scholarly journals Purification of phosphodiesterase II from rat and guinea-pig intestinal mucosa

1976 ◽  
Vol 155 (3) ◽  
pp. 607-613 ◽  
Author(s):  
P R Flanagan ◽  
S H Zbarsky
Keyword(s):  
Guinea Pig ◽  
Intestinal Mucosa ◽  
Deae Cellulose ◽  
Large Loss ◽  
Arrhenius Plots ◽  
The Poor ◽  
Km Value ◽  
Soluble Material

Phosphodiesterase II from extracts of intestinal mucosa of rat and guinea pig was purified by chromatography on DEAE-cellulose, CM-cellulose and agarose. The rat enzyme was purified 350-550-fold, with recoveries ranging up to 46%. The best purification of the guinea-pig enzyme was 15-fold, and the recovery was only 2.6%, the large loss occurring during chromatography on DEAE-cellulose and agarose. The poor results with the guinea-pig enzyme reflect the difficulty in obtaining a truly soluble material. Repeated sonication of the crude guinea-pig preparations yielded material that was initially soluble but tended to re-aggregate quickly. Purification of the rat phosphodiesterase II increased its thermostability, the temperature of half-inactivation being increased from 54degrees to 60degreesC. Both enzymes had a Km value of 4 × 10(-5) M with thymidine 3′-(2,4-dinitrophenyl) phosphate as substrate and showed similar pH optima for activity. Both enzymes were inhibited slightly in 0.1 M-MgC12 or 2M-urea and much more strongly in 2M-(NH4)2SO4 or 6M-NaC1. The guinea-pig enzyme was usually inhibited more than the rat enzyme. The Arrhenius plots of the two enzymes differed slightly in slope, but both were biphasic, showing breaks between 30degrees and 40degreesC. It was concluded that the two enzymes were markedly similar in behaviour and that the differences found were related to the different degrees of purification attained by the procedures described.

scholarly journals Glucose metabolism in the mucosa of the small intestine. A study of hexokinase activity

1968 ◽  
Vol 109 (1) ◽  
pp. 35-42 ◽  
Author(s):  
L M Srivastava ◽  
P. Shakespeare ◽  
G. Hübscher
Keyword(s):  
Small Intestine ◽  
Lactate Dehydrogenase ◽  
Glucose Metabolism ◽  
Guinea Pig ◽  
Intestinal Mucosa ◽  
Brush Border ◽  
Deae Cellulose ◽  
Mitochondrial Fraction ◽  
Type Ii ◽  
Hexokinase Activity

1. The intracellular distribution of hexokinase activity was studied in the mucosa of rat and guinea-pig small intestine. In the rat 60% and in the guinea pig 45% of the hexokinase activity of homogenates were recovered in a total particulate fraction that contained only 5–17% of the homogenate activity of hexose phosphate isomerase, pyruvate kinase, lactate dehydrogenase and overall glycolysis (formation of lactate from glucose). 2. Fractionation of homogenates from guineapig small intestine showed that the particulate hexokinase activity was chiefly in the mitochondrial fraction with a small proportion in the nuclei plus brush-border fraction. 3. After chromatography of the particle-free supernatants on DEAE-cellulose, hexokinase types I and II were determined quantitatively. No evidence was obtained for the presence of hexokinase type III or glucokinase. In the preparations from guinea pigs, hexokinase types I and II amounted to 69% and 31% respectively of the eluted activity; the corresponding values for preparations from rats were 5·8% and 94·2%. 4. Total and specific hexokinase activities decreased significantly in homogenates and particle-free supernatants prepared from the intestinal mucosa of rats starved for 36hr. and increased again after re-feeding. The decrease in hexokinase activity in the particle-free supernatant from starved rats was chiefly due to a decrease in the type II enzyme.

