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The four structural proteins of bacteriophage PM2 have been related to thc morphological structures in the virion. Protein I can be selectively removed from the virus by degradation with bromelain, a proteolytic enzyme. From electron microscopic studies and from experiments showing that the virus cannot attach to its host cell after bromelain treatment, we conclude that protein I forms the outer spikes at the vertices of the icosahedron. Protein II can be labeled with [35S]sulfanilic acid diazonium salt. As shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, proteins I and II can be removed if the virus is treated with 1 M urea. From the above studies and from packing considerations, we conclude that protein II forms the outer shell of the virus. The dissociation of the virus in 1 M urea leads to: (a) a nucleocapsid core containing viral lipid, DNA, protein III and IV, (b) a second fraction which consists of the same elements obtained by treatment of the virus with bromelain. Both panicles have been identified by electron microcopy. The dissociation of the virus in 1 M urea plus 0.5% Triton X-100, a nonionic detergent, leads to a third fraction representing an empty shell which contains all of the lipid, proteins II and III, and about 50% of protein IV, but no DNA. We have also been able to isolate a second type of nucleocapsid core by dissociation of the virus in the presence of 4.5 M urea. This core, which has an approximate s20.w = 133 S and which has been identified in the electron microscope as a core with icosahcdral shape, contains proteins III and IV, as well as the DNA, but not phospholipid. In the presence of 4.5M urea this core is resistant to Triton X-100 concentrations up to 0.1%. Higher concentrations of the nonionic detergent dissociate the core into DNA and the solubilized proteins HI and IV. In the absence or in the presence of Iow concentrations of urea (1M) the core is resistant to TritonX-100 concentrations up to 0.5%. The DNA in the core is digestible by pancreatic DNAase I and the core structure is broken down after DNAase treatment. Protein IV interacts with the DNA as shown by transformation of non-covalent interactions between DNA and protein into covalent bonds induced by a nucleophilic reagent. In conclusion, protein I forms the spikes, protein II the outer icosahedral shell and protein III the inner icosahedral shell around the DNA, which is closely associated with protein IV. The phospholipid bilayer is sandwiched between these two shells. Protein III may also form the protein bridges to protein I which connect the inner and outer shells through the bilayer. [ABSTRACT FROM AUTHOR]

The lipid-containing bacteriophage PM2 has four distinct structural proteins, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate or electrophoresis on cellulose acetate strips in the presence of 6-8 M urea. The molecular weights of these four polypeptides determined in several polyacrylamide gel systems are: I = 43000, II = 26000-27000, III = 12500, IV = 4700. The isoelectric points of the four viral proteins were determined on disrupted virus particles by electrophoresis on cellulose acetate strips in the presence of 6 M urea at various pH values. The mobilities were extrapolated to zero mobility, a method preferable to isoelectric focusing where the denatured viral proteins aggregate and precipitate near the isoelectric point. The method is well suited for the determination of the isoelectric point of proteins in thc native or only partially denatured state. Whereas proteins I, III and IV have isoelectric points slightly under neutrality (pH 6.2, 5.8 and 5.5, respcctively), thc isoelectric point of protein II is around 12.3. Calcium ions are tightly bound to protein II. After removal of the calcium ions in the presence of 6 M guanidine hydrochloride and EDTA, the pi of protein II is 9. The isoelectric point of thc intact virus, determined on cellulose strips, is around 7.3. Protein I, the spike protein, is the only structural polypeptide which is soluble in aqueous solutions around pH 7. All of the proteins are soluble in 6-8 M urea or 6 M guanidine hydrochloride at pH 7-8.5. Proteins III and IV. the nucleocapsid core proteins, have the solubility properties of proteolipids or highly lipophilic polypeptides as shown by their solubility in nonionic detergents and by their distribution in a Bligh and Dyer fractionation. Neither glycolipids nor glycoproteins are present in the virus. The distribution of phospholipids has been determined by chemical modification of the outer lamella or outer and inner lamellae of the bacteriophage lipid bilayer. The viral lipid bilayer is asymmetric; most of the phosphatidylglycerol residues form the outer lipid layer, most of the phosphatidylethanolamine residues the inner lipid layer. A complete model of the virus, including the assembly process, is discussed at the end of this report. [ABSTRACT FROM AUTHOR]

