A Comparative Study of the Distribution of Soluble and Particulate Glycyl-l-Leucine Hydrolase in the Small Intestine

1974 ◽  
Vol 46 (4) ◽  
pp. 501-510
Author(s):  
Manjusri Das ◽  
A. N. Radhakrishnan
Keyword(s):  
Enzyme Activity ◽  
Guinea Pig ◽  
Comparative Study ◽  
Specific Activity ◽  
Maximal Activity ◽  
Total Activity ◽  
Soluble Enzyme ◽  
Km Value ◽  
Specific Activities ◽  
Very High

1. A comparative study has been made of glycyl-l-leucine hydrolase activity in the soluble and particulate fractions of intestinal mucosa from monkey, guinea-pig, rabbit and rat. The specific activity of the soluble enzyme is very high in monkey and guinea-pig, and lower in rabbit and rat. The particulate enzymes from all the four species show low specific activities and form 1–10% of the total activity. 2. The pH optima in all cases lie in the range 7·6–7·8. The Km values of the substrate were similar for both soluble and particulate enzyme from monkey and guinea-pig, but in the rabbit and rat the Km value with the particulate enzyme was higher than with the soluble enzyme. 3. The particulate enzyme activity in all cases was the highest in the distal regions of the intestine, whereas the soluble enzyme showed maximal activity in the proximal and middle regions.

Isolation and characterization of electrophoretic variants of human prostatic acid phosphatase.

1983 ◽  
Vol 29 (11) ◽  
pp. 1886-1889 ◽  
Author(s):  
M D Hibbard ◽  
R C McCarthy ◽  
H Markowitz
Keyword(s):  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Specific Activity ◽  
Prostatic Acid Phosphatase ◽  
Ph Gradient ◽  
Electrophoretic Variants ◽  
Isolation And Characterization ◽  
Immunochemical Determination ◽  
Km Value ◽  
Specific Activities

Abstract Prostatic acid phosphatase (EC 3.1.3.2) purified from benign hypertrophic prostate tissue was fractionated by preparative slab isoelectric focusing over a pH gradient of 3.16 to 7.16. Twenty-two of 29 fractions contained enzyme activity. We further examined each active fraction by determining the Michaelis-Menten constant and specific activity. The protein concentration used in the latter determination was estimated either spectrophotometrically or immunochemically by three different radioimmunoassays for the enzyme. Determination of specific activities for each fraction directly correlated enzyme activity with an immunochemical determination, which indicated the immunochemical relationships among different molecular species of the enzyme. We found that the Michaelis-Menten constants for the isolated fractions were similar to the Km value for purified, unfractionated enzyme. Most fractions analyzed by each immunoassay had similar specific activities; the few fractions with discrepant specific activities were found at either end of the pH gradient. The similarity in specific activities among the fractions indicates that RIAs involving polyclonal antisera detect all of the electrophoretic variants of the enzyme.

Estrogen sulfotransferase distribution in tissues of mouse and guinea pig: steroidal inhibition of the guinea pig enzyme

1992 ◽  
Vol 70 (8) ◽  
pp. 712-715 ◽  
Author(s):  
Ronald Hobkirk ◽  
Mary Ann Glasier
Keyword(s):  
Guinea Pig ◽  
Enzyme Inhibition ◽  
Specific Activity ◽  
Total Activity ◽  
Fetal Membrane ◽  
Estrogen Sulfotransferase ◽  
Structural Specificity ◽  
Specific Activities

The highest total activity of estrogen sulfotransferase in guinea pig is in liver and the highest specific activities are in the adrenal and the midgestational chorion. Guinea pig gonads contain scarcely detectable activities. In CD-1 mice the highest specific activity is in testis and the highest total activity is in late placenta. Adrenals from both sexes and ovaries contain minimal activities, while liver and fetal membrane activities are remarkably low. In CD-1, DBA, C57BL, and BALB mice, qualitative patterns are similar. Purified or partially purified estrogen sulfotransferase from guinea pig adrenal and chorion were used to study the effect of a number of possible steroidal inhibitors. Considerable structural specificity is evident within a range of steroids, even among some which are not substrates. Pregnenolone is the most effective 21-carbon inhibitor and, in general, more highly hydroxylated forms are less inhibitory. Within a series of 21-, 19- and 18-carbon steroids, the structure of the A ring appears to be extremely important in regard to inhibitory effects.Key words: estrogen sulfotransferase, mouse, guinea pig, enzyme inhibition, steroids.

