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This study describes the structure of the N-linked oligosaccharide chains of bile-salt-dependent lipase isolated from the pancreatic juice of a normal donor. After hydrazinolysis, neutral sugar chains were separated from acidic chains by paper electrophoresis and were fractionated using serial column chromatography with immobilized lectins and Bio-Gel P-4 filtration. Structural analysis was performed by means of sequential glycosidase digestion and revealed that the neutral sugar chains are mainly of the biantennary complex type. Fucose residues were identified for some trimannosyl core structures and were α(1–6) or α(1–2) linked to the innermost GlcNAc residue and a terminal Gal residue, respectively. Sialyl residues were also involved in the oligosaccharide structures. Most of these structures have no linear N-acetyllactosamine repeats. Evidence from several approaches suggests that the sugar chains of the human pancreatic bile-salt-dependent lipase possess a blood-group-related antigenic determinant. [ABSTRACT FROM AUTHOR]

1. Desmosine and isodesmosine were separated by ion-exchange and paper chromatography, after acid hydrolysis of purified elastin from beef ligamentum nuchae. The fractions obtained by ion- exchange chromatography were clearly mixtures of related compounds. The desmosine fraction could be resolved into seven compounds and the isodesmote into four by paper charmatography. 2. Desmosine was maximally degraded by irradiation at 274 nm and isodesmosine at 285 nm. These wavelengths did not correspond to the absorption maxima of the cross links, but to shoulders of the main absorption peaks. 3. When irradiated at their optimum wavelengths, but at various pH, both desmosine and isodesmosine seemed quite stable at pH greater than 8.5. Between pH 8 and 5, the photolytic rate was maximum and decreased slightly at more acidic pH. Below pH 4.0, one of the products of photolysis was free lysine. 4. In analogy to the mechanism of the photolytic degradation of N-methyl pyridinium chloride, it appears that the (iso)desmosines were degraded via the formation of an open amino aldehyde, which was hydrolysed at acid pH to give free lysine and a substituted glutaconic aldehyde. [ABSTRACT FROM AUTHOR]

Abnormally high amounts of low molecular weight mannose-rich carbohydrate material were found in the urine of an Angus calf with mannosidosis. At least five oligosaccharide fractions were detected by paper chromatography. The most abundant compound was purified by gel chromatography, zone electrophoresis, and two consecutive preparative paper chromatographic steps. The yield was 10 mg/liter of urine. From structural studies including nuclear magnetic resonance spectroscopy, optical rotation, sugar analysis, methylation analysis, and partial enzymatic degradation the following structure was deduced: α-D-Manp- (1 → 6)-β-D-Manp-(1 → 4)-β-D-GlcNAcp-(1 → 4)-β-D-GlcNAcp-(1 → 4)-D-GlcNAc. This oligosaccharide is distinct from all the oligosaccharides previously described which are excreted by patients with mannosidosis. [ABSTRACT FROM AUTHOR]

A polyol was released from the lipopolysaccharide of Proteus mirabilis, strain D52, during alkaline hydrolysis and its phosphate ester was isolated after acid hydrolysis. This polyol has been identified as ribitol by comparison of the free polyol, its phosphate ester and its anhydro derivative formed after acid treatment with authentic xylitol, D- and L-arabitol, ribitol and their corresponding derivatives on paper and gas-liquid chromatography. [ABSTRACT FROM AUTHOR]

Procedures for the stepwise addition of one or more deoxyribonucleotide residues to the 3′ end of an oligodeoxyribonucleoside phosphate acceptor using commercially available terminal deoxynucleotidyl transferase is described. 2–80 nmol of acceptors with a chain length of four, five or nine monomer units were elongated with a single 2′-deoxyribonucleoside 5′-triphosphate in yields of 20-30 %. The monomers carried no protecting groups and were used both radioavtively labelled and unlabelled. The elongated oligodeoxynucleoside phosphates were isolated by reverse-phase (Nucleosil C18) high-performance liquid chromatography or paper chromatography. The isolated products were sequenced by the fingerprint method. Advantages and disadvantages of this new methodology for the enzymatic synthesis of defined oligodeoxynucleotides are discussed. [ABSTRACT FROM AUTHOR]

The light chain fraction was separated from myosin extracted from chicken cardiac muscle. Two light chain components, L-1 and L-2 in the fraction were isolated by chromatography on a column of DEAE-cellulose (DE-52) in the presence of 4 M urea. After performic acid oxidation, the L-1 chain was digested with trypsin and the resulting peptides were isolated. The amino acid sequences of the peptides were established. The ordering of these tryptic peptides in the L-1 chain was deduced from the amino acid compositions and the partial sequences of peptic peptides from S-carboxymethylated L-1 chain. Comparing the whole sequence of the L-1 chain thus established with that of alkali light chain of rabbit skeletal muscle myosin, 67 amino acid substitutions and two insertions were recognized. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that Corynebacterium cyclohexanicum degrades cyclohex-anecarboxylic acid, a bacteriocide, through a pathway including the aromatization of 4-oxocyclohex-anecarboxylic acid to 4-hydroxybenzoic acid [Kaneda, T. (1974) Biochem. Biophys. Res. Commun. 58, 140–144]. Aromatization has now been shown to be catalysed by two desaturase enzymes. Under the action of desaturase I, 4-oxocyclohexanecarboxylic acid is converted to (+)-4-oxocyclohex-2-enecarboxylic acid which is then aromatized by desaturase II to 4-hydroxybenzoic acid. The latter reaction is presumed to occur via the unstable intermediate, 4-oxocyclohex-2,5-dienecarboxylic acid, which is spontaneously isomerized to 4-hydroxybenzoic acid. Desaturase I has been purified in an electrophoretically homogeneous form. It is monomeric with a molecular mass of 67 kDa and contains one tryptophan, one histidine and two cysteine residues per enzyme molecule. The enzyme produces an equivalent amount of 4-oxocyclohex-2-enecarboxylic acid and hydrogen peroxide from 4-oxocyclohexanecarboxylic acid. The properties of desaturase I have been studied in detail. Desaturase II is unstable and has been partially purified. Its characterization is therefore limited. However, the molecular mass of desaturase II was estimated to be 43 kDa by gel filtration chromatography. The characterization of both desaturase enzymes is described in this paper. The possible environmental importance of microbial aromatization in the biodegradation of compounds with the cyclohexane structure is discussed. [ABSTRACT FROM AUTHOR]

From bronchial mucins of cystic fibrosis patients with blood group O, the carbohydrate chains were released in the form of oligosaccharide-alditols by alkaline borohydride treatment. Application of high-performance liquid chromatography directly on the pool of neutral oligosaccharides afforded 23 fractions. Twenty oligosaccharide structures were characterized by employing 500-MHz 1H-NMR spectroscopy in conjunction with sugar analysis. Thirteen among these had been revealed before to occur in human bronchial nucins [Van Halbeek, H., Dorland, L., Vliegenthart, J. F. G., Hull, W. E., Lamblin, G., Lhermitte, M., Boersma, A., and Roussel, P. (1982) Eur. J. Biochem. 127, 7–20], when paper-chromatographic fractionation of this pool of neutral oligosaccharides was employed. High-performance liquid chromatography enabled to obtain another seven pentassacharide and hexasaccharide alditols; the largest-size representatives are: [This symbol cannot be presented in ASCII format] Thereby, this approach afforded deeper insight into the structural heterogeneity displayed by the carbohydrate chains of bronchial mucins. [ABSTRACT FROM AUTHOR]

Purified extracellular endo-1,4-β-xylanase (EC 3.2.1.8) of the yeast Cryptococcus albidus was found to catalyze not only the known l,4-β-transfer, but an alternative transglycosylation reaction leading to the formation of 1,3-β-glycosidic linkages. From a mixture of products of β-xylanase degradation of phenyl β-D-xylopyranoside three xylooligosaccharide fractions, differring chromatographically from the 1,4-β-linked products, were isolated by preparative paper chromatography. Their structure was elucidated by mass spectrometry, 13C-NMR spectroscopy and enzymic hydrolysis by β-xylanase and β-xylosidase. The isomeric xylotriose was identified as 3-O-β-D-xylopyranosyl-4-O-β-D-xylopyranosyl-D-xylose. The fraction of isomeric tetrasaccharides was found to be represented mainly by 4-O-β-D-xylopyranosyl-3-O-β-D-xylopyranosyl-4-O-β-D-xylopyranosyl-D-xylose. The xylooligosaccharides containing one 1,3-β-linkage were also produced on the enzyme treatment of 1,4-β-xylotriose and 1,4-β-xylan. When treated with the enzyme responsible for their synthesis, the isomeric xylooligosaccharides were hydrolyzed at the 1,3-β-linkage, despite the fact the enzyme does not attack 1,3-β-xylan. The results are interpreted in the relation to the characterized four-subsite substrate-binding site of the enzyme. [ABSTRACT FROM AUTHOR]