Motion of acyl chains in the phospholipid bilayer of bacteriophage PM2 was studied by the technique of electron spin resonance. The spin labels used were derivatives of stearic acid with the nitroxide radical in several different positions. The spectra were measured over the temperature range of 0°C to 60°C, within which the spectral changes were reversible. Spectral analysis by means of pertubation theory was carried out over the temperature range of 30°C to 60°C. The spectra, characteristic of a fluid bilayer, indicated that the hydrocarbon chains are in a state of rapid motion; close to the polar region there is a highly ordered environment in which motion is restricted, whereas the bilayer interior is disordered; the strong temperature dependance of the order parameter S3, in the absence of a phase change, and the behaviour of the hyperfine splitting constant aN, suggest that there are strong lipid-protein interactions. [ABSTRACT FROM AUTHOR]

Yeast DNA polymerases A and B were purified 5000- 10000-fold by chromatography on DEAEcellulose, phosphocellulose, DNA-agarose and DEAE-Sephadex. In acrylamide gel electrophoresis in the presence of sodium dodecylsulfate, both enzymes give rise to three main bands. The enzymes are equally susceptible to inhibition by the - SH reagents N-ethylmaleimide and p-chloromercuribenzoate but differ in their sensitivity to cytosine-arabinoside triphosphate, polymerase A being considerably more sensitive to this nucleotide analog. Whereas DNA polymerase A prefers nicked DNA and poly[d(A-T)] as template-primer, polymerase B is most active with poly(dA) · (dT)10 · Km values for deoxyribonucleoside triphosphates were determined as 3.7-3.9 μM for enzyme A and 1.8-2.4 μM for enzyme B. During active growth of cells polymerase activity increases about 1.4-fold over the amount found in resting cells, which is mainly if not exclusively due to an increased level of DNA polymerase A. This together with other properties suggests a role of this enzyme m DNA replication. [ABSTRACT FROM AUTHOR]

Bence-Jones protein DEL was isolated from the urine of a patient with multiple myeloma by ammonium sulfate precipitation. Ion-exchange chromatography provided no further purification of the protein; the ammonium sulfate precipitate consisted of only one protein moiety as tested by polyacrylamide electrophoresis. Protein DEL contains 213 amino acids and has a molecular weight of 22500. Eighteen peptides have been isolated by ion-exchange chromatography after tryptic digestion of the completely reduced and aminoethylated protein. Sequence studies were performed mainly on these tryptic peptides. In addition, 14 chymotryptic pepfides were isolated and characterized to obtain overlapping peptides for the whole V-region and for some tryptic peptides of the C-region. Protein DEL is related to subgroups III and IV of the γ-chains as indicated by a deletion of three amino acids after position 27, a common pattern of these two subgroups. Furthermore, position one is deleted in protein DEL as in the other proteins of subgroups IH and IV but in contrast to subgroups I and II of the γ-chains. However, marked differences exist between protein DEL and the proteins of subgroups IH and IV. a) The deletion of two amino acids in positions 95 and 96, characteristic of subgroup IV, was not found in protein DEL. b) 19 of the 27 linked exchanges specific for subgroup IV are substituted in protein DEL. Although the insertion of two amino acids after position 94 is the same as in subgroup III, a high exchange rate was observed for proteins DEL and Sh. Therefore it is concluded that protein DEL does not belong to either subgroups III or IV, but is the first example of a new subgroup of the γ-chains. [ABSTRACT FROM AUTHOR]

