scholarly journals Selective depletion of small basic non-glycosylated proteins in diabetes

1981 ◽  
Vol 198 (1) ◽  
pp. 149-157 ◽  
Author(s):  
F C Samaniego ◽  
F Berry ◽  
J F Dice
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Cytosol ◽  
Physiological Importance ◽  
Subunit Molecular Weight ◽  
Uncontrolled Diabetes ◽  
Streptozotocin Induced Diabetes ◽  
Protein Classes ◽  
Rat Liver Cytosol ◽  
Glycosylated Proteins

Degradative rates of small basic non-glycosylated proteins are preferentially enhanced in rat liver cytosol during severe streptozotocin-induced diabetes. Synthetic rates of these classes of proteins are not selectively enhanced in diabetes, so small basic non-glycosylated proteins should be depleted from liver cytosol as a consequence of this disease. To test this hypothesis, proteins were analysed from normal animals, from diabetic animals receiving insulin and from diabetic animals after insulin withdrawal for 3 days. The proteins were separated according to subunit molecular weight by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, according to isoelectric point by isoelectric focusing and according to carbohydrate content by affinity chromatography with concanavalin A linked to agarose. Severe uncontrolled diabetes is associated with the predicted depletion of small basic non-glycosylated proteins from liver cytosol. The preferential degradation and loss of these protein classes may be of considerable physiological importance to the animal.

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.

Novel Catalatic Proteins of Bakers' Yeast. I. An Atypical Catalase

1973 ◽  
Vol 51 (11) ◽  
pp. 1551-1555 ◽  
Author(s):  
Tony C. M. Seah ◽  
A. R. Bhatti ◽  
J. G. Kaplan
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Native Protein ◽  
Wild Type ◽  
Major Band ◽  
Subunit Molecular Weight ◽  
Purification And Properties

At any stage of growth of a wild-type bakers' yeast, some 20% of the catalatic activity of crude extracts is not precipitable by means of antibody prepared against the typical catalase (catalase T), whose purification and properties have been previously described. Some of this catalatic activity is due to the presence of an atypical catalase (catalase A), a heme protein, with a molecular weight estimated as 170 000 – 190 000, considerably lower than that of the usual catalases (225 000 – 250 000). Preparations of catalase A were found to be homogeneous in the analytical ultracentrifuge and in polyacrylamide gel electrophoresis. Its subunit molecular weight, determined from its iron content, was 46 500, virtually the same as that of the major band obtained in gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native protein is tetrameric. Its specific activity is in the range of those reported for other typical catalases.

scholarly journals Increased membrane binding of erythrocyte catalase in hereditary spherocytosis and in metabolically stressed normal cells

Blood ◽  
1977 ◽  
Vol 49 (1) ◽  
pp. 113-123 ◽  
Author(s):  
DW Allen ◽  
S Cadman ◽  
SR McCann ◽  
B Finkel
Keyword(s):  
Molecular Weight ◽  
Hereditary Spherocytosis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Membrane Binding ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Normal Cells ◽  
Calcium Dependent ◽  
Subunit Molecular Weight ◽  
Erythrocyte Catalase

Abstract Normal red blood cell (RBC) membranes were compared with (1) RBC membranes from six patients with hereditary spherocytosis (HS), (2) normal membranes after hemolysis of the RBC in the presence of calcium, or (3) normal membranes after incubation of RBC for 24 hr in phosphate- buffered saline containing calcium without added glucose. When compared with normal controls, polyacrylamide gel electrophoresis with sodium dodecyl sulfate (PAGE SDS) of all three preparations showed an increase in membrane binding of globin and protein band 4.5 (60,000 molecular weight). In an attempt to identify band 4.5, 14 enzymes were assayed in the RBC membranes. Of these, catalase and lactate dehydrogenase were increased in membranes from HS RBC and from normal cells exposed to calcium. Only catalase, however, was present in sufficient quantity and had the correct subunit molecular weight on PAGE SDS and calcium- dependent membrane binding to account for an appreciable portion of 4.5. Caralase was further identified with a component of band 4.5 by double immunodiffusion using a specific anti-catalase antibody.

scholarly journals Purification and properties of a new glutathione-dependent thiol:disulphide oxidoreductase from rat liver