The specific polysaccharide was obtained from the lipopolysaccharide of Shigella newcastle by mild acid hydrolysis and further purified by permeation chromatography on Sephadex G-50. It was found to consist of L-rhamnose, 2-acetamido-2-deoxy-D-galactose, D-galacturonic acid residues and O-acetyl groups in the molar ratios of 2: 1: 1: 1. On the basis of 1H and 13C nuclear magnetic resonance spectroscopy, methylation analysis, partial acid hydrolysis, Smith degradation, and chromium trioxide oxidation, the following structure can be assigned to the repeating oligosaccharide unit of the polysaccharide :-4)DgalA(βI-3)Dga1NAc-(βl-2)LAc3Rha(αl-2)LRha(αl-, where GalA = galacturonic acid, GalNAc = N-acetylgalactosamine, Ac3Rha = 3-O-acetylrhamnose. The structural and immunochemical data presented prove that Sh newcastle lipopolysaccharide belongs to a ‘non-classical’ type of somatic antigens with acidic O-specific polysaccharide chains. [ABSTRACT FROM AUTHOR]

The lytic enzyme from bacteriophage T7 has been purified from T7-infected Escherichia coli B cells and characterized. In the purification procedure advantage was taken of the reversible binding of the enzyme to chitin. Molecular weight determinations of the enzyme by Sephadex G-75 chromatography and sodium dodecyl sulphate gel electrophoresis gave estimates of 21000 and 18000 respectively. Because the activity of the enzyme falls rapidly to 1-3% of that observed immediately after lysis of the cells, various means to stabilize the enzyme have been tried. Of the methods attempted only binding to the substrate analogue chitin was successful. The enzymic activity was found to be markedly dependent on ionic strength and pH, maximum activity was observed using 5 mM phosphate buffer pH 7.5. MgCl2 was found to enhance the lytic activity 2–3-fold, the optimal concentration being about 0.5 mM. The enzyme was not sensitive to penicillin G or spermine, but was specifically inhibited by p-chloromercuribenzoate. Following digestion of E. coli peptidoglycan the cell wall peptides L-Ala-D-Glu(A2pm), L-Ala-D-Glu(A2pm-D-Ala) and the dimer of the latter were formed, together with paper chromatographically immobile glycan strands. The enzyme, therefore, acts as an N-acetylmuramyl-L-alanine amidase by cleaving the bond between N-acetylmuramic acid and L-alanine in the E. coli peptidoglycan. The enzyme has apparently no effect on the peptidoglycan from Micrococcus luteus, and seems to have a preference for high-molecular-weight substrates. Thus the disaccharide-tetrapeptide GlcNAc-MurNAc-L-Ala-D-Glu(A2pm-D-Ala) and its dimer are not substrates for the enzyme. [ABSTRACT FROM AUTHOR]

From Escherichia coli 0124 two lipopolysaccharide preparations were obtained with phenol/water extraction and cetavlon precipitation. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and chemical analysis showed that the two preparations differed only in the extent of the O-specific polysaccharide moiety. In passive haemagglutination and gel precipitation the two lipopolysaccharide preparations from E. coli 0124 and the corresponding preparations from Shigella dysenteriae type 3 reacted alike. The O-specific polysaccharide moiety was characterized with proton magnetic resonance spectroscopy, optical rotation and paper electrophoresis. The constituents were determined by gas chromatography and ion-exchange chromatography. The polysaccharide contained glucose (Glc), gatactose (Gal), galactosamine (GalN) and 4-O-(1′-carboxyethyl)-D-glucopyranose (glucolactilic acid, GIcLA) in the molar ratios of 1 : 2 : 1 : 1. Glucolactilic acid, which has a structure similar to muramic acid, was first found in Sh. dysenteriae. The polysaccharide from E. coli 0124 and oligosaccharides obtained from it by partial acid hydrolysis were subjected to methylation analysis using the method of combined gas chromatography—mass spectrometry. The results indicated that the pentasaccharide repeating unit of the polysaccharide is[This symbol cannot be presented in ASCII format] In the polysaccharide the repeating units are joined through galactofuranosidic linkages. This structure is identical with that of the somatic polysaccharide of Sh. dysenterae type 3. [ABSTRACT FROM AUTHOR]

Two isoenzymes of phospholipase A (fraction DE-I and DE-JI) were isolated from the venom of Hemachatus haemachatus (Riughals) by gel filtration on Sephadex 0-50 and by ion-exchange chromatography on DEAE-cellulose. Fraction DE-I was homogeneous by various physicochemical criteria. The enzyme comprises 119 amino acid residues and is cross-linked by seven disulphide bridges. The values of the molecular weight of fraction DE-I from gel filtration in the presence of calcium ions, also by the dodecylsulphate gel method and calculated from the amino acid composition were close to 13500. The complete amino acid sequence of phospholipase A (fraction DE-I) was elucidated. The reduced and S-carboxymethylated enzyme was digested with trypsin, chymotrypsin, thermolysin and subtilisin and the peptides purified by DEAE-cellulose chromatography, gel filtration and chromatography or electrophoresis on paper. The amino acid sequences of the intact enzyme and the pure peptides were determined by the Edman procedure, either through the use of the automatic sequencer or by manual manipulation. The chymotryptic digest provided the necessary overlapping peptides for aligning the tryptic peptides. The amino acid sequence of Hemachatus haemachatus phos- pholipase A (fraction DE-I) shows a high degree of homology with the three isoenzymes of phospho- lipase A from Naja melanoleuca and is also homologous with the phospholipase A from Bitis gabonica and porcine pancreas. [ABSTRACT FROM AUTHOR]

The positions of the disulfide bridges in toxin Il of Androctonus australis Hector were investigated using proteolytic enzymes. The resulting peptides were separated by column and paper chromatography and high-voltage electrophoresis. Determination of the amino acid compositions of the cystinecontaining peptides or their oxidized derivatives and, when necessary, dansyl Edman degradation allowed to locate the disulfide bonds in the toxin. The four disulfide bridges were found to link the half-cystine residues number 12 and 63, 16 and 36, 22 and 46, 26 and 48. These positions are probably the same in all scorpion neurotoxins. [ABSTRACT FROM AUTHOR]

In this paper, data are presented on purification and properties of a new serine endopeptidase (duodenase) isolated from bovine duodenum mucosa. The enzyme has been purified to homogeneity by combinations of ammonium sulphate fractionation,carboxymethyl-cellulose 52 chromatography, and affinity chromatography on Sepharose 4B with Kunitz soybean trypsin inhibitor as a ligand. Some physicochemical properties of this protease have been investigated. The molecular mass of the purified duodenase was determined to be 29±0.5 kDa by SDS/PAGE and G-2000 SW column chromatography. The enzyme molecule is a single chain and the native enzyme is a monomeric protein. Its isoelectric point was estimated to be 10 ± 0.2. Duodenase has two forms (I and II) which possess similar properties but differ in their amino acid composition. The new protease is a glycoprotein and contains approximately 3.5% sugars. The enzyme displays trypsin-like and chymotrypsin-like activities and hydrolyzes the amide bonds of substrates having Lys, Arg, Tyr, Phe and Leu residues at the P1 position. Duodenase is most active at pH 7.9–8.2. Duodenase was irreversibly inhibited by diisopropylphosphofluoridate and phenylmethanesulphonyl fluoride, indicative of an activ-site serine in this protease, α-N-Tosyl-L-lysine chloromethane and α-N-tosyl-L-phenylalanine chloromethane, which react with an active His, caused marked inhibition of trypsin-like and chymotrypsin-like activities of duodenase. The enzyme activity was strongly supressed by trypsin inhibitors from different sources (soybeans, bovine lungs and Lima beans). Chicken egg white ovomucoid had no effect on the duodenase activity. The N-terminal sequence of the native duodenase (24 amino acid residues) shows high similarity with those of human and murine cytotoxic T-lymphocyte granzymes, human leukocyte cathepsin G and rat mast cell chymases. The biological role of duodenase is discussed. [ABSTRACT FROM AUTHOR]

