scholarly journals Interaction of soluble glucosyl- and mannosyl-transferase enzyme activities in the synthesis of a glucomannan

1972 ◽  
Vol 129 (3) ◽  
pp. 645-655 ◽  
Author(s):  
J. S. Heller ◽  
C. L. Villemez
Keyword(s):  
Enzyme Activity ◽  
Enzyme Activities ◽  
Enzyme Preparation ◽  
The Other ◽  
Acceptor Molecule ◽  
Soluble Enzyme ◽  
Phaseolus Aureus ◽  
Polymeric Product ◽  
Sugar Nucleotide ◽  
Solubilized Enzyme

A neutral-detergent-solubilized-enzyme preparation derived from Phaseolus aureus hypocotyls contains two types of glycosyltransferase activity. One, mannosyltransferase enzyme activity, utilizes GDP-α-d-mannose as the sugar nucleotide substrate. The other, glucosyltransferase enzyme activity, utilizes GDP-α-d-glucose as the sugar nucleotide substrate. The soluble enzyme preparation catalyses the formation of what appears to be a homopolysaccharide when either sugar nucleotide is the only substrate present. A β-(1→4)-linked mannan is the only polymeric product when only GDP-α-d-mannose is added. A β-(1→4)-linked glucan is the only polymeric product when only GDP-α-d-glucose is added. In the presence of both sugar nucleotides, however, a β-(1→4)-linked glucomannan is formed. There are indications that endogenous sugar donors may be present in the enzyme preparation. There appear to be only two glycosyltransferases in the enzyme preparation, each catalysing the transfer of a different sugar to the same type of acceptor molecule. The glucosyltransferase requires the continual production of mannose-containing acceptor molecules for maintenance of enzyme activity, and is thereby dependent upon the activity of the mannosyltransferase. The mannosyltransferase, on the other hand, does not require the continual production of glucose-containing acceptors for maintenance of enzyme activity, but is severely inhibited by GDP-α-P-glucose. These properties promote the synthesis of β-(1→4)-linked glucomannan rather than β-(1→4)-linked glucan plus β-(1→4)-linked mannan when both sugar nucleotide substrates are present.

scholarly journals Solubilization of a mannose-polymerizing enzyme from Phaseolus aureus

1972 ◽  
Vol 128 (2) ◽  
pp. 243-252 ◽  
Author(s):  
J. S. Heller ◽  
C. L. Villemez
Keyword(s):  
Enzyme Activity ◽  
Enzyme Preparation ◽  
Specific Activity ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Soluble Enzyme ◽  
Phaseolus Aureus ◽  
Original Activity ◽  
Solubilized Enzyme ◽  
High Concentrations

A soluble enzyme preparation, which catalyses the polymerization of mannose, was obtained by Triton X-100 extraction of a particulate fraction derived from Phaseolus aureus hypocotyls. The product that resulted when GDP-α-d-mannose was used as a substrate was a β-(1→4)-linked mannan, about three-quarters of which was alkali-insoluble. The mannose-polymerizing enzyme activity was at least as great in the soluble preparation as in the particulate preparation, and the specific activity of the solubilized enzyme was greater by a factor of at least 3.5. Kinetic studies of the soluble enzyme indicate that the apparent Km is 55–62μm, and a disproportionate increase in rate is observed at high concentrations. GDP-α-d-glucose is a strong competitive inhibitor of the mannose-polymerizing reaction, with an apparent Ki of 6.2μm. The soluble enzyme is relatively unstable, losing about two-thirds of its original activity in 5h at 0°C or in 24h at −20°C. A solvent (acetone, butanol, diethyl ether)-extracted particulate preparation, which also exhibits the same enzyme activity, is more stable, retaining full activity for at least 5 days at −20°C. There was no polymerizing-enzyme activity in the soluble enzyme preparation when UDP-d-glucose, UDP-d-galactose, UDP-d-xylose, UDP-l-arabinose or UDP-d-glucuronic acid were used as substrates. However, the soluble enzyme preparation would catalyse the polymerization of glucose, with GDP-d-glucose as substrate.