THE RELATIVE DISTRIBUTION OF THYROXINE, TRIIODOTHYRONINE AND 3,3′,5′-(REVERSE)-TRIIODOTHYRONINE IN VARIOUS FRACTIONS OF THYROGLOBULIN

1978 ◽  
Vol 88 (2) ◽  
pp. 298-305 ◽  
Author(s):  
Peter Laurberg
Keyword(s):  
Ionic Strength ◽  
Guinea Pig ◽  
Sucrose Gradient ◽  
Deae Cellulose ◽  
High Ionic Strength ◽  
Relative Distribution ◽  
Thyroid Extract ◽  
Reverse Triiodothyronine ◽  
Thyroid Secretion ◽  
Stable Iodine

ABSTRACT Thyroglobulin fractions rich and poor in new thyroglobulin were separated by means of DEAE-cellulose chromatography of dog thyroid extracts and by zonal ultracentrifugation in a sucrose gradient of guinea pig thyroid extract incubated at low temperature. The distribution of thyroxine, triiodothyronine and 3,3′,5′-(reverse)-triiodothyronine in hydrolysates of the different fractions was estimated by radioimmunoassays. Following DEAE-cellulose chromatography there was a small but statistically significant increase in the T4/T3 ratio in thyroglobulin fractions eluted at high ionic strength - that is fractions relatively rich in stable iodine but poor in fresh thyroglobulin. There were no differences in the T4/rT3 ratios between the different fractions. The ratios between iodothyronines were almost identical in the various thyroglobulin fractions following zonal ultracentrifugation in a sucrose gradient of cold treated guinea pig thyroid extract. These findings lend no support to the possibility that a relatively high content of triiodothyronines in freshly synthesized thyroglobulin modulates the thyroid secretion towards a preferential secretion of triiodothyronine and 3,3′,5′-(reverse)-triiodothyronine at the expense of the secretion of thyroxine.

scholarly journals EVIDENCE THAT THE L-ASPARAGINASE OF GUINEA PIG SERUM IS RESPONSIBLE FOR ITS ANTILYMPHOMA EFFECTS

1963 ◽  
Vol 118 (1) ◽  
pp. 99-120 ◽  
Author(s):  
J. D. Broome
Keyword(s):  
Guinea Pig ◽  
Guinea Pigs ◽  
Serum Proteins ◽  
Deae Cellulose ◽  
Salt Precipitation ◽  
Asparaginase Activity ◽  
Pig Serum

A number of the properties of the L-asparaginase present in guinea pig serum have been examined and shown to be indistinguishable from those of the agent responsible for inhibiting cells of lymphoma 6C3HED in vivo. The patterns of instability of the enzyme to changes in temperature and pH were found to parallel closely those of the antilymphoma agent. L-Asparaginase activity was essentially absent from the serum of newborn guinea pigs and this failed to inhibit 6C3HED cells. On separating guinea pig serum proteins by salt precipitation, electrophoresis, and chromatography on DEAE cellulose, antilymphoma activity was found only in fractions which contained L-asparaginase.

scholarly journals Purification and characterization of acid phosphatase from a germinating black gram (Vigna mungo L.) seedling

2011 ◽  
Vol 63 (3) ◽  
pp. 747-756 ◽  
Author(s):  
A.K.M. Asaduzzaman ◽  
Habibur Rahman ◽  
Tanzima Yeasmin
Keyword(s):  
Acid Phosphatase ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Black Gram ◽  
Km Value

An acid phosphatase has been isolated and purified from an extract of a germinating black gram seedling. The method was accomplished by gel filtration of a germinating black gram seedling crude extract on sephadex G-75 followed by ion exchange chromatography on DEAE cellulose. The acid phosphatase gave a single band on SDS-polyacrylamide slab gel electrophoresis. The molecular weight of the acid phosphatase determined by SDS-polyacrylamide slab gel electrophoresis was estimated to be 25 kDa. The purified enzyme showed maximum activity at pH 5 and at temperature of 55?C. Mg2+, Zn2+ and EDTA had an inhibitory effect on the activity of the acid phosphatase. Black gram seedling acid phosphatase was activated by K+, Cu2+ and Ba2+. The Km value of the enzyme was found to be 0.49 mM for pNPP as substrate.