Microsomal coumarin 7-hydroxylase activity is regulated differently from several other monooxygenase enzymes, at least in mice [Wood, A. W. and Conney, A. H. (1974) Science (Wash. DC) 612-614]. Recently we found that in D2 mice this activity is strongly and selectively induced by pyrazole [Juvonen, R. O., Kaipainen, P. K. and Lang, M. A. (1985) Eta. J. Bloc/tern. 152, 3-8]. This paper describes the purification of the pyrazole- inducible cytochrome P450 isoenzyme. Because of the lability of the protein, a special procedure for the purification was developed. The procedure is based on a combination of hydrophobic and ion-exchange chromatography and the presence of 100 μM coumarin in the preparations throughout the whole purification. Coumarin effectively protected the P-450 from degradation and also converted the pyrazole-inducible P-450 to its high-spin state. This enabled us to choose only those fractions for further purification where the P-450(s) was in its high-spin state (rather than measuring the content of the total P-450). As a result the purified protein had an apparent molecular mass of 49.7 kDa, a specific content of 19.9 nmol/mg protein and a very high affinity and metabolic capacity for coumarin. [ABSTRACT FROM AUTHOR]

A 200-fold purification of the maturation-promoting factor or MPF from unfertilized eggs of Xenopus laevis is reported for the first time. Purification was achieved by three successive column chromatographies on hydroxyapatite, trisacryl blue and L-arginine-agarose. The presence of MPF was assessed by the usual maturation criteria after injections of test material into immature stage VI unstimulated X. laevis oocytes: the precocious appearance of the maturation spot (within 45–120 min), the germinal vesicle breakdown, the presence of the first polar body and the second metaphase spindle. Purification was monitored by the decrease of the minimal amount of protein injected in a constant volume (50 nl) required to induce 50% frequency of germinal vesicle breakdown. This amount decreased from 500 ng in the crude extract to 2.5 ng in the 200-fold purified material. Analysis by SDS-PAGE of the etude extract showed about 40 Coomassie-blue-stained polypeptides with molecular masses ranging from 300 kDa to 20 kDa, whereas in the 200-fold purified MPF only 5 stained polypeptides were revealed, with molecular masses of 62, 53, 49, 39 and 37 kDa. In vitro phosphorylations for the detection of kinase activities for endogenous and exogenous substrates were monitored by analysis of autoradiograms of SDS-PAGE, after treatment of fractions with [γ-32P]ATP. Only inactive fractions eluted from columns ahead of MPF, and fractions containing MPF activity were tested. Phosphorylation of numerous stained polypeptides was demonstrated in the crude MPF extract and exogenous substrates such as phosvitin, casein and histone type II-AS were also strongly phosphorylated. In the MPF fraction, purified on hydroxyapatite, a polypeptide of 53 kDa was more highly and specifically phosphorylated and the presence of kinase activities was observed for the above three exogenous substrates. In the 100-fold and 200-fold purified MPF, phosphorylation of endogenous substrates could not be shown and kinase activities for the above three substrates were drastically decreased as compared with the crude and purified MPF obtained after hydroxyapatite column chromatography. However, neither endogenous phosphorylations nor kinase activities with the above exogenous substrates could be shown in inactive fractions eluted ahead of MPF at the different purification steps. Some characteristics of the purified material are also described in this paper. [ABSTRACT FROM AUTHOR]

1. In Escherichia coli ML308 isocitrate dehydrogenase is partially inactivated during growth on acetate [Bennett, P. M. and Holms, W. H. (1975) J. Gen. Micribiol. 87, 37-51]. 2. The active form of isocitrate dehydrogenase was purified to homogeneity from cells grown on glycerol. The key step in the procedure was chromatography on procion-red-Sepharose, from which the enzyme was specifically eluted with NADP+. 3. Two forms of isocitrate dehydrogenase were purified to homogeneity from cells grown on acetate. One form did not bind to procion-red-Sepharose and was essentially inactive; this form could be resolved from the active form by non-denaturing gel electrophoresis. The other form was specifically eluted from procion-red-Sepharose and was partially active; analysis of this form by non-denaturing gel electrophoresis suggested that it was a mixture of the active and inactive forms. 4. The three forms comigrated on denaturing gel electrophoresis and were identical by the criterion of onedimensional peptide mapping. 5. Analysis of the active and inactive forms by sedimentation equilibrium centrifugation and non-denaturing gel electrophoresis showed that they differed in charge but not in size. Amino acid analysis and two-dimensional peptide mapping showed that both forms were directs of identical subunits. 6. The active form of the enzyme contained no detectable alkali-labile phosphate, the inactive form contained 0.8 molecule subunit and the partially active form contained an intermediate amount. 7. The data suggest that the active and inactive forms of isocitrate dehydrogenase differ only in the presence of one phosphate group per subunit in the latter form; this is consistent with our results from phosphorylation of isocitrate dehydrogenase in vitro (Following paper in this journal). 8. The nature of the partially active form of isocitrate dehydrogenase and the significance of the results are discussed. [ABSTRACT FROM AUTHOR]

The partial ammo acid sequence of dihydrofolate reductase (DHFR. EC 1.5,1.3) from human KB/6b cells hi been determined by using 3.5 mg of protein. Peptides covering the entire polypeptide chain were recovered from preparative peptide maps generated by the combination of paper chromatography and electrophoresis at pH 4.4 Peptide maps from mouse L1210 DHFR were also generated for comparison. Amino acid sequence of 75% of the 186 amino acid residues in the polypeptide chain of human KB/6b DHFR was obtained from Edman degradation and the remaining sequence was deduced from the amino acid compositions, from electrophoretic mobilities of related peptides and from the sequence homologies with other known mammalian DHFR sequence. A comparison of the proposed human DHFR sequence with the previously known sequent of mouse enzyme [Stone, et al. (1979) J. Biol. Chem. 245, 480-488] indicates that 18 differences are located in the established sequence of 139 residues and that 5 additional differences are in the tentative sequence of the remaining 47 amino acids. Kinetic properties of human KB/6b and mouse L1210 DHFR, which were determined in parallel experiments, are also compared. The possible structural-functional relationships between human and mouse DHFR are discussed. [ABSTRACT FROM AUTHOR]

From lysozyrne digests of N-acetylated cell walls of Bacillus cereus AHU 1030, two acidic polymer fractions with molecular weights of about 24000 and 45000 were isolated by ion-exchange chromatography and gel chromatography. These polymer fractions, containing glycerol, phosphorus and glucose in a molar ratio of 1.00: 1.00 : 0.85 together with small amounts of glycopeptide components and mannosamine were characterized as teichoic-acid - glycopeptide complexes with one and two teichoic acid chains made of 60-65 repeating glycerol phosphate units that were mostly glucosylated. Mild alkali treatment of the complexes yielded a disaccharide-Linked glycopeptide. The disaccharide was liberated from the glycopeptide by mild acid treatment and identifled as N-acety1rnarniosaminyl(β 1 →4)N-acetylglucosamine. On the other hand, the same disaccharide linked to the teichoic acid chain was obtained by direct heating of the cell walls at pH 2.5. These results lead to a conclusion that in the cell walls of this strain the glycerol teichoic acid chain is attached to the glycan chain of peptidoglycan through this disaccharide unit. The disaccharide is linked at its reducing and nonreducing ends to the glycan chain and the teichoic acid chain, respectively, through phosphodiester bridges. [ABSTRACT FROM AUTHOR]

