone or in combination with a 6-amino or 6-hydroxyl group inhibits catalysis. 2. The replacement of one or both hydrogen atoms of the 6-amino group with methyl groups does not have a large effect on either binding or catalysis. 3. The replacement of the nitrogen at position 7 with a carbon atom has virtually no effect. 4. The movement of the glycosidic bond from the N9 to the N³ position has only a moderate effect on both Km and V. 5. The presence of both of the 2'- and 3'-cis hydroxyls in the ribose is important for catalysis. 6. The replacement of ribose with arabinose or glucose produces a large decrease in catalysis. experiments were carried out with the ATP analog containing glucose instead of ribose. It was found to be competitive for ATP and uncompetitive for glucose under the conditions used. These results are consistent with an ordered reaction mechanism. [ABSTRACT FROM AUTHOR]

1. Cytoplasmic aspartate aminotransferase from pig heart catalyses β-elimination of L-serine-o-sulphate and β-chloro-L-alanine. The rates of these reactions have been compared with the rates of the same reactions catalysed by free pyridoxal 5'-phosphate and CuCl2. 2. The effect of formate on the two enzyme-catalysed reactions has been studied and it has been shown that this anion competitively inhibits the serine sulphate reaction and activates the chloroalanine reaction by increasing kcat considerably. 3. The β-elimination of both these compounds is accompanied by progressive inactivation of the enzyme but whereas formate decreases the rate of inactivation by serine sulphate it increases the rate of inactivation by chloroalanine, 4. It is proposed that formate binds to the active site of the enzyme in a position normally occupied by the β-carboxyl group of aspartate, the carboxyl group of glutamate or the sulphate group of serine sulphate and that binding of the appropriate anion induces a change in the active site which accounts simultaneously for increased catalytic activity and increased inactivation. [ABSTRACT FROM AUTHOR]

Ion-exchange chromatography of the total cytochrome c extracted from a wild-type laboratory strain of Saccharomyces cerevisiae shows the presence of two minor cytochrome c peaks, besides the already known iso-1 and iso-2 cytochromes c. Their amino acid composition is very similar to that of iso-1, but one of them lacks the ε-N-trimethylated lysine residue characteristic of yeast cytochromes c, thus excluding, at least for this peak, an artifactual origin. [ABSTRACT FROM AUTHOR]

It has been found that purified phenylalanyl- and valyl-tRNA synthetases from yeast catalyse the mischarging of numerous heterologous Escherichia coli and even of homologous yeast tRNAs when special aminoacylation conditions are used. For instance E. coli tRNAAla, tRNAVal, tRNALys and yeast tRNAAla, tRNAVal can be incorrectly aminoacylated by yeast phenylalanyl-tRNA synthetase, whereas E. coli tRNAAla, tRNAMet, tRNAIle, tRNAThr and yeast tRNAAla, tRNAPhe can be mischarged by yeast valyl-tRNA synthetase. The production of errors is highly dependant on the experimental conditions used, as their number and their level is increased when the Mg2+/ATP ratio or the enzyme concentration in the medium is increased or when the reactions are performed in the presence of dimethylsulfoxide. In the ease of phenylalanyl-tRNA synthetase, conditions can be found where practically all E. coli tRNAs are wrongly aminoacylated, although at different levels. On the other hand, in the case of homologous systems some errors can only be detected when purified tRNAs are used, thus suggesting, when total tRNA is used, a competition between the cognate and non-cognate tRNAs which minimises the mischarging. The comparison of the sequences of the cognate and non cognate tRNAs which are aminoacylated either by the yeast phenylalanyl- or valyl-tRNA synthetase led us to ascribe some importance to several regions inside of these tRNAs, for instance (a) the dihydrouracil arm, (b) the terminal part of the amino-acid acceptor stem and (c) the extra-loop. These regions should be essentially necessary for the establishment of the correct tri-dimensional conformation necessary for the recognition by the aminoacyl-tRNA synthetases. [ABSTRACT FROM AUTHOR]

The study undertaken shows the existence of water-soluble complexes of sterols, sterol esters and their precursors (triterpenes and 4α-methylsterols) in a higher plant, Zea mays L. The leaves, roots and culture medium were treated separately in order to investigate the role and nature of these complexes. The sterols found in the water-soluble form represent 0.1-1% by weight of the total sterols in the plant. The composition of the hydrosoluble fraction of the sterols and their precursors is different from that of the lipid-soluble fraction, cholesterol, sitosterol and isofucosterol being preferentially complexed compared to campesterol and stigmasterol. This complex was found in the microsomal supernatant after homogenisation and also in the culture medium. The sterols are released from the complex by a treatment with pyrogallol under basic conditions or after repeated extraction with petroleum ether. The complex is precipitated with ethanol but not with trichloroacetic acid. The behaviour of several sterols on treatment with a solution of starch was investigated. Starch was able to complex with a large number of phytosterols unspecifically. The implication of this complex for the transport of sterols, for the biosynthesis of steroids and the ecological impact is discussed. [ABSTRACT FROM AUTHOR]

