摘要:

Cell-free extracts of mixed rumen microorganisms were incubatedin vitroin order to determine the existence and nature of metabolic conversions leading from succinate to glutamate. When succinate was incubated as the substrate in the presence of reduced ferredoxin (FDH), a product was formed, which reacted with 2,4-dinitrophenylhydrazine to give the hydrazone of succinic semialdehyde as determined by paper chromatography. When succinic semialdehyde was incubated in the presence of FDH and14C-bicarbonate, fixation of label was catalyzed by the extracts. The labelled product was isolated as an organic acid and identified as 2-hydroxyglutaric acid by means of paper chromatography. In the presence of ammonia and NAD, 2-hydroxyglutarate was aminated to a ninhydrin-reactive compound that was identified as glutamate by paper chromatography.From the information obtained, a new pathway for the synthesis of glutamate in rumen microbes was proposed. This pathway entails the reduction of succinate to succinic semialdehyde, followed by reductive carboxylation of succinic semialdehyde to yield 2-hydroxyglutarate, which is then aminated to glutamate. The pathway would agree with the labelling pattern in glutamate produced in the presence of14C-bicarbonate by mixed rumen microorganisms.

ISSN:0008-4018    

DOI:10.1139/o72-001

出版商:NRC Research Press    

年代:1972

数据来源: NRC

摘要:

The cytoplasmic carboxylesterases of bovine, ovine, equine and human liver were fractionated by starch gel electrophoresis and by gel filtration on Sephadex. While species-specific, heterogeneous bands were observed in starch gel, the esterases of the bovine, ovine and equine liver were eluted from Sephadex G-100 as single peaks of activity, each with a characteristic elution volume. Gel filtration of human liver extracts yielded two peaks of activity, one containing electrophoretically slow esterases, the other electrophoretically fast esterases. Extracted equine and human hepatic carboxylesterases aggregated readily on storage or concentration, forming larger units which could be dissociated by a combination of acidic pH and high salt concentration. Molecular weight estimates of the hepatic esterases by gel filtration on Sephadex G-100 and G-200 yielded values of 65 000 for ovine, 55 000 for bovine, 96 000 and 70 000 for equine variants and 180 000 and 65 000 for human variants. The observations suggested that the cytoplasmic enzymes in relatively crude hepatic extracts had a lower molecular weight than those in concentrated or partially purified preparations which formed stable dimers or trimers.

ISSN:0008-4018    

DOI:10.1139/o72-002

出版商:NRC Research Press    

年代:1972

数据来源: NRC

摘要:

The incorporation of various labelled precursors intoE.colilipids was studied. In growing cells, incorporation of glycerol, doubly labelled with14C and3H, occurs without a change in isotope ratio. In cell homogenates incorporation ofsn-glycero-3-phosphate-U-14C was greater in the polyglycerophosphatide fraction than in phosphatidylethanolamine and higher in the unacylated than in the acylated glycerol moiety of phosphatidylglycerol. This uneven distribution of label in phosphatidylglycerol was not affected by addition of unlabelled dihydroxyacetone phosphate and occurred to the same extent when glucose-3,4-14C was the precursor. Our results obtainedin vivoindicate that glycerophosphate is acylated without prior oxidation. Furthermore, labelling patterns obtainedin vitrosuggest an extensive dilution by endogenous diacyl glycerol precursors not formed by acylation of dihydroxyacetone phosphate.

ISSN:0008-4018    

DOI:10.1139/o72-003

出版商:NRC Research Press    

年代:1972

数据来源: NRC

摘要:

Increases in the levels of four lysosomal enzymes were measured during the induction of tyrosine transaminase in rat liver by cortisone acetate. Tyrosine transaminase showed a maximum specific activity 2 h after the injection of the steroid hormone whereas lysosomal enzyme levels reached a maximum specific activity at 4 h. The maximum increase in specific activity for 15 injected animals compared to 15 controls was 100% for tyrosine transaminase; 40% for cathepsin A, cathepsin D, and β-N-acetylglucosaminidase; and 10% for acid phosphatase. Increased specific activities from livers of cortisone-acetate-treated rats were observed when lysosomal enzymes were released both by detergent treatment and by freezing and thawing.The increased specific activities were found in the readily solubilized lysosomal enzyme fractions and not in those lysosomal enzyme fractions which remain associated with particulate matter after lysosomal disruption. Similar increased specific activities for acid phosphatase and β-N-acetylglucosaminidase were observed in cultures of Morris hepatoma cells from rat liver when incubated with cortisone acetatein vitro. Thus the response appears to be typical of single cell types.