The amino acid sequence of the heme-binding domains of rat liver cytochromes b5 from outer mitochondrial membranes and from microsomes has been determined by a combination of automatic and manual degradation of fragments generated by trypsin digestion and by cleavage at tryptophan. Tryptic peptides were separated by high-pressure liquid chromatography. The sequence of microsomal cytochrome b5 is identical with the one published by Owls and Heinemann after completion of this study [Biochim. Biophys. Acta (1982) 704. 163- 173]. The sequence of outer membrane cytochrome b5 differs from the microsomal one at 38 positions out of 91. There are 40 positions invariant between this sequence and the eight micrasornal sequences published thus far. The non-conservative substitutions are located at the surface of the known three-dimensional structure of calf microsomal cytochrome b5 except for the substitution of histidine- 15 by arginine. This paper brings the final proof that two iso-cytochromes b5- exist in the same cell. Their high degree of similarity as well as their differential cellular localization raise some questions which are briefly discussed. [ABSTRACT FROM AUTHOR]

Rabbit reticulocyte lysates were passed through 2'5'ADP-Sepharose columns under conditions in which the gel- filtration effect was negligible and low-molecular-weight compounds were retained in the flow-through lysate. Glucose-6-phosphate dehydrogenase. 6-phosphogluconate dehydrogenase and glutathione reductase were quantitatively adsorbed by the column and removed from the lysate.. but isocitrate dehydrogenase and thioredoxin reductase were retained in the flow-through lysate. The initial rate of protein synthesis in lysates treated in this way was normal, but synthesis stopped after about 20 mm of incubation. This shut-off could be prevented by the addition of dithiothreitol or by providing a means of NADPH generation, which could be achieved either by adding isocitrate or glucose-6-phosphate dehydrogenase. Further experiments used lysates which were first gel-filtered to remove low- molecular-weight metabolites and then passed through 2'5'ADP-Sepharose columns. Under these conditions thioredoxin reductase was efficiently adsorbed by the affinity column, in addition to the three enzymes already listed. The maintenance of full protein synthesis activity in these lysates required the addition of both a sugarphosphate and a reducing agent. The sugar phosphate requirement could be satisfied by glucose 6-phosphate. or 2-deoxyglucose 6- phosphate, or fructose 1,6-bisphosphate, but not by 6-phosphogluconic acid. The requirement for reducing agent could be met by the addition of dithiothreitol, or by an NADPH-generating system together with rabbit thioredoxin ceductase. Purified thioredoxin reductase from Escherichia coli was also effective provided E. coli thioredoxin was also added, but the addition of glutathione with glutathione reductase did not activate protein synthesis. It is concluded that there is a dual requirement for the maintenance of high rates of protein synthesis in reticulocyte lysates: certain sugar phosphates must be present. in addition to an NADPI-I-generating system and a functional thioredoxin/thioredoxin reductase system. [ABSTRACT FROM AUTHOR]

During short-term labeling experiments, cells of Methanohacterium thermoautolrophicum incorporated a substantial part of 14CO2 in a compound with a bright yellow fluorescence on dry thin-layer chromatography plates and called yellow fluorescent compound (YFC) [Daniels, L, and Zeikus, J. G. (1978) J. Baeteriol. 136, 75 - 84]. This compound was extracted and purified by ion-exchange column chromatography with formic acid gradients up to 0.3 M. Out of 325 g wet cells of M. thermoautotrophicum about 4 mg of the compound were isolated. This material and some degradation products obtained from it were studied by means of chemical decomposition, ultraviolet-visible- light spectroscopy and preliminary 1H-NMR spectroscopy. It has structural elements in common with methanopterin (see preceding paper in this journal); these elements are a pterin group, glutamate, a hexosamine. The pterin in this compound is present in a reduced form, presumably as 5,6,7,8-tetrahydromethanopterin, and the additional one-carbon unit is probably present as a carboxy group. Probably the first step of methanogenesis implies a carboxylation of methanopterin and a concomitant reduction of the pterin. The trivial name carboxy-5,6,7,8-tetrahydromethanopterin is introduced for the compound. [ABSTRACT FROM AUTHOR]

The results reported in the present paper and N-terminal sequence homologies established by other authors strongly support the assumption that the gastric protease previously called bovine pepsin I or bovine pepsin B belongs to the aspartate protease group and corresponds to gastricsin (or pepsin C) (EC 3.4.23.3) from other species. Bovine gastricsin was prepared from commercial extracts of adult bovine veils by a procedure involving DEAE-cellulose chromatography, gel filtration on Sephacryl S-200 and a further DEAE-cellulose chromatography. The preparation thus obtained was shown to be free of chymosin and bovine pepsin A by immunodiffusion, selective inactivation in urea and isoelectric focusing. Us molecular weight was estimated by gel filtration to be 32800. Bovine gastricsin displayed microheterogeneity on isoelectric focusing with pi values of the components ranging from 3.5 to 4.0. Chromatography of bovine gastricsin on hydroxyapatite separated three fractions, none of them being homogeneous by isoelectric focusing. Concanavalin-A—Sepharose 4B bound bovine gastricsin to some extent, but without any significant fractionation. Proteolytic activity could be detected directly on the isoelectric focusing gel for all the components of gastricsin and its fractions from hydroxyapatite and concanavalin-A—Sepharose 4B. Bovine gastricsin and its fractions from hydroxyapatite have similar amino acid compositions, different from those of bovine chymosin and pepsin A but obviously related to those of human, simian and porcine gastricsins. Bovine gastricsin which is inactivated by reaction with diazoacetyl-DL-norleucine methyl ester and with 1,2-epoxy-(p-nitrophenoxy)propane in a 1:1 and 1:2 stoichiometry, respectively, is able to hydrolyse a synthetic hexapeptide, Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe, used as reference substrate for aspartate proteases, and exhibits a low activity towards N-acetyl-L-phenylalanyl-L-diiodotyrosine. Its specific clotting activity with χ-casein as substrate is only half of that of chymosin and pepsin A. [ABSTRACT FROM AUTHOR]

Blood-group-specific glycoproteins obtained from ovarian cyst fluids of A1 and A2 persons were degraded with NaOH/NaBH4. The oligosaccharides released were de-N-acetylated with Ba(OH)2 and then hydrolysed with dilute H2SO4. The products were fractionated on columns of ion-exchange resin and the components isolated were re-N-acetylated with 14C-labelled acetic anhydride; further purification was effected by paper chromatography. The following trisaccharides: type 1, GaINAc(αl - 3)Gal(βl - 3)G1cNAc; type 2, Ga1NAc(αl - 3)- Gal(βl -4)GlcNAc; type 3 (reduced), GalNAc(αl - 3)Gal(β1 - 3)GalNAcOH (where Gal is galactose, GalNAc is N-acetylgalactosamine, G1cNAc is N-acetylglucosamine and Ga1NAcOH is N-acetylgalactosaminitol) were isolated and characterised from both the A1 and A2 materials. The type 3 (reduced) trisaccharide has not previously been obtained from human glycoproteins. Chromatographic evidence indicated that the three trie saccharide structures were also present in other A1, A2, A1B and A2B ovarian cyst glycoproteins and in A1 and A2 salivary glycoproteins. These findings are not indicative of structural differences between the A determinants of A1 and A2 glycoproteins. [ABSTRACT FROM AUTHOR]

Surface glycoproteins on plasma membranes from Dictyostelium discoideum were labeled by sodium metaperiodate oxidation and sodium boro[³H]hydride reduction. The amount of incorporation of tritium from NaB³H4 reached a plateau after 10 min at a periodate concentration of 20 mM. The density analysis carried out by sucrose density-gradient centrifugation showed that the plasma membranes were selectively labeled by this technique. About 84% of the radioactivity incorporated was released by hydrolysis with 0.25 M H2SO4 for 3 h at 100 °C. The released materials were eluted at a bed volume after chromatography using a Sephadex G-50 column. From the paper chromatographic analysis of the eluate, four radioactive spots were detected. Two of them were glycerol and glyceraldehyde and the other two spots seemed to be oligosaccharides. Using the above method, the plasma membranes from aggregation-phase cells were labeled four times more than those from growth-phase cells. The labeled plasma membrane fraction during the aggregation phase was separated into at least 39 distinct glycoprotein bands on a one-dimensional dodecylsulfate/polyacrylamide gel. Six of the 39 bands increased markedly in density and two new bands appeared. In contrast, four bands decreased in density in the aggregation phase. Using this method, the surface glycoproteins can be analyzed directly by gel electrophoresis of the lysate of labeled whole cells without preparing the plasma membranes. Changes during development of glycoproteins on outer cell surfaces were also confirmed by O'Farrell's method of two-dimensional polyacrylamide gel electrophoresis. Utilizing this technique, glycoproteins on plasma membranes of D. discoideum were separated into 63 individual spots; 45 of these 63 spots changed during the early developmental course of D. discoideum. This is in contrast to the slight change in the soluble and membrane proteins during this phase. This fact also suggests that the glycoproteins have important roles in the process of aggregation. [ABSTRACT FROM AUTHOR]