5α-Reductase activity in stroma and epithelium of rat prostate and epididymis. A contribution to elucidation of the mechanism for development of hyperplastic growth of prostatic tissue

1983 ◽  
Vol 103 (2) ◽  
pp. 273-281 ◽  
Author(s):  
Ole Djøseland ◽  
Nicholas Bruchovsky ◽  
Paul S. Rennie ◽  
Navdeep Otal ◽  
Sian Høglo
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Reductase Activity ◽  
Major Change ◽  
Growth Stimulation ◽  
Total Activity ◽  
Prostatic Tissue ◽  
Ventral Prostate ◽  
Total Enzyme Activity ◽  
Abnormal Growth

Abstract. The 5α-reductase activity was assayed in homogenates of stroma and epithelium in the rat ventral prostate and epididymis. Samples consisting of 0.3 mg/ml tissue protein in TES buffer, pH 7.0 were incubated at 37°C for 30 min in the presence of 50 nm [1,2-3H]testosterone and a NADPH-generating system started with 5 × 10−4 m NADP. The yield of 5α-reduced metabolites, as established by using thin-layer chromatography, gave an estimate of enzyme activity. Whereas the specific activity of 5α-reductase was highest in prostatic stroma and epididymal epithelium, most of the total enzyme activity was associated with the epithelium in both the prostate and epididymis. The effect of dihydrotestosterone on specific activity of 5α-reductase was studied by administering the hormone to 7-day castrated rats. In prostate, the specific activity of both stromal and epithelium forms of the enzyme reached a maximum after 4 days of treatment. In epididymis only the epithelial form of 5α-reductase underwent a major change in specific activity, the latter peaking after 8–12 days of treatment. Furthermore, while the total activity of 5α-reductase in the prostatic tissue fractions could be induced by as much as 4-fold the normal control values, the epididymal enzyme could not be induced above the normal level either in the stroma or the epithelium. This may explain the relative resistance of epididymis to abnormal growth stimulation under the influence of hormones.

scholarly journals Comparative study on glucocerebrosidase in spleens from patients with Gaucher disease

1990 ◽  
Vol 269 (1) ◽  
pp. 93-100 ◽  
Author(s):  
J M F G Aerts ◽  
W E Donker-Koopman ◽  
S Brul ◽  
S Van Weely ◽  
M C Sa Miranda ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Comparative Study ◽  
Molecular Mass ◽  
Gaucher Disease ◽  
Specific Activity ◽  
Normal Subjects ◽  
Accurate Assessment ◽  
Residual Enzyme Activity ◽  
Immunological Analysis ◽  
Immunological Method

In Gaucher disease (glucosylceramide lipidosis), deficiency of glucocerebrosidase causes pathological storage of glucosylceramide, particularly in the spleen. A comparative biochemical and immunological analysis has therefore been made of glucocerebrosidase in spleens from normal subjects (n = 4) and from Gaucher disease patients with non-neuronopathic (n = 5) and neuronopathic (n = 5) phenotypes. The spleens from all Gaucher disease patients showed markedly decreased glucocerebrosidase activity. Discrimination of different phenotypes of Gaucher disease was not possible on the basis of the level of residual enzyme activity, or by measurements, using the immunopurified enzyme, of kinetic constants, pI or molecular mass forms. A severe decrease was found in the specific activity of glucocerebrosidase purified to homogeneity from the spleen of a patient with the non-neuronopathic phenotype of Gaucher disease, as compared with that of the enzyme purified from the spleen of a normal subject. This finding was confirmed by an immunological method developed for accurate assessment of the relative enzyme activity per molecule of glucocerebrosidase protein. The method revealed that the residual enzyme in the spleens of all investigated patients with a non-neuronopathic course of Gaucher disease had a more than 7-fold decreased activity of glucocerebrosidase (measured in the presence of taurocholate) per molecule of enzyme, and that the concentration of glucocerebrosidase molecules in the spleens of these patients was near normal. Observations made with immunoblotting experiments were consistent with these findings. In contrast, in the spleens of patients with neuronopathic phenotypes of Gaucher disease, the concentration of glucocerebrosidase molecules was severely decreased.