1. Sialic acids were released by dilute acids or by neuraminidase from glycoproteins of bovine and equine submandibular glands, from glycoproteins of equine blood plasma and from equine erythrocyte membranes. They were purified by ion-exchange chromatography and fractionated on cellulose. 2. The following methods have been adapted to the structural analysis of sialic acids with regard to the nature and position of the N-acyl and O-acyl groups. a) Thin-layer chromatography in two dimensions with intermediate ammonia treatment to remove the O-acyl groups leading to the identification of the basic N-acylneuraminic acids. b) Thin-layer chromatography in two dimensions with intermediate hydroxylamine treatment allowing the identification of the O-acyl groups of sialic acids as hydroxamates. c) Gas-liquid chromatography for identification of the different N-acylated and O-acylated sialic acids. 3. The structure of the following new sialic adds have been established: a) N-glycoloyl-8-O-acetylneuraminic acid isolated from glycoproteins of bovine submandibular glands; b) N-acetyl-4-O-glycoloylneuraminic acid isolated from glycoproteins of equine submandibular glands and also occurring in equine blood plasma and equine erythrocyte membranes; c) N-acetyl-7 or 8-O-glycoloylneuraminic acid detected in glycoproteins from bovine submandibular glands. 4. The O-acetylated N-glycoloylneuraminic acid first detected in horse erythrocyte membranes by Hakomori and Saito and in horse serum by Pepper, was determined to be N-glycoloyl-4-O-acetylneuraminic acid; it was also found to occur in submandibular glands of horse. 5. The sialic acid patterns from the different biological materials investigated are compared. [ABSTRACT FROM AUTHOR]

1. DNA of DNase-treated mitochondria of Acanthamoeba castellanii bands at a buoyant density of 1.690 g/cm³. Up to 80% of the molecules were circular, with a circumference of 12.7 μm. Two circular conformations, the closed twisted and the open form, were separated in preparative CsCIethidium bromide gradients. 2. When mtDNA was renatured at a temperature Tm; - 23 °C, deviations from second-order kinetics were observed. At Tm; -10 °C, however, it renatured as a single kinetic class without indications of sequence repetition. The coincidence of the kinetic complexity (2.57 x 107) and the circle contour length within the limits of error of the methods suggests that each circular molecule represents a chondriome. 3. The main DNA component, which is of nuclear origin and has a peak density of 1.716 g/cm³ in CsC1, displays heterophasic renaturation kinetics. Its slow-reassociating fraction shows a kinetic complexity corresponding to a molecular weight of 2.1 x 1010. Approximately 22% of the main component appears to be repetitive at a renaturation temperature of Tm - 23 °C. [ABSTRACT FROM AUTHOR]

Micrococcin inhibited the binding of aminoacyl-tRNA at 13 mM Mg2+, but not at 6 mM Mg2+ . The elongation factor Tu (EF-Tu)-dependent binding of aminoacyl-tRNA was inhibited appreciably by 7 μM micrococcin. The EF-G-dependent puromycin reaction of the ribosome-bound aminoacyltRNA, release of unesterified tRNA concomitant with translocation, and the EF-G-dependent evacuation of aminoacyl-tRNA from the acceptor site of the ribosomes were all sensitive to this antibiotic. It is concluded that micrococcin interacts with the acceptor site specifically and this interaction results in the dual action of this antibiotic, namely inhibition of translocation and the binding of aminoacyl-tRNA. [ABSTRACT FROM AUTHOR]

α-Galactosidase from Lens esculanta (red lentils) was purified 4090-fold through a multistep process involving acidification, ammonium sulphate fractionation, Sephadex gel filtration, DEAEcellulose and CM-cellulose chromatography. The purified enzyme is stable at 4°C for nearly six months with only a small loss of activity. It showed a pH optimum of 5.5 with raffinose as the substrate and 6.0 with p-nitrophenyl-α-D-galactoside. The Km for the former substrate was 40 mM and for the latter, 0.26 mM. The enzyme failed to liberate galactose from guar gum, tara gum and locust bean gum galactomannans. A study of the hydrolyzability of α-D-galactoside, α-D-fucoside and β-L-arabinoside showed that the presence of primary alcoholic group in the sugar was important for faster hydrolysis and a stronger binding. The substrate specificity studies indicated the absence of any related glycosidase activity in the α-galactosidase preparation. [ABSTRACT FROM AUTHOR]

1. The oxidation of benzyl alcohols by liver alcohol dehydrogenase under conditions [S]o » [E]o is a biphasic process with exponential rise to a steady state. The rate constants for the transient phase were determined by a curve-fitting procedure. 2. A large isotope effect was obtained for p-methoxybenzyl alcohol, indicating that hydrogen transfer is rate-determining for the transient phase. Rate constants for the hydrogen transfer step, k3, were obtained for the various alcohols and correlated with the Hammett σ constant by plotting log k3 against σ. The slope of this line (q = -0.76) is consistent with rate-limiting hydride transfer; a mechanism is proposed to account for the low magnitude of q. 3. The pre-steady-state rate constants, kb, for ali benzyl alcohols (<20 s -1) are lower than that for ethanol. 2-Methylpropan-l-ol and cyclohexylmethanol have pre-steady-state rate constants of about 150 s--1, indicating that the lower kb values for benzyl alcohols are due to the electronwithdrawing effect of the benzene ring, rather than to steric effects. [ABSTRACT FROM AUTHOR]