scholarly journals Purification, properties and comparative specificities of the enzyme prolyl-transfer ribonucleic acid synthetase from Phaseolus aureus and Polygonatum multiflorum

1965 ◽  
Vol 97 (1) ◽  
pp. 112-124 ◽  
Author(s):  
PJ Peterson ◽  
L Fowden
Keyword(s):  
Carboxylic Acid ◽  
Enzyme Preparation ◽  
Enzymic Activity ◽  
The Other ◽  
Asparagus Officinalis ◽  
Acid Activation ◽  
Phaseolus Aureus ◽  
Substrate Specificities ◽  
Imino Acids ◽  
Ammonium Sulphate Fractionation

1. A prolyl-s-RNA synthetase (prolyl-transfer RNA synthetase) has been purified about 250-fold from seed of Phaseolus aureus (mung bean), a species not producing azetidine-2-carboxylic acid, and more than 10-fold from rhizome apices of Polygonatum multiflorum, a liliaceous species containing azetidine-2-carboxylic acid. The latter enzyme was unstable during ammonium sulphate fractionation. 2. The enzymes exhibited different substrate specificities towards the analogue. That from Phaseolus, when assayed by the ATP-PP(i) exchange, showed azetidine-2-carboxylic acid activation at about one-third the rate with proline. Both labelled imino acids gave rise to a labelled aminoacyl-s-RNA. The enzyme from Polygonatum, however, activated only proline. 3. The enzyme from Polygonatum also formed a labelled prolyl-s-RNA with Phaseolus s-RNA but at a lower rate than when the Phaseolus enzyme was used. No reaction occurred when the Phaseolus enzyme was coupled with Polygonatum s-RNA, and only a very slight one was observed when both enzyme and s-RNA came from Polygonatum. 4. Protein preparations from seeds of Pisum sativum, another species not producing azetidine-2-carboxylic acid, also activated the analogue in addition to proline, whereas those from rhizome and seeds of Convallaria, the species from which the analogue was originally isolated, failed to activate it. However, a liliaceous species not producing the analogue, Asparagus officinalis, activated it. 5. Of the other proline analogues investigated, only 3,4-dehydro-dl-proline and l-thiazolidine-4-carboxylic acid were active with the enzyme preparation from Phaseolus. 6. pH optima of 7.9 and 8.4 were established for the enzymes from Phaseolus and Polygonatum respectively. 7. The Phaseolus enzyme was specific for ATP and PP(i). Mn(2+) partially replaced the requirement for Mg(2+) as cofactor. Preincubation with p-chloromercuribenzoate at a concentration of 0.5mm or higher produced over 99% inhibition of the Phaseolus enzyme. One-half the enzymic activity was destroyed by preheating for 5min. at 62 degrees in tris-hydrochloric acid buffer, pH7.9. 8. All experimental evidence supports the hypothesis that azetidine-2-carboxylic acid and proline are activated by the same enzyme in Phaseolus preparations, whereas the analogue was inactive in all Polygonatum preparations. The possible nature of this different substrate behaviour is discussed.

CHARACTERIZATION OF HUMAN OVARIAN OESTRADIOL-17β OXIDOREDUCTASE ACTIVITY

1977 ◽  
Vol 85 (3) ◽  
pp. 624-635 ◽  
Author(s):  
Donald E. Pittaway ◽  
Richard N. Andersen ◽  
James R. Givens
Keyword(s):  
Enzyme Activity ◽  
Enzyme Activities ◽  
Enzyme Preparation ◽  
Phenolic Hydroxyl ◽  
Supernatant Fraction ◽  
Oxidoreductase Activity ◽  
Sulfhydryl Reagent ◽  
Michaelis Constants ◽  
Human Ovaries