scholarly journals The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Temperature-dependence of microsomal enzyme activity and thermotropic changes in membrane structure

1978 ◽  
Vol 175 (1) ◽  
pp. 115-124 ◽  
Author(s):  
D T Pechey ◽  
A B Graham ◽  
G C Wood
Keyword(s):  
Guinea Pig ◽  
Phospholipid Bilayer ◽  
Permeability Barrier ◽  
Uridine Diphosphate ◽  
Membrane Bilayer ◽  
Arrhenius Plots ◽  
Acceptor Activity ◽  
Microsomal Membranes ◽  
Acyl Chains ◽  
Phospholipid Acyl Chains

Arrhenius plots of the non-latent UDP-glucuronyltransferase (p-nitrophenol acceptor) activity of guinea-pig microsomal membranes prepared with 154 mM-KCl were linear from 5 to 40 degrees C. Arrhenius plots for other microsomal preparations from guinea pig and rat liver that show various degrees of transferase latency, exhibited two linear regions intersecting at a sharp transition point near 20-25 degrees C. This discontinuity was abolished or greatly decreased when transferase latency was removed by treating the membranes with perturbants of phospholipid bilayer strucutre. The fluorescent probe N-phenyl-1-naphthyl-amine detected a thermotropic change in the fluidity of the phospholipid acyl chains of all the microsomal membrane preparations studied, at temperatures close to those of the Arrhenius-plot transitions. It is concluded that the thermotropic change in the structure of the membrane bilayer probably is a ‘phase separation’ or clustering of phospholipids, which affects a permeability barrier that restricts access of substrate to the transferase molecules.

scholarly journals Purification and properties of a new testosterone 17β-dehydrogenase (NADP+) from guinea-pig liver

1977 ◽  
Vol 163 (3) ◽  
pp. 401-407 ◽  
Author(s):  
E Kaguera ◽  
S Toki
Keyword(s):  
Guinea Pig ◽  
Column Chromatography ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Supernatant Fraction ◽  
Pig Liver ◽  
Ring Junction ◽  
Purification And Properties ◽  
Androstane Derivatives ◽  
Guinea Pig Liver

As a result of studies of guinea-pig live testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), a new testosterone 17beta-dehydrogenase was discovered. The new enzyme was purified to a single homogeneous protein from the 105 000 g-supernatant fraction of guinea-pig liver by (NH4)2SO4 fractional precipitation and two gel-filtration stages, DEAE-cellulose column chromatography and hydroxyapatite column chromatography. It was characterized by many properties. The enzyme has almost the same properties as the classical testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), with respect to cofactor requirement, pH optima for dehydrogenation, effect of phosphate ion on the NAD+-dependent reaction and molecular weight, but characteristic differences were observed in substrate-specificity between the two dehydrogenases. With various androstane derivatives, the configuration of the A/B-ring junction was closely connected with enzyme activity. 5alpha-Androstanes, such as 5alpha-androstane-3alpha,17beta-diol, 5alpha-androstane-3beta,17beta-diol and 17beta-hydroxy-5alpha-androstan-3-one, and 5beta-congeners, such as 5beta-androstane-3alpha,17beta-diol, 5beta-androstane-3beta,17beta-diol and 17beta-hydroxy-5beta-androstan-3-one, served as substrates for both the EC 1.1.1.64 enzyme and the new enzyme. The EC 1.1.1.64 enzyme oxidized testosterone more rapidly than did the new enzyme. These comparisons were based on the relative activities, apparent Km values and apparent Vmax values.

Bile acid uptake by isolated intestinal mucosa cells of guinea pig

1982 ◽  
Vol 721 (3) ◽  
pp. 247-252 ◽  
Author(s):  
Michael Schwenk ◽  
Victor Lopez Del Pino ◽  
Esmail Hegazy
Keyword(s):  
Bile Acid ◽  
Guinea Pig ◽  
Intestinal Mucosa ◽  
Acid Uptake ◽  
Bile Acid Uptake
Sign in / Sign up

Export Citation Format

Share Document