The work comprises: 1. Determination by nuclear magnetic resonance method of the conformation of the imino group of pyridoxal-P oxime in solution. It has been found to be nearly coplanar with the aromatic ring. 2. Theoretical calculation by the method of atom-atom potentials of the permitted conformations of pyridoxal-P imine and its analogs: 6-methylpyridoxal-P and 5'-methylpyridoxal-P. There have been found seven favourable conformations for pyridoxal-P imine, some of which were prohibited in the analogs mentioned. 3. Experimental estimation of the conformation of pyridoxal-P imine in the active site of aspartate aminotransferase with the use of the above sterically hindered analogs and 5'-deoxy-5'-carboxymethylenepyridoxal analog. In result, the number of the possible conformations for pyridoxal-P imine in the active site has been reduced to a pair of symmetrical conformations. [ABSTRACT FROM AUTHOR]

1.An O-methyltransferase catalyzing the transfer of the S-methyl group of S-adenosyl-L-methionine to the 4'-hydrozyl group of isoflavones has been detected in seedlings of Cicer arietinum L. and in cell suspension cultures of this plant.2.After transfer of the cell cultures to fresh medium the activity of the methyltransferase rose after a lag period of about 2 and reached a maximum after about 20 h and then declined. Illumination of the cell cultures had no effect on the enzyme activity. 3.The enzyme has been purified 360-fold by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and hydroxyapatite and Sephadex G-200 gel filtration. 4.The S-adenosylmethionine : isoflavone 4'-0-methyltransferase was isolated as a soluble enzyme with a pH optimum of 9 in glcine-NaOH buffer. It requires - SH-protecting agents for activity. The elution volume from a Sephadex G-200 column to be about 110000 estimated the molecular weight of the enzyme. 5.The enzyme is specific for the methylation of 4'-hydrozyisoflavones. Intermediates in the biosynthetic pathway to isoflavones, e.g. p-hydroxycinnamic acid and 5,7,4'-trighdroxyflavone, were not methylated. The Km values for daidzein (7,4'-dihydroxyisoflavone) and S-adenosylmethionine were determined to be 80μM and 0.16mM, respectively. Because of the high specificity of the methyltransferase for isoflavones it can be concluded that methylation is the last step in the biosynthesis of 4'-methoxyisoflavones. 6.The enzyme was inhibited by S-adenosylhomocysteine. The Ki values for S-adenosylhomocysteine versus daidzein and S-adenosylmethionine were determined to be 0.3 mM and 30μM, respectively. From the inhibition kinetics it can be concluded that the reaction follows an Ordered BiBi mechanism with S-adenosylmethionine and S-adenosylhomocysteine as leading reaction partners. [ABSTRACT FROM AUTHOR]

A potato particulate preparation, which is constituted mainly by proplastids, was capable of glucose transfer from micromolar concentration of uridine diphosphate [14C]glucose into an endogenous protein molecule to form a 14C-labelled glucoprotein. This glucoprotein had short radioactive glucan chains and could act as acceptor for the synthesis of larger radioactive glucan chains from labeled uridine diphosphate glucose, either at millimolar concentration or in the presence of glucose 1-phosphate or adenosine diphosphate glucose at micromolar concentrations. This last step required Mn2+ or Mg2+ ions. The enzyme responsible for the formation of the glucoproteic acceptor was named uridine diphosphate glucose-protein transglucosylase I and its properties were studied. The uridine diphosphate glucose-protein transglucosylase activity, that was detected at micromolar uridine diphosphate glucose concentrations in the presence of glucose 1-phosphate, could be distinguished from uridine diphosphate glucose-protein transglucosylase I by the following criteria: The glucose 1-phosphate dependent activity required Mn2+ or Mg2+ ions, was sensitive to temperature and formed radioactive glucan chains totally hydrolyzed by β-amylase. Conversely, uridine diphosphate glucose - protein transglucosylase I was active in the absence of Mn2+ or Mg2+ ions, was resistant to temperature and formed radioactive glucan chains scarcely attacked by β-amylase. Sequential analysis of the glucose incorporation from uridine diphosphate [3H]glucose and uridine diphosphate [14C]glucose into the glucoprotein and sodium dodecylsulphate - acrylamide gel electrophoresis of the labeled glucoprotein formed in the presence and in the absence of glucose 1-phosphate were carried out.On the basis of the data obtained a simple scheme of reactions is proposed to explain the involvement of the uridine diphosphate glucose - protein transglucosylase I and glucose 1-phosphate dependent activities in the synthesis of α-1,4 glucan chains of the starch type. [ABSTRACT FROM AUTHOR]