ABSTRACT Oestradiol-17β oxidoreductase activity, which catalyzes the interconversion of oestrone and oestradiol, was investigated in preparations of human ovaries. The enzyme activities were localized primarily in the 105 000 × g supernatant fraction; dialyzed supernatant preparations were used in subsequent studies. The pH optima were 6.9 for reduction and 8.1 for 17β-dehydrogenation. The apparent Michaelis constants for oestrone and oestradiol were 1 × 10-7 m and 5 × 10-7 m, respectively. The enzyme activity was present with either NADP(H) or NAD(H), though NADP(H) were the preferred cofactors. Non-aromatic steroids androstenedione, dehydroepiandrosterone, testosterone and 5-androstene-3β,17β-diol were poor substrates for the enzyme preparation. Methylation of the phenolic hydroxyl of oestrone and oestradiol resulted in slightly enhanced activities. The sulfhydryl reagent, N-ethylmaleimide, inhibited the reduction of oestrone. A dialyzed supernatant preparation retained approximately 79 % of the original enzyme activity when stored at −20°C for 6 weeks.

scholarly journals The effect of tsushimycin on the synthesis of lipid-linked saccharides in aorta

1981 ◽  
Vol 193 (2) ◽  
pp. 477-484 ◽  
Author(s):  
A D Elbein
Keyword(s):  
Enzyme Preparation ◽  
The Other ◽  
Dolichyl Phosphate ◽  
Other Hand ◽  
Solubilized Enzyme ◽  
Antibiotic Inhibition

The antibiotic, tsushimycin, inhibits the formation of dolichyl phosphate mannose, dolichyl phosphate glucose and dolichyl pyrophosphate N-acetylglucosamine in the particulate enzyme preparation from pig aorta. Although this antibiotic also inhibits the incorporation of mannose and glucose into lipid-linked oligosaccharides, these reactions are less sensitive to antibiotic than those involved in the synthesis of lipid-linked monosaccharides. In the presence of tsushimycin, most of the mannose incorporated into lipid-linked oligosaccharides is into one oligosaccharide that has the properties of the heptasaccharide Man5GlcNAc2, whereas in the absence of antibiotic most of the mannose is in larger-sized oligosaccharides. On the other hand, the glucose-labelled lipid-linked oligosaccharides appear to be similar in size in the presence or absence of antibiotic. Tsushimycin also inhibits the formation of lipid-linked monosaccharides by the solubilized enzyme preparation of aorta. Various concentrations of dolichyl phosphate or the detergent, Nonidet P40, had no effect on antibiotic inhibition. Some evidence indicates that tsushimycin binds to the particulate enzyme.

Enzymatic sulfation of steroids. XIX. Cortisol sulfotransferase activity, glucocorticoid sulfotransferases, and tyrosine aminotransferase induction in chicken, gerbil, and and hamster liver

1985 ◽  
Vol 63 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Sanford S. Singer ◽  
Edward G. Galaska ◽  
Theresa A. Feeser ◽  
Robert L. Benak ◽  
Anne Z. Ansel ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Sex Differences ◽  
Sexual Dimorphism ◽  
Enzyme Activities ◽  
Tyrosine Aminotransferase ◽  
The Other ◽  
Chicken Liver ◽  
Molecular Weights ◽  
Low K

Radioisotopic, pH 6.8 assays were designed to measure hepatic cortisol sulfation in chickens, gerbils, and hamsters of both sexes. Enzyme levels with 40 μM cortisol were similar in males of all three species and due mostly to low Km enzymes with 10–30 μM cortisol Km's. Maximum enzyme activity in male chickens required 40 μM Cortisol. In the other species, the much higher maximum enzyme activity observed required 500 μM cortisol owing to sulfotransferases with Km's for the hormone near 300 μM. Coenzyme 3′-phosphoadenosine-5′-phosphosulfate requirements also varied between species. Sex differences of the enzyme levels were found only in hamsters. There, males possessed only 24–33% of the enzyme levels found in females. Cortisol 21-sulfate was the reaction product in all of the species. Sexual dimorphism in hamsters appeared to be due to repressive effects of androgens. pH optima of enzyme activities in the three species ranged from pH 6 to 7. Routine use of pH 6.8 assays allowed representative interspecies comparisons. DEAE-Sephadex fractionation of cytosol showed that chicken liver contained mostly two enzymes with different pH optima that catalyzed cortisol sulfation. These differed from the enzymes that catalyzed dehydroepiandrosterone and estradiol sulfation. In the gerbil four enzymes with similar pH optima catalyzed cortisol sulfation. The second of these to elute from DEAE-Sephadex columns was the low Km form. In hamsters most glucocorticoid sulfotransferase activity appeared to be due to one enzyme. The molecular weights of the low Km gerbil enzyme and the main hamster enzyme were 98 300 ± 6100 and 105 000 ± 8100. Hamsters and gerbils responded to injection of cortisol by hepatic tyrosine aminotransferase induction.