The lysosomal acid α-glucosidase has been purified more than 10000-fold from rat and mice liver and from human placenta. The kinetic properties of the human enzyme are similar to those previously described for the rodent enzyme, with the main exception of the absence of inhibition by an excess of maltose. Antibodies were obtained by injection of the pure rat liver, mouse liver or human placental α-glucosidase to rabbits. The property of the enzyme of hydrolysing glycogen or to catalyse transglucosylation from maltose to glycogen could be nearly completely inhibited by the antibodies whereas the hydrolysis of small molecular substrates was much less inhibited. Within the same species, the same amount of enzyme was inhibited by a given amount of antibodies, whatever the tissue used as a source of enzyme. A partial cross-reactivity was observed from species to species. The presence of an immunologically reacting protein in tissues from patients with type-II-glycogenosis could not be demonstrated either by double-diffusion test or by antibodies consumption. Similar negative results were also obtained with normal human liver in which acid α-glucosidase has been inactivated at alkaline pH. [ABSTRACT FROM AUTHOR]

The binding of saccharide inhibitors to lysozyme has been studied using a microcalorimetric method. Measurements have been performed with N-acetyl-D-glucosamine (pH 5 and 25 and 40 °C), tri[N-acetyl-D-glucosamine] (pH 2, 5 and 7.6 at 25 °C and pH 5 at 40 °C) and with tetra[N-acetyl-D-glucosamine] (pH 2, 5 and 7.6 at 25 °C). From the results of the calorimetric measurements values for ΔG0, ΔH0, ΔS0 and ΔCp0 were derived for the binding processes. In addition to the saccharide-binding reactions, some model experiments were performed. [ABSTRACT FROM AUTHOR]

The effects of parathyroid hormone, adenosine 3':5'-monophosphate (Ado-3':5'-P) and oleate on renal metabolism have been studied in isolated kidney-cortex tubules from starved rats. Substrate uptake and glucose formation have been determined with L-lactate, pyruvate, glutamate, and glycerol as substrates. The results indicate that parathyroid hormone and Ado-3':5'-P increase renal gluconeogenesis by the same mechanism, which however, differs from the effects brough about by free fatty acids: 1. Parathyroid hormone and Ado-3':5'-P increased glucose formation from L-lactate, pyruvate and glutamate. The stimulatory effect of parathyroid hormone was always smaller compared with that of Ado-3':5'-P. Glucose formation from L-lactate was shown to be stimulated with hormone doses as low as 1-10 nM. 01cate had an increasing effect on glucose formation from L-lactate and glycerol but no effect on glucose formation from 10 mM pyruvate and inhibited gluconeogenesis from glutamate. 2. The stimulatory action of parathyroid hormone and Ado-3':5'-P was accompanied by an increase in substrate uptake, whereas oleate decreased substrate uptake. 3. The effects of parathyroid hormone and Ado-3':5'-P on kidney-tubule metabolism were not additive, when both substances were added together. 4. All effects of oleate were found to be unchanged in the presence of parathyroid hormone and Ado-3':5'-P or both. The results suggest two different mechanisms which may regulate renal gluconeogenesis additively. [ABSTRACT FROM AUTHOR]

Rat liver polysomes, single ribosomes, EDTA-produced subparticles and free ribosomal RNA were treated with formaldehyde and their properties were studied. The following results were obtained. 1. RNA can be isolated from fixed polysomes or subparticles by exhaustive digestion with pronase. Such RNA shows a normal electrophoretic behaviour in agar gel. 2. In free RNA treated with formaldehyde the mobility of 18-S and 28-S RNA fractions is reduced; latent breaks are not revealed by heating; sensitivity towards ribonuclease is increased. No such effects are observed with RNA treated with formaldehyde while in the particles. 3. In fixed polysomes or single ribosomes the two subparticles appear to be covalently bound through their proteins. No intermolecular bonding occurs between 18-S and 28-S RNA either fixed polysomes or in free RNA. The fixed ribosomes and subparticles can be fractionated by agar gel electrophoresis and give profiles similar to those of untreated particles. These resets may prove useful in combining CsCl density gradient fractionation with gel electrophoretic methods. [ABSTRACT FROM AUTHOR]

