scholarly journals Purification and immunological quantification of rat liver lysosomal glycosidases

1989 ◽  
Vol 264 (1) ◽  
pp. 115-123 ◽  
Author(s):  
P C de Groen ◽  
G D LeSage ◽  
P S Tietz ◽  
N F LaRusso
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Lysosomal Enzyme ◽  
Enzyme Activities ◽  
Molecular Sieve ◽  
Polyacrylamide Gel Electrophoresis ◽  
Enzyme Concentration ◽  
Regulatory Control ◽  
Beta Galactosidase ◽  
Lysosomal Glycosidases

Although lysosomal enzyme activities are known to vary in response to numerous physiological and pharmacological stimuli, the relationship between lysosomal enzyme activity and enzyme concentration has not been systematically studied. Therefore we developed radioimmunoassays for two lysosomal glycosidases in order to determine lysosomal enzyme concentration. beta-Galactosidase and beta-glucuronidase were purified from rat liver 2780-fold and 1280-fold respectively, by using differential centrifugation, affinity chromatography, ion-exchange chromatography and molecular-sieve chromatography. Polyclonal antibodies to these enzymes were raised in rabbits, and two radioimmunoassays were established. Antibody specificity was shown by: (i) selective immunoprecipitation of enzyme activity; (ii) identical bands of purified enzyme on SDS/polyacrylamide-gel electrophoresis and immunoelectrophoresis; (iii) single immunoreactive peaks in molecular-sieve chromatography experiments. Sensitivities of the assays were such that 15 ng of beta-galactosidase and 45 ng of beta-glucuronidase decreased the ratio of bound to free radiolabel by 50%; minimal detectable amounts of immunoreactive enzymes were 2 ng and 10 ng respectively. The assays were initially used to assess the effects of physiological perturbations (i.e. fasting and age) on enzyme concentrations in rat liver; these experiments showed that changes in enzyme concentrations do not always correlate with changes in enzyme activities. This represents the first report of radioimmunoassays for lysosomal glycosidases. The results suggest that these radioimmunoassays provide useful technology for the study of regulatory control mechanisms of the concentrations of lysosomal glycosidases in mammalian tissues.

scholarly journals Proteolytische Aktivitäten der lysosomalen Enzyme bei Milchrindern* – 3. Mitteilung: Beziehungen der Energie- und Eiweißversorgung zur lysosomalen Enzymaktivität im Blutplasma beim Milchrind

2000 ◽  
Vol 43 (1) ◽  
pp. 17-26
Author(s):  
L. Panicke ◽  
J. Weingärtner ◽  
M. Schmidt ◽  
T. Król
Keyword(s):  
Enzyme Activity ◽  
Food Intake ◽  
Dairy Cows ◽  
Milk Production ◽  
Lysosomal Enzyme ◽  
Enzyme Activities ◽  
Correlation Coefficients ◽  
Food Ration ◽  
Milk Performance ◽  
Relationship Of

Abstract. Title of the paper: Relationship between lysosomal blood activity and milk content» of urea and protein in different phases of milk production in dairy cows Relationship of lysosomal enzyme activities in blood and supply of energy and protein in dairy cattle were investigated. Closed correlation coefficients were calculated for lysosomal enzyme activity and content of protein and urea in milk. Especially a high or a low content of protein in the food ration affects the lysosomal enzyme activities considerably. A different lysosomal response to equal food supply was gained after deviding the cow stock into different groups regarding performance at a different lactation status. Growth, breed, age, capacity of food intake and milk performance might be influencing factors.