1982 ◽  
Vol 207 (1) ◽  
pp. 133-138 ◽  
Author(s):  
M G Battelli ◽  
E Lorenzoni
Keyword(s):  
Rat Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Xanthine Dehydrogenase ◽  
Enzymic Activity ◽  
Oxidized Glutathione ◽  
Adult Rats ◽  
Purification And Properties ◽  
Rat Liver Cytosol

A new GSSG-dependent thiol:disulphide oxidoreductase was extensively purified from rat liver cytosol. The enzymic protein shows molecular weight 40 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and 43 000 as determined by thin-layer gel filtration on Bio-Gel P-100. The pI is 8.1. This enzyme converts rat liver xanthine dehydrogenase into an oxidase, in the presence of oxidized glutathione. Other disulphide compounds are either inactive or far less active than oxidized glutathione in the enzymic oxidation of rat liver xanthine dehydrogenase. The enzyme also catalyses the reduction of the disulphide bond of ricin and acts as a thioltransferase and as a GSH:insulin transhydrogenase. The enzymic activity was measured in various organs of newborn and adult rats.

scholarly journals Increased membrane binding of erythrocyte catalase in hereditary spherocytosis and in metabolically stressed normal cells

Blood ◽  
1977 ◽  
Vol 49 (1) ◽  
pp. 113-123
Author(s):  
DW Allen ◽  
S Cadman ◽  
SR McCann ◽  
B Finkel
Keyword(s):  
Molecular Weight ◽  
Hereditary Spherocytosis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Membrane Binding ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Normal Cells ◽  
Calcium Dependent ◽  
Subunit Molecular Weight ◽  
Erythrocyte Catalase

Normal red blood cell (RBC) membranes were compared with (1) RBC membranes from six patients with hereditary spherocytosis (HS), (2) normal membranes after hemolysis of the RBC in the presence of calcium, or (3) normal membranes after incubation of RBC for 24 hr in phosphate- buffered saline containing calcium without added glucose. When compared with normal controls, polyacrylamide gel electrophoresis with sodium dodecyl sulfate (PAGE SDS) of all three preparations showed an increase in membrane binding of globin and protein band 4.5 (60,000 molecular weight). In an attempt to identify band 4.5, 14 enzymes were assayed in the RBC membranes. Of these, catalase and lactate dehydrogenase were increased in membranes from HS RBC and from normal cells exposed to calcium. Only catalase, however, was present in sufficient quantity and had the correct subunit molecular weight on PAGE SDS and calcium- dependent membrane binding to account for an appreciable portion of 4.5. Caralase was further identified with a component of band 4.5 by double immunodiffusion using a specific anti-catalase antibody.

STUDIES OF A THYROID HORMONE AND ANDROGEN DEPENDENT PROTEIN IN RAT LIVER CYTOSOL

1977 ◽  
Vol 84 (3) ◽  
pp. 548-558 ◽  
Author(s):  
Wolfgang H. Dillmann ◽  
Enrique Silva ◽  
Martin I. Surks ◽  
Jack H. Oppenheimer
Keyword(s):  
Molecular Weight ◽  
Thyroid Hormone ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Female Rats ◽  
Male Rats ◽  
Male Rat ◽  
Liver Cytosol

ABSTRACT Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of the liver cytosol of euthyroid male rats revealed a prominent band (molecular weight, 26 000 daltons), designated Protein II, which was virtually absent in the cytosol of hypothyroid animals. Injection of 500 μg triiodothyronine (T3) per 100 g body weight resulted in a maximal increase in the level of Protein II, reaching 90% of the euthyroidal level 3 days after hormone administration. Concomitant studies with the liver mitochondrial enzyme alpha-glycerophosphate dehydrogenase (α-GPD) indicated that this T3 dose also resulted in a maximal enzyme response in this time period. Since we have estimated that 500 μg of T3 will saturate nearly all nuclear T3 binding sites, these results support the concept that the synthesis of both proteins is limited by nuclear binding. Protein II was absent in the liver cytosol of female rats but could be induced in ovariectomized female rats by androgens. Treatment of male rats with oestradiol resulted in disappearance of Protein II. Since administration of testosterone to hypothyroid male rats caused only a minimal increase in the amount of Protein II, the absence of the protein in hypothyroid animals was not due to androgen deficiency. Similarities in the molecular weight and the response to hormonal manipulation of Protein II and of the urinary α2uglobulin, previously reported by Roy (1973) raise the possibility that these proteins are the same. The high concentration of Protein II in male rat cytosol and the relative ease in its identification by SDS polyacrylamide gel electrophoresis make it a potentially useful model protein for the study of thyroid hormone action at the cellular level.