Mouse reticulocyte messenger (9-S) RNA has been prepared by treatment of polysomes with sodium dodecylsulphate. On 6% polyacrylamide gels, two sharp and one more diffuse component can be resolved in the 9-S region, Messenger RNA prepared from 14-S mRNA. protein from EDTA-treated polysomes contains only the diffuse component. This component has priming activity for both α- and β-globin chains, and evidence is presented that it can be partially resolved into separate messengers for each chain. The two sharp components, which migrate more slowly than the globin mRNA, are associated with the 30-S ribosomal subunit after EDTA treatment, have no translational activity in a duck reticulocyte cell-free protein-synthesis system, and (unlike the diffuse component) are not destroyed by gentle sonication of polysomes to monosomes. The non-coding components comprise approximately one-third of the RNA in the 9-S region of most RNA preparations from mouse reticulocytes. The diffuse (coding) 9-S component from 14-S mRNA · protein runs as a sharp symmetrical peak in formamide-acrylamide gel electrophoresis, suggesting that there may be variation in secondary structure in aqueous solution rather than in molecular weight. [ABSTRACT FROM AUTHOR]

We have isolated a form of Qβ replicase lacking subunit α and shown that it is deficient in the utilization of Qβ-plus strand RNA as template but competent to utilize Qβ-minus strands, poly(C) and "6-S" RNA. The functional identity of subunit α and protein-synthesis-inhibition factor i was proven by their equivalent capacity to restore full Qβ RNA-directed activity of α-less replicase. [ABSTRACT FROM AUTHOR]

The ionic strength, concentration, and temperature effects on the association-dissociation properties of lysozyme in aqueous solution have been studied by gel chromatography. The following results were obtained: 1. Increasing ionic strength leads to an increase m inter-conversion rate between the monomeric and associated forms. Between distilled water and 0.01 ionic strength, lysozyme shows a low velocity of interconversion, whereas between 0.01 and 0.2 rates are observed which are rapid as compared with the column fractionation time. 2. Analysis of concentration dependence indicates the formation of more highly associated forms than the darner. The data can be fitted within experimental error by an indefinite association with the same equilibrium constant for the addition of each successive monomer. 3. The equilibrium ratio for the slow polymerization process measured at temperatures between 15 ° and 40 °C yields the apparent thermodynamic quantities, ΔG°, ΔH0, and ΔS0 for the association reaction. This data is consistent with a hydrophobic interaction between lysozyme monomers and Sephadex gel matrix. It is suggested that the adsorption involves the tryptophan residues of the active centre. The enthalpy of the polymerization process is interpreted in terms of a conformational change of the same order of magnitude as on the bonding of N-acetylglucosamine to the enzyme. [ABSTRACT FROM AUTHOR]

The specific activities expressed as µmol×g wet wt-1min-1 of D-triokinase and NADlinked glycerol dehydrogenase, with D-glyceraldehyde as substrate, were determined in the 27 500 × g supernatant fraction of livers from rats subjected to various dietary and hormonal programs. The specific activity of triokinase varied over a three-fold to four-fold range, being lowest in livers of diabetic rats fed a Purina Chow arid highest in livers of intact rats fed 72% fructose diets containing casein; total triokinase activity per liver was five-fold to six-fold greater in the latter group than in the former group. The specific activity of triokinase also increased in livers of rats fed diets of 72% glucose or 72% dextrin plus 18% casein. Fructose feeding caused a decrease in specific activity of glycerol dehydrogenase. Glucagon had no effect on the specific activity of either enzyme in livers of fasted rats. Cortisone tended to increase the specific activity of triokinase as well as its total activity in livers of rats fed the Purina Chow. After only 48 h of a 72% fructose-18% casein diet, liver weight was increased 50% relative to livers of rats fed the Purina Chow. Triokinase is subject to regulation in livers of adult male rats. The data indicate that the NAD-linked glycerol dehydrogenase is also subject to regulation but to a lesser extent. If glucagon does stimulate the flux of carbon in the triokinase or the NAD-linked glycerol dehydrogenase reactions, it is not as the result of increases iii enzyme concentrations as determined by assayable activity measurements. [ABSTRACT FROM AUTHOR]

The theory of alternative substrates has been thoroughly investigated. The reciprocal rate equations, for ordered and random kinetic mechanisms, in the presence of an alternative substrafe, have been developed. The conditions in which those equations give rise to either hyperbolic or linear reciprocal plots have been established. Degeneracy and constraint conditions of the various kinetic models have also been derived. Definite conclusions concerning the relevant mechanism can be obtained only if the Dalziel coefficients pertaining to the substrate are different from those corresponding to the alternative substrate. It follows from this, that the method termed "isotope competition", cannot be used to make a choice among various two-substrate mechanisms, and appears to be unfounded. The theory of alternative substrates has been applied to the study of yeast hexokinase. The results obtained are at variance with a random mechanism. They do agree, on the other hand, with a compulsory order of substrate binding on the enzyme, glucose being bound first. [ABSTRACT FROM AUTHOR]