1. A glycogen synthase kinase was partially purified from rabbit skeletal muscle by precipitation with ammonium sulphate, chromatography on DEAE-cellulose and chromatography on hydroxy- apatite. 2. The enzyme was highly specific for glycogen synthase. In the standard assay, the relative rates of phosphorylation were: glycogen synthase (100), phosvitin (2.5), phosphorylase kinase ( < 1). casein (0.3), protein phosphatase inhibitor-1 (< 0.1), phosphorylase (< 0.01), mixed histones (< 0.01). The enzyme was separated from virtually all phosvitin kinase and casein kinase activity by the chromatography on DEAE-cellulose. 3. The Km values for ATP and GTP were 0.02 mM and 0.5 mM respectively, and a similar maximum reaction velocity was obtained with each nucleoside triphosphate. The activity of the enzyme was unaffected by cyclic AMP, cyclic GMP, calcium ions, calcium ions plus calmodulin, and the specific protein inhibitor of cyclic-AMP-dependent protein kinase. 4. The properties of the enzyme demonstrated that it was distinct from both cyclic-AMP- dependent protein kinase and phosphorylase kinase, the two well characterized glycogen synthase kinases in skeletal muscle. This enzyme was therefore termed glycogen synthase kinase-3. 5. The phosphorylation of glycogen synthase by glycogen synthase kinase-3 reached a pleateau near 1.5 molecule phosphate incorporated per subunit under optimal conditions. The activity of glycogen synthase measured in the absence of glucose 6-phosphate was decreased fivefold and the apparent Ka for glucose 6-phosphate was increased 15-fold, when 1,2 molecule phosphate per sub-unit had been introduced into the enzyme. Phosphorylation to a similar extent with either cyclic-AMP-dependent protein kinase or phosphorylase kinase produced smaller changes in activity. 6. Glycogen synthase was phosphorylated by cyclic-AMP-dependent protein kinase, phosphorylase kinase and glycogen synthase kinase-3, using conditions where the phosphorylation by any one protein kinase reached a plateau near one molecule of phosphate incorporated per subunit, The different protein kinases were used separately and in combination to generate seven different phosphorylated species of glycogen synthase. The phosphorylation of glycogen synthase approached two molecules per subunit when any two protein kinases were combined, and three molecules per subunit when all three protein kinases were combined, and the inactivation produced by the different protein kinases was essentially additive. The results imply that each protein kinase preferentially phosphorylates a different site(s) on glycogen synthase, and this is confirmed by the amino acid sequence analysis described in the following paper in this journal. [ABSTRACT FROM AUTHOR]

In this paper evidence is provided that one of the protein components of the water-soluble fraction of the call lens binds specifically to deoxyribonuclease I (DNAse I). On the basis of this property, the polypeptide could be purified by applying DNAse I affinity chromatography. Concomitantly a protein of Mr 55000 and a rather large amount of α-crystallin copurify with this polypeptide, which has a molecular weight of 42000. Highly purified 42000-Mr protein was also obtained by extraction of the water-insoluble fraction of the calf lens with 2-{(tris(hydroxyniethyl)methyl]amino}ethanesulfonic acid followed by gel filtration. Amino acid analyses, peptide mapping and electron microscopy show that the protein obtained from both lens fractions is identical to non-muscle actin. Furthermore the amino acid composition of the 55 000-Mr protein is identical to hog stomach skeletin and very similar to calf brain desmin. [ABSTRACT FROM AUTHOR]

The production of 1,4-β-glucosidase activity by the rot fungus Sporotrichum pulverulentum has been investigated. It was found that cellobiose or cellulose is necessary to cause induction. With cellobiose as sole carbon source only cell-wall-bound enzymes are produced. For extracellular excretion cellulose seems to be a necessary carbon source. The purification procedures, for purification of enzyme activity in the culture solution, involve the following steps: (a) ultrafiltration, (NH4)2SO4 precipitation and dialyses; (b) preparative slab gel isoelectric focusing I, pH range 3–10; (c) phenyl-Sepharose chromatography; (d) preparative slab gel isoelectric focusing II, pH range 3–5. On the phenyl-Sepharose column the β-glucosidase activity was associated with two separable enzyme peaks, enzyme A and B. When these enzymes were subjected to further purification on preparative isoelectric focusing II, enzyme A was split into two peaks, A1 and A2, and enzyme B was split into three peaks, B1, B2 and B3. The significance of the separations is discussed. The isoelectric points of all the enzymes have been determined and found to vary between 4.52 to 5.15. The molecular weights, determined by dodecylsulphate-polyacrylamide gel electrophoresis, vary between 165000 and 182000. The kinetic constants Km and Ki have been determined for enzyme A and B as well as for the cell-bound β-glucosidase activity. Km for cellobiose was for both enzyme A and B higher than Km for p-nitrophenyl β-D-glucoside. Km/Ki of the free enzymes for gluconolactone is approximately 2500 (enzyme B) to 13000 (enzyme A) with cellobiose as substrate. [ABSTRACT FROM AUTHOR]

From lysozyme digests of the N-acetylated cell walls of Bacillus cereus AHU 1356, a polysaccharide fraction and a teichoic acid fraction were isolated by ion-exchange chromatography and gel chromatography. The former fraction, accounting for 50% of the walls, contained N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine and glucose in a molar ratio of 4: 1: 1: 1 together with a small amount of the peptidoglycan constituents. The latter fraction accounted for 5% of the walls and was composed of N-acetylglucosamine, galactose, glycerol and phosphorus in a molar ratio of 1:1.4: 1:1 and a small amount of the peptidoglycan constituents. The molecular weight of the polysaccharide fraction was about 33000 as estimated by gel chromatography. After acid hydrolysis of the polysaccharide fraction, a stoichiometric amount of muramic acid 6-phosphate was detected which was probably involved in the linkage between the polysaccharide and peptidoglycan moieties. The polysaccharide moiety detached from the peptidoglycan components by mild acid hydrolysis was isolated as material with an approximate molecular weight of 28000. Structural studies on the polysaccharide, involving Smith degradation, CrO3 oxidation and proton magnetic resonance spectrometry of the polysaccharide fraction together with the analysis of oligosaccharides obtained from partial acid hydrolysis of the polysaccharide fraction, led to the tentative formulation of the most likely structure of the repeating unit as follows ... [ABSTRACT FROM AUTHOR]

In this paper we present a procedure for the isolation of the native bovine α chain. The method is based on affinity chromatography. The results show that the ligand-binding properties of the bovine α chain are almost identical to those of the human α chain. The hybrid α2B β2H prepared by mixing bovine α chains a human β chains shows ligand binding properties similar to those of human hemoglobin and different from those of bovine hemoglobin. [ABSTRACT FROM AUTHOR]

Three toxins (CM-8, CM-11, and CM-13 a) were purified from the venom of Naja haje annulifera by gel filtration on Sephadex G-50 and by ion-exchange chromatography on CM-cellulose, Whereas toxin CM-8 and CM-11 comprise 60 amino acid residues, toxin CM-13a contains 6t residues. All three toxins are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of these toxins have been elucidated. The reduced and S-carboxymethylated toxins were digested with trypsin and chymotrypsin and the peptides purified by ion-exchange chromatography, gel filtration and chromatography or electrophoresis on paper. The Edman procedure, either through the use of the automatic sequencer or by manual manipulation, was employed to obtain the sequence of the intact toxins and the pure peptides. The chymotryptic digests provided the necessary overlapping peptides which allowed the alignment of the tryptic peptides. The properties of the three toxins were compared with those of the cytotoxin group. The toxicities the serological properties, the sequences and the invariant amino acid residues of toxin CM-8 and CM-tt resemble the corresponding properties of the cytotoxin group, Tile sequence and serological properties of toxin CM-13a show that it is related to the cytotoxin group, but its toxicity is much lower than those encountered in the cytotoxin group, [ABSTRACT FROM AUTHOR]