Early effect of thyroid hormone on cytosolic protein phosphorylation in rat anterior pituitary

1985 ◽  
Vol 109 (3) ◽  
pp. 326-329 ◽  
Author(s):  
Hirotoshi Nakamura ◽  
Toshihiko Yokota ◽  
Hiroo Imura ◽  
Leslie J. DeGroot
Keyword(s):  
Protein Phosphorylation ◽  
Gel Electrophoresis ◽  
Anterior Pituitary ◽  
Polyacrylamide Gel Electrophoresis ◽  
Early Effect ◽  
Liver Cytosol ◽  
Intracellular Process ◽  
Previous Demonstration ◽  
Rat Liver Cytosol

Abstract. The effect of T3 was studied on phosphorylation of cytosolic proteins in rat anterior pituitary. Cytosols obtained from hypothyroid (H) and T3 (50 μg/100 g body weight) injected H rats (T) were phosphorylated in vitro. Although total incorporation of 32P into proteins did not differ between H and T, analysis of phosphoproteins by SDS polyacrylamide gel electrophoresis and autoradiography revealed that T3 injection significantly increased phosphorylation of 2 proteins within 1 h. These results and our previous demonstration of T3 effects on phosphorylation in rat liver cytosol and cultured human skin fibroblasts suggest that protein phosphorylation is an important early intracellular process in T3 action.

scholarly journals Identity of purified monoacylglycerol lipase, palmitoyl-CoA hydrolase and aspirin-metabolizing carboxylesterase from rat liver microsomal fractions. A comparative study with enzymes purified in different laboratories

1985 ◽  
Vol 232 (2) ◽  
pp. 479-483 ◽  
Author(s):  
R Mentlein ◽  
R K Berge ◽  
E Heymann
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Monoacylglycerol Lipase ◽  
Enzyme Preparations ◽  
Nitrophenyl Phosphate ◽  
Liver Microsomal Fraction ◽  
Coa Hydrolase ◽  
Rat Liver Cytosol

Two purified carboxylesterases that were isolated from a rat liver microsomal fraction in a Norwegian and a German laboratory were compared. The Norwegian enzyme preparation was classified as palmitoyl-CoA hydrolase (EC 3.1.2.2) in many earlier papers, whereas the German preparation was termed monoacylglycerol lipase (EC 3.1.1.23) or esterase pI 6.2/6.4 (non-specific carboxylesterase, EC 3.1.1.1). Antisera against the two purified enzyme preparations were cross-reactive. The two proteins co-migrate in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Both enzymes exhibit identical inhibition characteristics with Mg2+, Ca2+ and bis-(4-nitrophenyl) phosphate if assayed with the two substrates palmitoyl-CoA and phenyl butyrate. It is concluded that the two esterase preparations are identical. However, immunoprecipitation and inhibition experiments confirm that this microsomal lipase differs from the palmitoyl-CoA hydrolases of rat liver cytosol and mitochondria.

Anomalous aggregation of Clostridium perfringens enterotoxin under dissociating conditions

1976 ◽  
Vol 22 (9) ◽  
pp. 1410-1414 ◽  
Author(s):  
George L. Enders Jr. ◽  
Charles L. Duncan
Keyword(s):  
Molecular Weight ◽  
Clostridium Perfringens ◽  
Gel Electrophoresis ◽  
Cetyltrimethylammonium Bromide ◽  
Equilibrium Dialysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Binding Studies ◽  
Clostridium Perfringens Enterotoxin ◽  
Subunit Molecular Weight

Polyacrylamide gel electrophoresis of highly purified Clostridium perfringens enterotoxin revealed electrophoretic microheterogeneity of the enterotoxin, apparently because of slight charge differences in the peptides. Detergent gel electrophoresis showed that purified enterotoxin formed high molecular weight aggregates in the presence of both sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide. No conditions capable of inhibiting this phenomenon were found. Although a molecular weight of 35 000 daltons has been reported in the literature, the experimentally determined molecular weight values in the presence of detergents corresponded to multiples of a theoretical subunit molecular weight of 17 500 daltons. Binding studies performed by equilibrium dialysis and ultracentrifugation methods revealed that the enterotoxin bound very small amounts of SDS per gram of protein. The evidence presented indicates possible detergent induced structural alterations of the protein.

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