Certain enzymatic properties of homogenous DNA polymerase I from Acinetobacter calcoaceticus have been investigated. The enzyme requires an initiator DNA repair synthesis. It is, however, also capable of de novo synthesis in the presence of dATP and dTTP or dGTP and dCTP. The enzyme was only partially inhibited by the - SH group-blocking reagent p-hydroxymercuribenzoate.DNA polymerase I from Acinetobacter calcoaceticus contained both a 3'→5' and a 5'→3' exonuclease activity. The products of the former was 5'-deoxymononucleotide phosphates. The 5'→3' nuclease gave mononucleotides with T7 DNA and with d(a-T)n the products were mono, di, and trinucleotides, the ratio depending on the concentration of Mg2+. The 3'→5' exonuclease activity appeared to be slightly more active on single-stranded than double-stranded DNA. The opposite was the case for the 5'→3' nuclease which had an absolute requirement for double-stranded DNA. The present work offers further evidence for the view that DNA polymerase I enzymes in microorganisms have similar structural features and enzymatic properties. [ABSTRACT FROM AUTHOR]

Dynamic quenching of native tryptophan fluorescence has been used to assess the fluidity of intact membrane suspensions. The photophysical quenching of tryptophan fluorescence by chloroform occurred by a diffusion-controlled process where one-half of all collision were effective. The bimolecular rate constant for quenching was directly proportional to the fluidity for a series of solvents. Estimation of the rate constant in the membranes from quenching data indicated that the viscosity of membrane protein (about 1cP) was at least an order-of-magnitude lower than the values reported for lipid bilayer regions of nerve membranes. [ABSTRACT FROM AUTHOR]

Hananelose (2-hydroxymethyl-D-ribose) is a common sugar of green land plants. However, until now, all experiments failed to show dissimilation of this sugar by its producers or by soil microorganisms. For investigation of its biological degradation, microorganisms were used which had been isolated from plant material (leaves of Primula clusiana Tausch). These microorganisms belong to the Enterobacteriaceae and are almost identical to those described by Asai et al. (1956, 1962) as Kluyvera citrophila. They differ from the ATCC strains of this organism by their ability to grow on hamamelose. Isotope studies using 14C-labelled hamamelose and glucose (for comparison) as substrates were carried out to elucidate the biochemical route of hamamelose catabolism. This degradation leads to CO2 under aerobic conditions and to lactate, succinate, formate, acetate and ethanol anaerobic ones. Glucose dissimilation proceeds via glycolysis and leads to the same end-products. As intermediates of the biological degradation of hamamelose, hamamelose 21-phosphate, fructose 1-phosphate, fructose 1,6-bisphosphate and phosphoglycerate were identified. Thus the biochemical route of hamamelose dissimilation appears to be rather similar to glycolysis. The following sequence is proposed: hamamelose→hamamelose21-phospphate→→fructose 1-phosphate→fructose 1,6-bisphosphate→phosphoglycerate→pyruvate→volatile end-products. Differences from the glycolytic pathway occur at step 2 in which there is a rearrangement of the carbon skeleton and step 3, in which fructose 1-phosphate instead of fructose 6-phosphate has to be phosphorylated. [ABSTRACT FROM AUTHOR]