Metabolism of tritium- and 14C-labeled alanine in rats

1981 ◽  
Vol 241 (2) ◽  
pp. E121-E128 ◽  
Author(s):  
S. Golden ◽  
M. Chenoweth ◽  
A. Dunn ◽  
F. Okajima ◽  
J. Katz
Keyword(s):  
Continuous Infusion ◽  
Transit Time ◽  
Bolus Injection ◽  
Specific Activity ◽  
Mean Transit Time ◽  
Vena Cava ◽  
Maximal Activity ◽  
Total Body Mass ◽  
Arterial Blood ◽  
Specific Activities

[3-3H]- and [U-14C]alanine were administered to starved rats by bolus injection and by continuous infusion. The specific activities of alanine, glucose, and lactate in blood were followed. The tracer kinetics of alanine depended on the site of tracer administration and sampling. Tracer was either administered into the aorta and blood sampled from the vena cava (A-VC mode) or tracer was administered into the vena cava and arterial blood sampled (V-A mode) (Katz, J. F. Okajima, and A. Dunn. Biochem J. 194: 513-524, 1981). When tracer was infused in the A-VC mode the plateau specific activity of alanine was about half that obtained in the V-A mode. The parameters of alanine turnover were calculated from the specific activities obtained in the A-VC mode. The calculated apparent replacement rate averaged 1.9 mg.min-1.kg-1 for [U-14C]- and 3.9 mg.min-1.kg-1 for [3-3H]alanine, indicating a carbon recycling of about 50%. The apparent contribution of alanine carbon to that of glucose is 15%. The maximal activity in plasma water is attained at about 5 min after bolus injection of [3-3H]alanine and that of [14C]glucose in blood is attained about 10 min after the injection of [U-14C]alanine. Maximal specific activity of [3H]- and [14C]lactate is attained within about 1 min after injection. The apparent mean transit time and alanine mass were calculated from the areas of washout curves after the continuous infusion was terminated. The mean transit time for [3H]alanine was 10 min and apparent total body mass of alanine of the order of 40 mg/kg. The apparent means transit time for [U-14C]alanine ranged from 33 to 66 min corresponding to a mass of the order of 100 mg/kg of alanine or 40 mg/kg of alanine carbon.

scholarly journals Inactivation of glucose 6-phosphate dehydrogenase during germination and outgrowth of Bacillus cereus T endospores

1975 ◽  
Vol 148 (2) ◽  
pp. 259-268 ◽  
Author(s):  
M Orlowski ◽  
M Goldman
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Total Activity ◽  
Cellular Protein ◽  
Metabolic Energy ◽  
Supernatant Fraction ◽  
Stage Of Development ◽  
Glucose 6 Phosphate Dehydrogenase

The specific activity and total activity of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) under conditions of complete cell breakage fall 10-20-fold during a 3h period of spore germination and outgrowth. The spores must germinate (lose refractility), but do not have to undergo outgrowth, for the loss of activity to occur. Glucose 6-phosphate dehydrogenase activity from cells as any stage of development is completely stable in extracts at 4 degrees C or 30 degrees C. All of the enzyme activity is found in a soluble (50000g supernatant) fraction and remains completely soluble throughout development. Soluble protein and total cellular protein remain constant for about 2h. Proteinases could not be detected or protein turnover demonstrated during the morphogenetic process. Phenylmethanesuophony fluoride and o-phenanthroline, inhibitors of proteolytic enzymes, do not prevent glucose 6-phosphate dehydrogenase inactivation when added to whole cells. Mixing experiments show no inhibitor of glucose 6-phosphate dehydrogenase to be present in late-stage cells. The enzyme is not excreted into the culture medium. Chloramphenicol and rifampicine immediately stop protein synthesis and development but not the inactivation of glucose 6-phosphate dehydrogenase. NaN3, 2,4-dinitrophenol or anaerobiosis immediately stop development and prevent the loss of enzyme activity. A requirement for metabolic energy is therefore probable. Extracts of spores pre-labelled with L[14C]leucine were made at various stages of morphogenesis and subjected to polyacrylamide-gel electrophoresis. Glucose 6-phosphate dehydrogenase, which was identified by a specific stain, did not lose 14C label, and therefore may not be degraded during the inactivation process.

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.

INDUCTION BY GONADAL HORMONES OF HEPATIC LIPOGENIC ENZYME ACTIVITY IN IMMATURE PULLETS

1974 ◽  
Vol 61 (1) ◽  
pp. 29-43 ◽  
Author(s):  
D. BALNAVE ◽  
J. PEARCE
Keyword(s):  
Enzyme Activity ◽  
Corn Oil ◽  
Specific Activity ◽  
Liver Lipid ◽  
Lipogenic Enzyme ◽  
Atp Citrate Lyase ◽  
Liver Lipid Content ◽  
Hormone Administration ◽  
Citrate Lyase ◽  
Specific Activities