scholarly journals The formation of a β-(1→4)-d-galactan chain catalysed by a Phaseolus aureus enzyme

1973 ◽  
Vol 133 (2) ◽  
pp. 263-271 ◽  
Author(s):  
N. Panayotatos ◽  
C. L. Villemez
Keyword(s):  
Enzyme Preparation ◽  
Insoluble Fraction ◽  
Labelling Pattern ◽  
Phaseolus Aureus ◽  
Trace Quantity ◽  
Sugar Nucleotide ◽  
Polysaccharide Chain ◽  
Soluble Polysaccharide ◽  
Hydrolysis Of ◽  
Derivatives Of

With a particulate enzyme preparation from Phaseolus aureus hypocotyls, UDP-α-d-[U-14C]galactose served as a precursor for a number of products. One of these products was characterized as a β-(1→4)-linked galactan. The ADP-, GDP-, TDP- and CDP- derivatives of α-d-galactose did not serve as biosynthetic precursors for any products insoluble in 70% ethanol, nor as substrates for a sugar nucleotide 4-epimerase which is present in the particulate enzyme preparation. The 14C-labelled β-(1→4)-galactan is alkali-insoluble and was characterized by analysis of partial acetolysis products. The labelling pattern of the [14C]oligosaccharides derived from acetolysis indicates that (1) only slightly more than two [14C]galactose moieties are added to the growing polysaccharide chain on average, and (2) these additions take place at the reducing end of the polysaccharide chain. The radioactive β-(1→4)-linked galactan chain represented 8.5% of the radioactivity initially added, and 20% of the water- and butanol-insoluble products derived from UDP-α-d-[14C]galactose. Total hydrolysis of the alkali-insoluble fraction of Phaseolus aureus hypocotyl yielded d-glucose and d-mannose in a 5:1 ratio but no detectable quantities of d-galactose. A trace quantity of a radioactive disaccharide, identified as (1→3)-linked galactobiose, was isolated from the partial acetolysate of the alkali-insoluble [14C]polysaccharide material. Also isolated from this partial acetolysate was a C-1 derivative of [14C]galactose, which could not be identified. An alkali-soluble galactose-containing polysaccharide was also synthesized in this enzymic reaction, and represented 20% of the water- and butanol-insoluble products derived from UDP-α-d-[14C]galactose. The spectrum of radioactive oligosaccharides produced by partial acetolysis of this alkali-soluble polysaccharide material was different from that obtained from the alkali-insoluble polysaccharide, indicating a different structure.

scholarly journals Purification and subunit structure of argininosuccinate lyase from Chlamydomonas reinhardi

1977 ◽  
Vol 167 (1) ◽  
pp. 71-75 ◽  
Author(s):  
R F Matagne ◽  
J P Schlösser
Keyword(s):  
Enzyme Activity ◽  
Crude Extract ◽  
Enzyme Preparation ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Activity Analysis ◽  
Disc Electrophoresis ◽  
Subunit Structure ◽  
Argininosuccinate Lyase

Argininosuccinate lyase (EC 4.3.2.1) was purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The final enzyme preparation was purified 46-fold compared with the crude extract. Electrophoresis of this preparation revealed three bands, the major one having the enzyme activity. Analysis of the enzyme by gel filtration and by disc electrophoresis (in two different concentrations of acrylamide) gave mol.wts. of 200000 (+/- 15000) and 190000 (+/- 20000) respectively. Treatment with sodium dodecyl sulphate and mercaptoethanol dissociated the enzyme into subunits of mol.wt. 39000 (+/-2000). The results are indicative of the multimeric structure of the enzyme, which is composed of five (perhaps four or six) identical subunits.

scholarly journals Rat uterine microbiochemistry: metabolic enzyme activities stimulated by 17-beta-estradiol are localized in epithelial cells.