ISSN:0008-4018    

DOI:10.1139/o72-004

出版商:NRC Research Press    

年代:1972

数据来源: NRC

摘要:

Lymphocytes from the mesenteric lymph nodes of pigs were cultured for 61 h with and without phytohemagglutinin (PHA). Every 12 h the activity of PC-cytidyl transferase (CTP: cholinephosphate cytidylyltransferase, EC 2.7.7.15) and PC-glyceride transferase (CDPcholine: 1,2-diglyceride cholinephosphotransferase, EC 2.7.8.2) was correlated with the incorporation of Me-14C-choline into total lipid. Maximum labelling of total lipid (of the label was incorporated into lecithin) occurred between 37 and 48 h of culture. The activity of PC-cytidyl transferase was stimulated up to 10-fold at 37 h while the activity of PC-glyceride transferase exhibited a maximal sixfold increase at 48 h. Addition of actinomycin D (0.10 μg/ml) or cycloheximide (1.0 μg/ml) inhibited simultaneously the induction of the activity of both enzymes and the increased incorporation of14C-choline into total lipid. It is postulated that the accelerated production of lecithin in PHA-stimulated lymphocytes is dependent on the increased synthesis and/or activity of both these enzymes.

ISSN:0008-4018    

DOI:10.1139/o72-005

出版商:NRC Research Press    

年代:1972

数据来源: NRC

摘要:

Increased serum Cholinesterase activity was observed in hyperlipidemic patients. When hyperlipidemia was induced in rabbits by injecting the lipopolysaccharide ofEscherichia coli, a significant rise in serum low density lipoproteins and Cholinesterase activity occurred.In vitroexperiments demonstrated that isoniazid produced proportionate decreases in serum low density lipoprotein concentration and in serum Cholinesterase activity.

ISSN:0008-4018    

DOI:10.1139/o72-006

出版商:NRC Research Press    

年代:1972

数据来源: NRC

摘要:

Isozymes of alcohol dehydrogenase (ADH) from yeast were separated by discontinuous electrophoresis. Three major bands were observed: ADH-I, ADH-II, and a new band designated mito-ADH. Work with isolated mitochondria showed that the mito-ADH band could be produced from physiologically competent mitochondria by mechanical disruption or by treatment with detergents.The isozyme pattern was determined for the various physiological states of yeast growing anaerobically and aerobically on 2% glucose and aerobically on 2% ethanol, and for catabolite de-repressed cells undergoing an anaerobic → aerobic transition in continuous culture.The changing isozyme pattern substantiates the thesis that ADH-I is produced constitutively and that ADH-II is regulated via catabolite repression. It appears from the data that mito-ADH is more closely related to mitochondriogenesis than is ADH-II.

ISSN:0008-4018    

DOI:10.1139/o72-007

出版商:NRC Research Press    

年代:1972

数据来源: NRC

摘要:

Commercially available horseradish peroxidase (RZ3.1) was characterized with respect to its homogeneity by (1) chromatography on CM-cellulose, (2) disc gel electrophoresis in alkaline and acidic buffers, (3) micro-scale sucrose gradient isoelectrofocusing in pH 3–10 and pH 8–10 gradients, (4) gel isoelectrofocusing in pH 3–10 and pH 8–10 gradients, and (5) amino acid and hexosamine analyses. The preparation was found to be highly homogeneous except by pH 8–10 gel isoelectrofocusing which resolved it into several very close bands. This heterogeneity has been assumed to reflect differences in carbohydrate composition rather than in the amino acid sequence or composition. Amino acid analyses after performic acid oxidation yielded eight cysteic acid residues per mole of enzyme. Since noS-carboxymethylcysteine was recovered after treatment of the protein in 8 Murea with iodoacetic acid, it was concluded that the enzyme has four disulfide bridges. Peptides resulting from a tryptic digest of the heme-free enzyme were purified by high-voltage paper electrophoresis and subjected to sequence analysis. Several half-cystine sequences were elucidated after isolation of the radioactive peptides from a tryptic digest of the reduced and14C-S-carboxymethylated protein. The complete sequences of 21 and partial sequences of three tryptic peptides were determined. These account for 203 of the approximately 300 amino acid residues of this protein. Several sites of carbohydrate attachment were observed.

ISSN:0008-4018    

DOI:10.1139/o72-008

出版商:NRC Research Press    

年代:1972

数据来源: NRC

摘要:

Horseradish peroxidase (HRP) was digested with thermolysin. On fractionation on Sephadex G-25, Fine Chromobeads type P (Dowex 50 type resin) and by high-voltage paper electrophoresis, we isolated about 120 thermolytic peptides. Some experimentation on the composition of the pyridine acetate gradient, used for elution of the cation exchanger, is reported. All peptides were characterized with respect to amino acid composition, N-terminal residue, and pH 6.5 mobility. Unknown peptides or peptides not corresponding unambiguously to previously established tryptic sequences were subjected to dansyl-Edman analysis. Thermolytic peptides accounting for all tryptic sequences except a dipeptide and a tripeptide, and unique thermolytic sequences accounting for about 100 amino acid residues, were obtained. Nine convincing and several indicative overlaps were established for known tryptic sequences. The sequences around all four disulfide bridges, the three histidine residues, and the only tryptophan residue have been elucidated. Eight sites of carbohydrate attachment have been identified. For seven of these sites we have evidence for attachment to asparagine, and for six of the sites the carbohydrate-bound asparagine was found in the well-known sequences Asn–X–Ser/Thr. The remaining two sequences, though incomplete, are compatible with this pattern. Tentatively we suggest a pyrrolidone carboxyl N-terminal for HRP. The specificity of trypsin implicates a sequence found in two varieties, differing only by a C-terminal serine residue at the C-terminus of HRP. A discussion of the possible complications of the acidic heme extraction on the results obtained is included.

ISSN:0008-4018    

DOI:10.1139/o72-009

出版商:NRC Research Press    

年代:1972

数据来源: NRC

摘要:

After subcutaneous injection of [U-14C]glucose into rats the amount of14C incorporatedin vivointo proteins was always higher than into lipids in brain, liver, and heart. The specific radioactivity of brain proteins was higher than those of liver and heart. Blood-brain comparisons show that protein carbon is derived continuously from glucose in the brainin situand not as a result of deposition of amino acids or proteins from the circulation. Seventy-two percent of14C in purified brain protein fractions was found in the amino acids of the hydrolysates of these fractions, mainly in alanine, glutamic, and aspartic acids. Maximum labelling was reached about 4 h after injection of [U-14C]glucose. Elimination of14C from three classes of brain proteins (high-speed supernatant, particulate deoxycholate extractable, and residual) followed a biphasic time-course. The extent of labelling of, and the rate of elimination of14C from, the three classes of rat brain proteins were very similar. The fate of14C in the other investigated tissue fractions of brain, liver, and heart was compared with the fate of14C in brain proteins.The results lend further support to the previously published suggestion that: (a) brain does not contain appreciable amounts of metabolically inert proteins or of proteins with turnover rates significantly higher than the mean for the bulk of brain proteins; (b) glucose carbon participates at a different rate and to a different extent in the metabolism of high-molecular-weight constituents of brain as compared to liver, heart, and plasma proteins; (c) the continuous conversion of glucose carbon into protein is an important part of the maintenance of the homeostasis of tissue proteinsin vivo.

ISSN:0008-4018    

DOI:10.1139/o72-010

出版商:NRC Research Press    

年代:1972

数据来源: NRC