An α-L-rhamnosidase from the seeds of Fagopyrum esculentum (saracen corn) has previously been identified, and the effect of the enzyme on rhamnosic bonds has been studied with various flavonoid glycosides. This α-L-rhamnosidase can be useful in structural studies, and a preliminary report of this study has appeared. The present paper describes the extensive purification of the enzyme and the determination of its properties. The purification involved extraction, ammonium sulfate fractionation and chromatography on Sephadex G 75, DEAE-Sephadex and Ultrogel AcA-44. The α-L-rhamnosidase was purified about 9600 fold and the final enzyme preparation was practically pure according to the criteria of disc electrophoresis. The molecular weight of this α-L-rhamnosidase, calculated from data obtained by disc gel electrophoresis and gel filtration, was about 70000. Isoelectric focusing established the isoelectric point to be 3.7. The behaviour of the enzyme on a concanavalin-A-Sepharose column suggests the presence of residues resembling α-D-mannose or α-D-glucose in the protein. The various kinetic parameters, Kcat, Km and the Kcat/Km ratio have been determined at pH 5 on the following substrates: p-nitrophenyl-α-L-rhamnoside and rutinose (6-O-α-L-rhamnosyl-D-glucopyranose). All kinetics exhibit a Michaelian behaviour and the Km for the former substrate was 0.33 mM and for the latter, 2.2 raM. The Kcat/Km ratio corroborates the greater specificity of the enzyme for p-nitrophenyl-α-L-rhamnoside. L-Rhamnose, L-lyxose, 6-deoxy-D-glucose and methyl-α-D-mannoside were shown to behave strictly as competitive inhibitors of α-L-rhamnosidase activity; it seems that the methyl group of L-rhamnose is important for substrate binding to the enzyme. [ABSTRACT FROM AUTHOR]

A low-molecular-weight and a high-molecular-weight 1,4-β-glucan glucanohydrolase (Cχ enzyme) have been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus THchoderma vMde. The purification method for the isolation of the low-molecular-weight enzyme is a three-step procedure including chromatography on Bio-Gel P-10, chromatography on a dipolar adsorbent (arginine-fiepharose 6 B) and isoelectric focusing. The starting material for the isolation of the high-molecular-weight enzyme was pre-fractionated by chromatography on Bio-Gel P-10, by DEAE-Sephadex chromatography and by SE-Sephadex chromatography as described previously by us. Further fractionation of this material was achieved by affinity chromatography and repeated isoelectric focusing. Free zone electrophoresis of the low-molecular-weight enzyme indicated a homogeneous protein. The high-molecular-weight enzyme was homogeneous in sedimentation equilibrium analysis. The molecular weights of the enzymes were 12 500 and 50 000 ± 2000 respectively. The former value was determined by chromatography on a calibrated column of Bio-Gel P-100 and the latter value by sedimentation equilibrium analysis. The low-molecular-weight enzyme was isoelectric at pH 4.60 (10°C) and contained 21% carbohydrate. The corresponding values for the high-molecular-weight enzyme were pH 3.39 and 12%. Both enzymes were active in releasing free fibers from filter-paper. The low-molecular-weight enzyme was estimated to be about twice as effective as the high-molecular-weight enzyme in this regard. [ABSTRACT FROM AUTHOR]

The phenylalanine-specific tRNAPhe from 13-day-old chick embryo muscle was purified. Its fluorescent and hydrophobic Y+ base was excised by mild acid treatment and characterized chromatographically and by its ultraviolet absorption and fluorescent spectra. The compound obtained after removal of the Y+ base from chick embryo tRNAPhe was characterized. 1. Chick embryo t RNAPhe looses by the removal of the Y+ base its phenylalanine acceptor capacity. 2. In this state it has a reduced sedimentation coefficient of 2.8 S. a low Tm value (22 °C), only rudimentary hyperchromicity, but unchanged electrophoretic properties. 3. The compound obtained after removal of the Y+ base from chick embryo tRNAPhe regains normal melting behaviour and phenylalanine acceptor capacity when exposed to special renaturation conditions. 4. The Km of the renatured form is 0.25 × 10-6 M and V = 220 pmol × min-1 × ml-1 as compared tO Km = 0.11 × 10-6 M and V = 370 pmol x min-1 × ml-1 for the native chick embryo tRNAPhe. [ABSTRACT FROM AUTHOR]

Debate continues over the physical characteristics and even the existence of arginase isoenzymes. This paper gives additional support for such multiplicity and reports differences in physical characteristics among the various forms. After 2500-5000-fold purification of rat liver arginase, three molecular forms were separated on carboxymethyl-cellulose columns and were purified 2500-5000-fold, 800-1000-fold and 600-1000-fold, respectively. The molecular forms have also been identified by chromatography in the supernatant of tissue extracts. The isolation of these molecular forms by affinity chromatography, using Sepharose-lysine as a competitive inhibitor of arginase, shows only one main form, however. Kinetic studies were done for two of the molecular forms isolated, specifically the activation energy (Ea, the energy of denaturatization (Ed), Km, pH and the effect of divalent cations were determined. Significant differences were found for the Ea between the two molecular forms. The isolated isoenzymes are cationic at pH 5.5 and pH 8.8. However, they show different mobilities in electrophoresis. The molecular weight determination by gel filtration yields a value of 110000 to 115000 for both forms. The use of thin-layer immunochromatography plates, a combination of molecular weight and immunodiffusion technique, gave only one peak with the same molecular weight as that determined by gel filtration. The immunological studies showed that the isoenzymes have similar antigenic determinants. [ABSTRACT FROM AUTHOR]

The urinary excretion of fucose-containing material was found to be highly increased in a patient with fucosidosis type 2. Three structurally related compounds, a disaccharide and two glycoasparagines, were isolated from the urine. The isolation procedure included ultrafiltration, gel chromatography on Sephadex G-25, preparative zone electrophoresis and paper chromatography. From structural studies including optical rotation, sugar analysis, methylation analysis, ninhydrin degradation, reduction with lithium aluminium hydride and partial hydrolysis, the following structures were deduced: (1) α-L-Fucp-(1→6)-D-GlcNAc (2) α-L-Fucp-(1 →6)-β-D-GlcNAcp-Asn (3) D-Galp-(1 →4)-D-GIcNAcp-(1 → 2)-D-Manp-(l &rar; 6)-D-Manp-(1 →4)-D-GlcNAcp-(1 →4)- [L-Fucp-(l → 6)]-D-GlcNAcp-Asn {or D-Galp-(1 → 4)-D-GlcNAcp-(l → 4)-D-G lcN Acp-(l → 2)-D-Manp-(1 → 6)-D-Manp-(1 &rarrr; 4)- [L-Fucp-(1 → 6)]-D-GlcNAcp-Asn}, where Fucp is fucopyranose, Manp is mannopyranose, Galcp is galactopyranose, GlcNAcp is 2-acetamido-2-deoxyglucopyranose and Asn is asparagine. The yields of these compounds were 1.7, 40, and 6 mg/1, respectively. The origin of the disaccharide and the two glycoasparagines is probably the core region of glycoprotein carbohydrate chains. [ABSTRACT FROM AUTHOR]

Eight different types of peptide mixtures from [14C]carboxymethylated yeast alcohol dehydrogenase were obtained using trypsin with or without prior maleylation of the substrate, chymotrypsin, pepsin, microbial proteases or CNBr. Each mixture was fractionated by exclusion chromatography and peptides were further purified on paper. From results of analyses of all fragments it seems possible to deduce a primary structure of 347 unique residues in three segments. Together, the segments can account for the whole protein monomer with the exception of a small connecting region. Many unfavourable structures complicated the determination and made single sequence conclusions tentative, but known data are consistent and for most segments of the monomer results are abundant. Several microheterogeneities in the protein are indicated and one apparent amino acid exchange is characterized, suggesting that different types of subunits occur. This may probably be correlated with genetic polymorphism in yeast. Multiple desamidations are also characterized and a few of these affect particularly labile structures. Many residues are unevenly distributed and unexpected patterns are shown. Elements of repetitive sequences occur, reducing the uniqueness of structures. Hydrophobic segments are found, and the uncharacterized region is, at least in some subunits, in a core-like tryptic segment These and other aspects of the structure may explain some properties of the monomer, and form the background for evolutionary, structural and functional correlations with related enzymes. [ABSTRACT FROM AUTHOR]