1.Alcohol dehydrogenase isoenzymes BB were isolated from human livers of both "normal" and "atypical" phenotypes [1]. Their pH-rate profiles, their behaviour on CM-cellulose and the number of hybrids formed with horse subunits A show that the "atypical" isoenzymes contain subunits B1 and B2, the "normal" one only subunits B1. These results indicate that most "atypical" individuals genetically represent heterozygotes. 2.A high degree of homology between horse subunit A human subunits B is found. However, the human isoenzymes have a molecular weight of 87000 and contain about 410 amino acid residues, 6 tyrosines and 3 tryptophans per subunits. 3.the sequence of one tryptic peptide from subunit B1 is Phe-Ala-Lys and is altered to Phe-Pro-Lys in the subunit B2. This substituted residue is located in a region which corresponds to the coenzyme-binding site of the horse enzyme. [ABSTRACT FROM AUTHOR]

The stoichiometries of phenylalanyl-tRNA-sythetase&midd;tRNAPhe complexes were found close to 2, as determined by the following methods; zone centrifugation in sucrose gradient, Sephadex filtration, sedimentation boundary technique and fluorescence quenching of Y base of tRNAPhe. The two former techniques allow the isolation of a complex composed of active enzyme (which is only a fraction of total enzyme used) and tRNA; the two last methods take into account the input protein. All the observed stoichiometries are consistent with the existence of two binding sites for tRNAPhe. Under the experimental conditions used (pH 7.0 and 120C) a dissociation constant of 3 x 10-8 M was calculated from the fluorescence quenching of tRNAPhe by the enzyme. tRNAPhe protects the enzyme against heat denaturation. A protection constant of 9 x 107 M was found at 550C, pH 7.0. The presence of phenylalanine and ATP did modify the strength of interaction of the enzyme-tRNA system. They induce, however, a synergistic effect of the protection afforded by tRNAPhe.The binding of phenylalanine was examined by equilibrium dialysis in presence of tRNAPhe. The stoichiometry obtained indicated two binding sites of phenylalanine. The two sites however do not possess the same affinity: the corresponding Kd values are 6 and 30 μM. The presence of more than one active site is also manifested by the curvature of the double-reciprocal plots of v. versus [S] for phenylalanine, when measuring the initial rates of phenylalanyl-tRNA formation. With regard to the sites having the highest affinity, a 20-fold higher Kd value was found, in the absence of tRNAPhe, when binding of the amino acid was followed by the phase partition technique. [ABSTRACT FROM AUTHOR]

Ferredoxin of Escherichia coli is an iron-sulfur protein of the molecular weight about 12000 which contains two iron and two labile sulfur atoms. The sequence of the amino terminus is HN-ProLys-Val-Ile-Val-Leu-Tyr-. The electron paramagnetic resonance spectrum of the reduced form shows a low-field signal with g11 = 2.02 and a single high-field signal with g⊥ = 1.94. From the circular dichroism spectrum in the peptide absorption region an amount of 10% helically ordered regions is computed. A new peak in the light absorption spectrum at 545 nm appears upon reduction. From the spectral data, which reflect the fine structure of the chromophore and the gross conformation of the polypeptide chain, a close relationship to the mitochondrial adrenodoxin is concluded. The midpoint potential of the protein is -0.38 V. It does not mediate electron transport in the NADP-photoreduction system of spinach and is incapable of replacing the Pseudomonas putida ferredoxin in the camphor hydroxylation. The physiological role of this ferredoxin, which is not a functional analogue of the Escherichia coli flavodoxin and which is synthesized by aerobically as well as anaerobically growing cells, is unknown. [ABSTRACT FROM AUTHOR]