1987 ◽  
Vol 35 (6) ◽  
pp. 657-662 ◽  
Author(s):  
J P Holt ◽  
E Rhe
Keyword(s):  
Enzyme Activity ◽  
Smooth Muscle ◽  
Epithelial Cells ◽  
Dose Response ◽  
Enzyme Activities ◽  
Time Course ◽  
Citrate Synthase ◽  
Ovariectomized Rats ◽  
Similar Response ◽  
Estradiol Treatment

Lactate dehydrogenase (LDH; EC 1.1.1.27), citrate synthase (CS; EC 4.1.3.7), and beta-hydroxyacyl-CoA-dehydrogenase (beta-OH-acyl-CoA-DH; EC 1.1.1.35) activities were determined in each of the three major cell types of rat uterus, i.e., epithelial, stromal, and smooth muscle, using quantitative microanalytical techniques. Adult ovariectomized rats were treated with 17-beta-estradiol to determine the time course and dose response (0.025-50 micrograms/300-g rat) effect of estrogen on enzyme activity of each type of uterine cell. The use of "oil well" and enzyme-cycling microtechniques to determine the time course and the dose responses of enzyme activity changes required microassays involving 1595 microdissected single cell specimens. Estradiol treatment increased epithelial LDH, CS and beta-OH-acyl-CoA-DH activity but had no effect on these enzymes in the stroma or in smooth muscle cells. The estradiol-stimulated peak enzyme activities on Day 4 in the intervention group are compared with those in the ovariectomized rat controls as follows: LDH, 44.5 +/- 3.5 vs 22.3 +/- 3.9; CS, 3.5 +/- 0.2 vs 1.5 +/- 0.6; beta-OH-acyl-CoA-H, 3.5 +/- 0.32 vs 2.2 +/- 0.2 (mean +/- standard deviation; mol/kg/hr). Stromal cell activities (LDH, 7.4 +/- 1.0; CS, 1.2 +/- 0.2; beta-OH-acyl-CoA-DH, 0.9 +/- 0.1) were significantly lower than epithelial cell levels and were similar to smooth muscle levels. Therefore, even in the ovariectomized animal epithelial cells have markedly higher metabolic activity compared with adjacent cells. The enzyme activities are expressed as moles of substrate reacting per kilogram of dry weight per hour. All three enzymes exhibited a 17-beta-estradiol-induced dose response between 0.025-0.15 micrograms/300-g rat. The three enzymes studied all had similar response patterns to estrogen. The effect of estradiol was restricted to epithelial cells, with enzyme activities increasing to maximal levels after approximately 96 hr of hormone treatment. This study therefore not only confirms the specific and differential metabolic responses of uterine cells to estradiol treatment, but clearly demonstrates that marked metabolic differences exist between epithelial cells and stromal or smooth muscle uterine cells.

Change Analysis of Enzyme Activity under Different Conditions of Film Remnant in Soil

2012 ◽  
Vol 518-523 ◽  
pp. 39-43
Author(s):  
Xiao Guang Zhao ◽  
Yuan Yuan Guan ◽  
Wen Yu Huang
Keyword(s):  
Enzyme Activity ◽  
Enzyme Activities ◽  
Scientific Basis ◽  
Soil Enzyme ◽  
Soil Microorganism ◽  
Soil Enzyme Activities ◽  
Change Analysis ◽  
Soil Material ◽  
The Relationship

In this paper, simulated experiments were performed in pots by using soil materials in different conditions of film remnant. Based on the research on soil microorganism quantity trends of soil enzyme activities were analyzed systematically: soil without film remnant, soil with film remnant for 5, 10, 15 and 20 years. By analyzing crop progress, the relationship with soil material was studied, in order to provide scientific basis for the variation laws between different conditions of film remnant and the activity of soil enzyme.

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