Responses of the phosphatase activity of the lichen Cladina rangiferina to various environmental factors including metals

1979 ◽  
Vol 57 (14) ◽  
pp. 1534-1540 ◽  
Author(s):  
I. Lane ◽  
K. J. Puckett
Keyword(s):  
Enzyme Activity ◽  
Environmental Factors ◽  
Phosphatase Activity ◽  
Enzyme Activities ◽  
Enzyme Concentration ◽  
Acidic Ph ◽  
Nitrophenyl Phosphate ◽  
Cladina Rangiferina ◽  
Reduced Activity ◽  
Menten Constant

The characteristics of phosphatase activity of Cladina rangiferina (L.) Harm, have been studied. Calculations of enzyme activities were based on the liberation of p-nitrophenol from p-nitrophenyl phosphate. The phosphatase activity was found to be linear both with increasing sample size (enzyme concentration) and increasing time, showed highest activity at acidic pH, and had a Michaelis–Menten constant of 8.9 × 10−3 M. The enzyme activity was maximal in the range 61 ± 10 °C, was independent of light, and was completely eliminated by boiling the thalli. Various cations and anions were tested for their effect; uranyl and vanadyl ions inhibited activity by 60% whereas copper, nickel, and silver enhanced activity by 10%. The anions biselenite, cyanide, fluoride, molybdate, phosphate, and vanadate all greatly reduced activity (≥ 50%). Phosphatase activity was demonstrated in other lichen species.

scholarly journals Purification and immunochemical characterization of monoamine oxidase from rat and human liver

1977 ◽  
Vol 161 (1) ◽  
pp. 167-174 ◽  
Author(s):  
R G Dennick ◽  
R J Mayer
Keyword(s):  
Enzyme Activity ◽  
Monoamine Oxidase ◽  
Enzyme Activities ◽  
Gel Electrophoresis ◽  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Triton X

1. Monoamine oxidase from rat and human liver was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 2. The enzyme activity was extracted from mitochondrial preparations by Triton X-100. The enzyme was purified by (NH4)2SO4 fractionation followed by chromatography on DEAE-cellulose, Sepharose 6B, spheroidal hydroxyapatite, and finally chromatography on diazo-coupled tyramine-Sepharose. 3. Distinct differences occur in the chromatographic behaviour of the two enzymes on both DEAE-cellulose and spheroidal hydroxyapatite. 4. It is unlikely that the purification of the enzymes on tyramine-Sepharose is due to affinity chromatography and reasons for this are discussed. 5. The purified enzymes did not oxidize-5-hydroxytryptamine and the relative activities of the enzymes with benzylamine were increased approx. 1.25-fold compared with the enzyme activities of mitochondrial preparations. 6. Immunotitration of enzyme activity in extracts of mitochondrial preparations from rat liver was carried out with 5-hydroxytryptamine, tyramine and benzylamine. The enzyme activities were completely immunoprecipitated by the same volume of antiserum. Similar results were obtained with the antiserum to the enzyme from human liver.

scholarly journals Studies on the purification of rat liver uridine diphosphate glucuronyltransferase

1977 ◽  
Vol 161 (3) ◽  
pp. 543-549 ◽  
Author(s):  
B Burchell
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Uridine Diphosphate ◽  
Enzyme Protein ◽  
Epoxide Hydratase

1. A stable, more highly purified, preparation of UDP-glucuronyltransferase was obtained than previously reported. 2. Enzyme activity towards o-aminophenyl and p-nitrophenyl was increased 43- and 46-fold respectively. 3. The final preparation contains only three staining polypeptide bands visible after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. The only known major accompanying protein appears to be epoxide hydratase. 5. The purified enzyme activity towards o-aminophenol can still be activated 3 fold by diethylnitrosamine. 6. On evidence from purification, o-aminophenol and p-nitrophenol appear to be glucuronidated by the same enzyme protein. The possible recognition of the UDP-glucuronyltransferase enzyme is discussed.

scholarly journals Biosynthesis of rat liver pyruvate kinase. Measurement of enzyme lifetime and the rate of synthesis at weaning

1980 ◽  
Vol 192 (2) ◽  
pp. 507-516 ◽  
Author(s):  
T J Hopkirk ◽  
D P Bloxham
Keyword(s):  
Pyruvate Kinase ◽  
Rat Liver ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Concentration ◽  
Liver Cytosol ◽  
Maximum Value ◽  
Rat Liver Cytosol ◽  
Slow Turnover

Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of immunoprecipitates of liver cytosol with anti-(L-type pyruvate kinase) serum revealed proteins of mol.wt. 56 000 and 42 000 in addition to the heavy and light chains. The ratio of the 56 000 mol.wt. to the 42 000 mol.wt. protein increased under dietary conditions that resulted in an increase in the apparent specific activity of hepatic pyruvate kinase. The 42 000 mol.wt. protein was removed from immunoprecipitates if the liver cytosol was partially purified by pH precipitation and (NH4)2SO4 fractionation before addition of the antiserum. This technique may be used to analyse the formation of pure L-type pyruvate kinase in liver. By using H14CO3-labelling, the t1/2 of L-type pyruvate kinase was estimated as 75 +/- 1.7 h in post-weaned high-carbohydrate-diet-fed rats. Before weaning there was little immunoreactive pyruvate kinase in rat liver cytosol. Induction began between 6 and 24 h after weaning and reached a maximum value 120 h after weaning. When clearly enhanced total pyruvate kinase activity was first observed at 24 h post-weaning, the apparent specific activity of hepatic pyruvate kinase was considerably lower than the specific activity of the pure isolated enzyme. When the induction of L-type pyruvate kinase was monitored by the incorporation of L-[4,5-3H]leucine, the maximum rate of synthesis occurred 24–48 h after weaning. After this period synthesis declined, indicating a relatively slow turnover of the enzyme once the enzyme concentration was established in the liver.

An Automated Method for Measuring Creatine Phosphokinase Activity in Serum

1970 ◽  
Vol 16 (4) ◽  
pp. 294-299 ◽  
Author(s):  
Ronald K Wright ◽  
Roy L Alexander
Keyword(s):  
Enzyme Activity ◽  
Enzyme Activities ◽  
Creatine Phosphokinase ◽  
Creatine Phosphate ◽  
Enzyme Concentration ◽  
Magnesium Ion ◽  
Manual Method ◽  
Automated Method ◽  
Automated Procedures

Abstract We describe a procedure for automating the determination of creatine phosphokinase (CPK) activity in serum by use of the AutoAnalyzer. Enzyme activity is determined by measuring the creatine phosphate formed from the CPK-catalyzed reaction of creatine with adenosine triphosphate (ATP). The sensitivity of the automated procedure was comparable to that of the manual method. By use of the most favorable concentrations of creatine, ATP, and magnesium ion in the substrate, a linear relationship was obtained between enzyme concentration and enzyme activities up to 600 mU, representing a sixfold improvement over that obtained by the manual method. The degree of correlation between results obtained by the manual and automated procedures is shown.

Cystathionine Synthase from Rat Liver: Partial Purification and Properties

1971 ◽  
Vol 49 (5) ◽  
pp. 484-491 ◽  
Author(s):  
F. Christine Brown ◽  
P. H. Gordon
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Pyridoxal Phosphate ◽  
Enzyme Concentration ◽  
Partial Purification ◽  
Sulfate Ions ◽  
Nitrobenzoic Acid ◽  
Assay Procedure ◽  
Purification And Properties ◽  
Rate Of Increase

Cystathionine synthase which has been purified about 1000-fold from rat liver has absorbance maxima at 280, 360, and 428 mμ. Treating the enzyme with cysteine apparently affects the removal of pyridoxal phosphate and destroys the enzyme activity. So does reduction with borohydride. However, in neither case is the spectrum affected. These observations suggest that pyridoxal phosphate may be bound to cystathionine synthase in an atypical fashion.Mercuric ions strongly inhibit the enzyme, but not in the presence of serine; hydroxylamine inhibits, but not in the presence of substrates. Other carbonyl reagents inhibit little if at all. Sulfate ions activate the enzyme.A new assay procedure for cystathionine synthase has been devised. In the presence of 5,5′-dithiobis-(2-nitrobenzoic acid), the enzyme catalyzes the degradation of cystathionine to serine and homocysteine. The rate of increase in absorbance at 412 mμ is a measure of enzyme concentration.

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