Sputum has been separated into a sol and a gel phase by ultracentrifugation and the components of these phases examined. A glycoprotein of high molecular weight as well as serum albumin, immunoglobulin A, α1 acid glycoprotein and lactoferrin were detected in the sol phase by gel filtration and immunodiffusion studies. A glycoprotein of similar chemical composition was isolated from the gel phase by solubilisation in 6 M urea/0.1 M NaCl followed by fractional precipitation with ethanol. The carbohydrate content of this glycoprotein, termed the bronchial glycoprotein, varied from 58 % (w/w) to 69 % (w/w) with sputum from different patients and was composed of fucose, galactose. galactosamine, glucosamine and N-acetylneuraminic acid. Serine, threonine and proline constituted 44 % (w/w) of the amino acid residues of the bronchial glycoprotein and an alkaline borohydride cleavage study indicated that O-glycosidic linkages exist between N-acetylgalactosamine and the hydroxyamino acids in the peptide core. Fractionation of the bronchial glycoprotein on DEAESephadex A-25 yielded fractions enriched in sulphate and sialic acid but separate fuco, sialo and sulpho glycoproteins were not obtained. Similarly fractionation of the oligosaccharides released by the action of alkaline borohydride yielded a complex mixture of oligosaccharides but sulphated oligosaccharides free of sialic acid were not obtained. Separation of the oligosaccharides into two fractions of different sizes showed that sulphate content was greatest in the larger oligosaccharides whereas sialic acid content was highest in the smaller oligosaccharides. A methylation study of the bronchial glycoprotein, neuraminidase-digested bronchial glycoprotein and mild acid-hydrolysed bronchial glycoprotein revealed that the N-acetylneuraminic acid was linked to C-3 of penultimate galactose units. Fucose occurs in three different linkages, approximately 50 % is linked to C-2 of penultimate galactose units, approximately 25 % is linked to C-4 of glucosamine residues already substituted at C-3 and the remainder is linked to C-3 of glucosamine residues already substituted at C-4. [ABSTRACT FROM AUTHOR]

A rapid purification method for tRNA nucleotidyltransferase from baker's yeast has been worked out. The enzyme, purified 900-fold with a yield of 20%, is electrophoretically pure. ATP and CTP protect the enzyme against heat inactivation and are competitive inhibitors for each other in the reaction of CCA attachment. Differences in the protection properties, as well as in the kinetic constants, suggest that the ATP and CTP sites on the enzyme are different. The enzyme is also protected by high concentrations of tRNA without the CCA terminus (tRNA-X), but not by the intact tRNA. Differences between tRNA's with or without the CCA triplet were also found when their inhibitory effect on the AMP attachment to tRNA-XCC, whereas tRNA-XCCA acts as a non-competitive inhibitor both for CMP attachment to tRNA-X and AMP attachment to tRNA-XCC. Two interpretations for these differences in the behaviour of tRA with or without the CCA terminus are discussed: (a) either a conformational change of the tRNA when the terminal A is reattached, or (b) the occurrence of a sequential mechanism involving the binding of ATP or CTP to the enzyme prior to the binding of tRNA. [ABSTRACT FROM AUTHOR]

HeLa cells, prelabeled with [³H[leucine for several generations, were pulse-labeled with [14c]-leucine. The 14C/³H ratio of the leucine in the medium, in the total amino acid pool and bound to transfer RNA was determined.It was found that the 14C/³H ratio of the leucyl-tRNA was identical with that of the extracellular medium while that of the amino acid pool was much lower. These results confirm previous reports that selection of amino acids for protein synthesis takes place before the amino acids are released intracellularly.Further, it is concluded that amino acids resulting from intracellular proteolysis are not reutilized directly under the conditions of our experiments.A possible model of intracellular amino acid pathways is discussed. [ABSTRACT FROM AUTHOR]

1. A technique is described, based on the distribution of rubidium, acetate and methylammonium ions, for the simultaneous estimation of membrane potential and pH gradient across the inner membrane of mitochondria. The technique requires less than 0.5 mg mitochondrial protein and is independent of many factors which interfere with electrode determinations of protonmotive force <Δp). 2. With a limiting matrix volume of 0.4 μl/mg mitochondrial protein, indicated value of Δp for rat liver mitochondria is 228 mV in sate 4,170 mV in state 3, and -0.6 mV in the presence of rotenone and oncoupler. The relative contributions of the pH gradient and membrane potential are dependent on the availability of electrophoretically and electroneutrally translocatable species in the incubation medium. 3. In a sucrose-based medium containing 0.5 mM KCl, rotenone and uncoupler, the technique indicated a membrane potential of + 85 mV and a pH gradient of + 1.46 (acidic in the matrix compartment). In state 4m under no conditions examined did the pH gradient contribute more than 50% of the total protomotive force. 4. The hydrolysis of ATP generates an optimal Δp of 220 mV. 5. The proton conductance of the inner membrane is potential dependent, increasing when Δp is greater than 200 mV. 6. The extra-mitochondrial phosphate potential sustainable by respiration was found to change in parallel to Δp, but to exceed the latter parameter when based upon a stoichiometry of two protons translocated per ATP synthesized. [ABSTRACT FROM AUTHOR]