SUMMARY The effect of gonadal hormone administration on hepatic lipogenic enzyme activity, and some physiological parameters was investigated in immature pullets. Pullets (aged 4 weeks) were allocated to treatment groups and received intramuscular injections of oestradiol, testosterone, progesterone or oestradiol + testosterone (all in 0·3 ml corn oil) or corn oil alone (control group). There was no evidence of any hormone-induced changes in the specific activity of hepatic ATP-citrate lyase 1, 3, 6 and 12 h after hormone administration. NADP-malate dehydrogenase exhibited significant variations in specific activity over this period of time and it is probable that these changes reflected an increased requirement for NADPH for synthetic purposes in hormone-treated birds. The effect of 1, 2, 4 and 9 days of hormone administration was also investigated. In testosterone-treated birds there were significant increases in the specific activities of both lipogenic enzymes after 1 day of hormone treatment whereas for birds receiving oestradiol the maximum specific activities were found on the second day. Similarly, the liver lipid content of oestradioltreated birds showed a substantial increase on day 2. After 9 days of hormone administration no significant differences in the specific activity of ATP-citrate lyase were observed between treatments but the specific activity of NADP-malate dehydrogenase was significantly reduced in oestradiol- or mixed hormone-treated birds; it is possible that the reduced enzyme activity is associated with a reduced requirement for NADPH and in this connexion there were no further increases in liver lipid content or liver weight after 4 days of hormone administration. The liver RNA:DNA ratio tended to be greatest in birds receiving oestradiol or oestradiol + testosterone. Studies utilizing inhibitors of RNA and protein synthesis showed that such compounds abolished the increases in lipogenic enzyme activity following hormone administration suggesting that these increases were hormone-induced effects. These results are discussed in relation to the effects of the various hormones on liver lipid metabolism and also in relation to the situation in the mature laying hen.

scholarly journals Isolation of β-N-acetylhexosaminidase from rabbit semen and its role in fertilization

1980 ◽  
Vol 191 (3) ◽  
pp. 827-834 ◽  
Author(s):  
A A Farooqui ◽  
P N Srivastava
Keyword(s):  
Enzyme Activity ◽  
Metal Ions ◽  
Seminal Plasma ◽  
Concanavalin A ◽  
Zona Pellucida ◽  
Specific Activity ◽  
Optimal Activity ◽  
Step Procedure ◽  
Km Value ◽  
Arylsulphatase A

Beta-N-Acetylhexosaminidase was purified from the rabbit seminal plasma by a three-step procedure involving hydroxyapatite, Sephadex G-200 and concanavalin A–Sepharose chromatography. The specific activity of the purified preparation was 56mu mol/min per mg of protein, which represented a 226-fold purification and a 54% yield of the enzyme activity. The purified enzyme was electrophoretically homogeneous. The homogeneous enzyme showed optimal activity at pH4.0. The apparent Km value and Vmax. were 1.4 mM and 56mu mol/min per mg of protein respectively. Metal ions such as Ag + and Hg2+ and p-chloromercuribenzoate strongly inhibited the enzyme activity. The treatment of rabbit ova with a mixture of Beta-N-acetylhexosaminidase and arylsulphatase A results in the swelling of the zona pellucida.

EXTRACTION OF ENZYMES AND ASSESSMENT OF METABOLISM IN BOVINE RUMEN EPITHELIUM

1982 ◽  
Vol 62 (2) ◽  
pp. 429-438 ◽  
Author(s):  
ROY S. BUSH
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Total Activity ◽  
Small Contribution ◽  
Bacterial Protein ◽  
Total Enzyme Activity ◽  
Rumen Epithelium ◽  
Bacterial Enzyme ◽  
Protein Contamination ◽  
Total Enzyme

Papillae collected from the rumens of freshly killed cows were used to estimate the most appropriate methods for enzyme extraction from rumen epithelium and the amount of enzymes in extracts which might be of bacterial origin. Extractions of enzymes from fresh and frozen papillae were compared for the Polytron homogenizer (PT), the Potter-Elvehjem homogenizer (PE), the Waring blender, sonication and acetone powdering plus PE. PE extraction yielded solutions with the highest specific activity for each enzyme. PT extraction released the most protein and total enzyme activity into solution. PT extraction was chosen for the remaining tests because of the high total activity released. Mixed rumen bacteria were homogenized by sonication. Electrophoretic examination of epithelial and bacterial extracts showed differential migration for malate dehydrogenase. Lactate dehydrogenase from the epithelium showed four distinct isozymes whereas the bacterial enzyme showed little distinct band development. Contamination of epithelial extracts by bacterial protein was estimated to be less than 5%. The specific activities of 10 enzymes were found to be similar in epithelial and bacterial extracts so that a small amount of protein contamination would result in only a small contribution to total enzyme activity. The presence of the enzymes assayed in this study plus a number reported in the literature showed that rumen epithelial metabolism is more diverse than previously recognized. Key words: Rumen epithelium, enzymes, extraction

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