Hydrophobic chromatography on alkyl-agarose columns has been applied to the fractionation of histones. This paper describes: (a) a two-column method for the resolution of whole histone from calf thymus into its five main components (H1, H2a, H2b, H3 and H4), (b) a rapid one-step procedure for the isolation of the H3 fraction from whole histone, (c) an alternative one-step procedure for the resolution of H3 and H2a (which co-elute during gel exclusion chromatography on Biogel P-60). These experiments are also used for gaining further insight into the mechanism of action of hydro- carbon-coated agaroses. [ABSTRACT FROM AUTHOR]

Poly(A)-specific ribonuclease was co-purified with poly(A) polymerase from Vigna unguiculata seedlings. Both activities were separated into two forms (enzymes 1 and II) by a final hydrophobic column chromatography. The enzyme I preparation, which was homogeneous as examined by SDS/PAGE, had both poly(A) polymerase and poly(A)-specific ribonuclcase activities. The antibody raised to the enzyme I preparation precipitated both enzyme activities. These indicate that a single polypeptide (Mr 63 000) is responsible for both poly(A)-polymerizing and poly(A)-hydrolyzing activities. The poly(A)-specific ribonuclease was a 3'-exonuclease specific to single-stranded poly(A), forming 5'AMP as the sole reaction product. The hydrolytic activity required either Mn2+ or Mg2+ with different optimum concentrations, whereas the polymenzing activity required Mn2+ but not Mg2+ ATP and PPi had little or no effect on the poly(A)-specific ribonuclease activity. [ABSTRACT FROM AUTHOR]

A polyol dehydrogenase was detected in cell extracts of the facultative phototrophic bacterium Rhodobacter sphaeroides strain Si 4 grown on D- glucitol (sorbitol) as the sole carbon source. The enzyme was purified 150-fold to apparent homogeneity by steps involving fractionated (NH4)2SO4 precipitation, chromatography on Q-Sepharose and phenyl- Sepharose, and FPLC on Superose 12. The relative molecular mass (Mr) of the native polyol dehydrogenase was 47,200 as calculated from its Stokes' radius (rs = 2.76 nm) and sedimentation coefficient (s20, w = 4.15 S). SDS/PAGE resulted in one single band representing a polypeptide with a Mr of 52,200, indicating that the native protein is a monomer. The isoelectric point of the polyol dehydrogenase was determined to be pH 4.3. The enzyme was specific for NAD+ and oxidized both D-glucitol and D-mannitol to D-fructose, as well as D-arabinitol to D-ribulose. The pH optimum of substrate oxidation was pH 9.0 in 0.1 M Tris/HCl and that of substrate reduction was pH 6.5 in 0.1 M potassium phosphate. The reactions exhibited normal Michaelis-Menten kinetics allowing the estimation of KM values for NAD+ (0.18 mM) in the presence of D-glucitol, and for D-glucitol (31.8 mM), D-mannitol (0.29 mM) and D-arabinitol (1.8 mM), respectively. The KM value for D- fructose was 16.3 mM and that for NADH 0.02 mM. The equilibrium constants determined for the conversion of D-mannitol, D-glucitol and D- arabinitol were 4.5 nM, 0.58 nM and 80 pM, respectively. Based on the catalytic preference of the polyol dehydrogenase for D-mannitol, an enzymatic assay for D-mannitol was elaborated. [ABSTRACT FROM AUTHOR]

The pathway for the de novo synthesis of a teichoic acid, poly(galactosylglycerol phosphate), in Bacillus coagulans AHU 1366 was studied by means of characterization and stepwise conversion of lipid-linked intermediates. Incubation of membranes with UDP-N-acetylglucosamine and UDP-glucose yielded a disaccharide-linked polyprenylpyrophosphate, whose sugar moiety was characterized as glucosyl(Β1 → 4)N- acetylglucosamine (Glc-GlcNAc). By incubation with membranes and CDP-glycerol, Glc-GlcNAc-PP-prenol was converted to a series of glycolipids characterized as (Gro-P)1-6-Glc-GlcNAc-PP-prenol (Gro = glycerol). Glc-[14C]GlcNAc-PP-prenol was converted to polymer by incubation with membranes, CDP-glycerol and UDP-galactose. Smith degradation of the polymer gave two radioactive fragments corresponding to (Gro-P)3- a Glc-GlcNAc and (Gro-P)4-Glc-GlcNAc. These results, together with data on gel chromatography of radioactive polymer synthesized from UDP-[3H]galactose, CDP-glycerol and Glc-[14C]GlcNAc-PP-prenol, led to the conclusion that in this strain poly(galactosylglycerol phosphate) is probably synthesized through the following pathway: GlcNAc-PP-prenol → Glc-GlcNAc-PP-prenol → (Gro-P)3-4-Glc-GlcNAc-PP-prenol → (Gro-P-Gal)n- (Gro-P)3-4-Glc-GlcNAc-PP-prenol → (Gro-P-Gal)n-(Gro-P)3-4-Glc-GlcNAc-P- peptidoglycan complex. [ABSTRACT FROM AUTHOR]

Homogenates of Perinereis cultrifera oocytes were found to transform GDP-D-mannose into another sugar nucleotide. Ultraviolet absorption spectra, chromatographic behaviour, gas-liquid chromatography coupled to mass spectrometry analysis revealed that GDP-D-mannose had been converted into GDP-L-fucose. This conversion is a multi-step reaction as proved by the involvement of two intermediates identified as GDP-4-oxo-6-deoxy-D-mannose and GDP-4-oxo-6-deoxy-L-galactose, this latter being reduced by NADPH to give GDP-L-fucose. It is shown that the enzymatic activities responsible for the conversion of GDP-D-mannose into GDP-L-fucose is recovered only in oocytes and is not present in the other coclomic cells (i.e. coelomocytes). More interesting is the fact that maximum activity is recovered at a well defined stage of the hormone-controlled oogenesis. Thus, this enzymatic system appears as a biochemical marker of the oocyte maturation in P. cultrifera. [ABSTRACT FROM AUTHOR]

Seryl-tRNA synthetase was purified 1800-fold from bovine liver extract by ultracentrifugation at 150000 x g, chromatography on DEAE-cellulose, fractional precipitation with ammonium sulfate, gel chromatography on Sephacryl S-300, adsorption chromatography on hydroxyapatite, affinity chromatography on blue-Sepharose and finally on M&asline;trex gel red A. The relative molecular mass, Mr, in the denatured state was estimated as 87000 by sodium dodecyl sulfate disc gel electrophoresis; in the active state the Mr was estimated as 170000 for the dimeric native enzyme (α2 type) by chromatography on Sephacryi S-300. The amino acid composition of the enzyme was determined. The Km values for ATP and serine were 0.49 mM and 30 μM, respectively. The Km values for tNAIGASer and tRNAcmCASer were 1.40 μM and 1.25 μM, respectively. Sequences common to the two isoaccepting tRNASer molecules are discussed in relation to the recognition mechanism of the purified seryl-tRNA synthetase. [ABSTRACT FROM AUTHOR]

Trimming glucosidase 1 and II have been solubilized from crude calf liver microsomes and partially enriched by a fractionated extraction procedure applying different concentrations of nonionic detergent and salt. The pH optimum of both enzymes was found to be close to 6.2., which discriminates them from hydrolases of lysosomal origin acting on p-nitrophenyl glycosides with the highest rate at more acidic pH. Glucosidase I and II and the nonspecific α-glucosidase(s) were inhibited by l-deoxynojirimycin with median inhibitory concentration 3 &muM, 20μM, 12μM, respectively. Discrimination between these enzymes was strongly enhanced by N-alkylation of l-deoxynojirimycin and formed the basis for the design of the affinity ligand. Glucosidase I has been purified to homogeneity by affinity chromatography on AH-Sepharose 4B with N-carboxypentyl-l-deoxynojirimycin as ligand. Sodium dodecyl sulfate gel eletrophoresis of the purified enzyme revealed a subunit molecular mass of about 85kDa. The molecular mass of the native enzyme, determined by gel chromatography, was&ap; 320– 350 kDa, pointing to the association of subunits to a tetramer. Glucosidase I is rather stable when stored at 4 °C in the presence of detergent (t½&ap; 20 days) and showed high specificity for the hydrolysis of the terminal (α: I,2)-linked glucose residue in the natural substrate Glc3-Man9-(GlcNAc)2. [ABSTRACT FROM AUTHOR]

1. Isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase were purified over 1000-fold from Escherichia coli ML308 by a procedure involving fractionation with (NH4)2SO4 and chromatography on DEAEcellulose, blue-dextran-Sepharose and Sephadex G150. 2. The kinase and phosphatase activities copurified, in agreement with the observation [Laporte, D. C. and Koshland, D. E. (1982) Nature (Lond.) 300, 458-460] that a single protein bears both activities. 3. Isocitrate dehydrogenase kinase catalysed the phosphorylation of homogeneous active isocitrate dehydrogenase with a stoichiometry of just under one phosphate group incorporated per subunit. This almost completely inactivated the dehydrogenase. There was a good correlation between phosphorylation and inactivation. 4. Analysis of a partial acid hydrolysate of phosphorylated isocitrate dehydrogenase showed that the only phosphoamino acid present was phosphoserine. 5. Isocitrate dehydrogenase phosphatase catalysed the release of 32P from 32P-phosphorylated isocitrate dehydrogenase; it required either ADP or ATP for activity. In the presence of ADP, or ATP plus an inhibitor of the kinase, the phosphatase catalysed full reactivation of isocitrate dehydrogenase and there was n good correlation between reactivation and the release of phosphate. In the presence of ATP alone the phosphatase catalysed the release of 32P from phosphorylated isocitrate dehydrogenase but the activity of the dehydrogenase remained low, indicating that the kinase and phosphatase were active simultaneously in these conditions. 6. The active and inactive forms of isocitrate dehydrogenase can be resolved by non-denaturing gel electrophoresis; the two forms of the enzyme were interconverted by phosphorylation and dephosphorylation in vitro. The extent of the interconversion correlated well with the changes in isocitrate dehydrogenase activity. [ABSTRACT FROM AUTHOR]

Protein phosphatase-2B was purified from extracts of rabbit skeletal muscle by a procedure that involved fractionation with ammonium sulphate, chromatography on DEAE-Sepharose, fractionation with poly(ethylene glycol), gel filtration on Sephadex 0-200 (Mr = 98000 ± 4000). chromatography on Affi-Gel Blue and affinity chromatography on calmodulin-Sepharose. The enzyme was purified 3500-fold in seven days with an overall yield of 0.5%. The α-subunit of phosphorylase kinase, protein phosphatase inhibitor-i and the myosin P-Light chain from rabbit skeletal muscle were dephosphorylated by protein phosphatase-2B with similar kinetic constants. The &alpha-subunit of phosphorylase kinase was dephosphorylated at least 100-fold more rapidly than the β-subunit, while glycogen phosphorylase, glycogen synthase, histones H1 and H2B. ATP-citrate lyase, acetyl-CoA carboxylase, L-pyruvate kinase and protein synthesis initiation factor elF-2 were not dephosphorylated at significant rates. Protein phosphatase-2B became activated 10-fold by calmodulin (A0.5 = 6 nM) after chromatography on DEAE-Sepharose and this degree of activation was maintained throughout the remainder of the purification. Calmodulin increased the Vmax of the reaction without altering the Km for inhibitor-I. The activity of protein phosphatase-2B was completely dependent on Ca2+ in the presence or absence of calmodulin. Half-maximal activation was observed at 1.0 μM Ca2 + in the absence, and at 0.5 μM Ca2+ in the presence, of 0.03 μM calmodulin. Protein phosphatase-2B was inhibited completely by trifluoperazine; half- maximal inhibition occurred at 45 μM in the absence and 35 μM in the presence of 0.03 μM calmodulin. The metabolic rote of protein phosphatase-2B in vivo is discussed in the light of the observation that this enzyme is probably identical to a major calmodulinabinding protein of neural tissue termed calcineurin or CaM-BP80 (Stewart, A. A., Ingebritsen, T. S., Manalan, A., Klee, C. B., and Cohen, P. (1982) FEBS Let!. 137, 80-84]. [ABSTRACT FROM AUTHOR]

Three rat α1-fetoprotein fractions were obtained by chromatography on concanavalin-A—Sepharose: one non-reactive, one weakly reactive and one reactive to concanavalin A. The non-reactive and reactive variants were found to vary in the structure of their carbohydrate chains while the conformation of the weakly reactive form may modulate the accessibility of these chains to the lectin. N-Glycosidically linked glycans from unfractionated α1-fetoprotein were isolated and chemically characterized. Particular attention was paid to develop sensitive methods based upon hydrazinolysis, quantitative re-N-acetylation of glycans with [14C]acetic anhydride and thin-layer chromatography of labeled compounds. With the aid of these methods two main kinds of glycans ( 1a and 2a) were obtained and fractionated on concanavalin-A—Sepharose into non-reactive (1a) and reactive (2a) molecules, Moreover it was demonstrated that each α1-fetoprotein variant contained either two glycans 1a or two glycans 2a, not randomly, but a pair of the identical carbohydrate chains at the two glycosylation sites. [ABSTRACT FROM AUTHOR]

3-Phosphoglycerate kinase is inactivated by 1-cyclohexyl-3-(2-morpholunoethyl)carbodiimide metho-p-toluenesulfonate and nitrotyrosine ethyl ester. The coupling of 1 mol nitrotyrosine/mol enzyme is sufficient to inactivate the protein completely. A weak protection against inactivation is observed with each substrate added separately. In contrast, the complex ATP—3-phosphoglycerate—enzyme of ATP—Mg—3-phosphoglycerate—enzyme affords a considerable protection. The critical residue is identified as a glutamyl residue after isolation by immuno-affinity chromatography of nitrotyrosyl peptide resulting from exhaustive proteolytic digestion of the modified protein. In addition, the determination of the primary sequence of the C-terminal part of the protein leads to the location of the glutamyl residue at position eight from the C-terminus. We conclude that this glutamyl residue is situated in the domain which does not bind the nucleotide substrates [Bryant, T.N., Watson, H. C. and Wendell, P. L. (1974) Nature (Lond.) 247, 14–17]. Its role in the catalysis process is discussed. [ABSTRACT FROM AUTHOR]

Four hexose-phosphorylating isozymes (hexokinases α, β, γ and δ) were separated by DEAE-cellulose column chromatography of high-speed supernatants from Neurospora crassa wild-type strains 77a and RL3-8A. The isozymes displayed broad sugar specificity and Michaelis constants for glucose in the micromolar range. The isozymes were not inhibited by glucose 6-phosphate nor activated by citrate. Molecular weights were estimated as 95000 by gel filtration. Glucose-specific kinases were not found. Marked quantitative variations in the relative proportion of the hexokinases were observed during growth of the fungus on minimal medium plus sucrose. Hexokinase β was the predominant form (82% of the total activity) during the period of hyphal growth. At maturation time all the isozymes were present at about the same level although a small preponderance of hexokinase β was still evident. The relative proportions of the hexokinases did not vary when sucrose was replaced by glucose, fructose or casamino acids. When sorbose was added to the minimal medium containing sucrose, a high proportion of hexokinase β was apparent concomitantly with the well known change of growth to a colonial mode. Several morphological mutant strains were examined with respect to the hexokinase profile. Strains col-16 and crisp showed a marked preponderance of hexokinase β. Extracts from the dapple and dingy strains lacked hexokinases α and δ. Rabbit antisera to commercial preparations of hexokinase PI and PII from Saccharomyces cerevisiae were able to inhibit partially the enzyme activity of Neurospora hexokinases γ and δ. No effect of the antisera on hexokinases α and β could be observed. [ABSTRACT FROM AUTHOR]