1,721,043 research outputs found
Phosphorylation of casein by mitochondrial protein kinase(s).
No full textAt least two protein kinase activities are bound to the rat liver mitochondrial membranes. Both activities are found to phosphorylate, besides endogenous proteins tightly bound to the membrane structures, also exogenous phosphoproteins such as casein and phosvitin. However one is able to phosphorylate both casein-bound serine and threonine residues, while the other is phosphorylating almost only serine residues
Phosphorylation of Cytosolic Proteins By Casein Kinases In Human-erythrocytes - Response To Ionic-strength and To 2,3-bisphosphoglycerate
No full textThe endogenous phosphorylation of human erythrocyte cytosolic proteins is markedly increased when the crude cytosol, prior to incubation in the presence of [y-32P] ATP, is submitted to DEAE-cellulose chromatography. Some proteins, including 22 and 23 kDa proteins, are preferentially phosphorylated by cytosolic casein kinase CS, whereas other proteins, including 42 kDa protein, are preferentially phosphorylated by casein kinase CTS. The CS-catalyzed phosphorylation is strongly inhibited by physiological ionic strength (150 mM KCl or NaCl) and by physiological levels (3 mM) of 2,3-bisphosphoglycerate, while CTS-catalyzed phosphorylation is unaffected. The very poor endogenous phosphorylation of these proteins in the crude cytosol may be due to the presence of other cytosolic inhibitors which are removed by DEAE-cellulose chromatography
Further purification and characterization of casein kinases from human erythrocyte hemolysate. Effect of Triton X-100.
No full textTwo cyclic AMP-independent casein kinases can be isolated from human erythrocyte hemolysate, one of which (referred to as 'casein kinase S') phosphorylates only serine residues of whole commercial casein, while the other (referred to as 'casein kinase TS') phosphorylates both serine and threonine residues of the same substrate. Moreover, the casein kinase S, unlike casein kinase TS, is able to phosphorylate the erythrocyte membrane proteins. The present paper deals with the further characterization of casein kinase S, freed from histone kinase activity by DEAE and subsequent phosphocellulose chromatography of the crude hemolysate in the presence of 0.2% Triton X-100. In particular, cytosol casein kinase S exhibits some physico-chemical and catalytic properties identical to those of the membrane-bound casein kinase, solubilised and purified as previously described. Both casein kinases display the same chromatographic behaviour, the same Sepharose elution volume, the same optimal pH range, the same Km for casein and ATP, the same response to NaCl, MgCl2 and CaCl2, and the same ability to phosphorylate serine but not threonine residues of beta-casein
Phosphorylation of Membrane-proteins by Cytosolic Casein Kinases In Human-erythrocytes - Effect of Mono-valent Ions, 2,3-bisphosphoglycerate and Spermine
No full textMembrane proteins of human erythrocytes can be phosphorylated not only by membrane casein kinase (MS) but also by cytosolic casein kinases CS and CTS, resembling casein kinase I and II, respectively. Casein kinase CS, like membrane casein kinase MS, preferentially phosphorylates membrane proteins such as band 2 (spectrin, beta-subunit) and band 3, which are the major phosphate-acceptor proteins in the endogenous phosphorylation of isolated ghosts in the presence of [gamma-32P]ATP. By contrast, cytosolic casein kinase CTS phosphorylates, in addition to band 2, some membrane proteins, whose endogenous phosphorylation in isolated ghosts under the same conditions is negligible, if any. The CS- and CTS-catalyzed phosphorylations exhibit different response to increasing NaCl (or KCl) concentrations up to physiological levels (140 mM KCl, 20 mM NaCl); i.e. CS- and MS-catalyzed phosphorylations are strongly inhibited by 75-150 mM KCl (or NaCl), while CTS-catalyzed phosphorylation is practically unaffected. In the absence of added NaCl, CS- and MS-catalyzed phosphorylations are markedly inhibited by 1.5-3 mM 2,3-bisphosphoglycerate, whereas CTS-catalyzed phosphorylation appears to be practically unaffected. Finally, CS- and MS-catalyzed phosphorylations are slightly inhibited also by 1 mM spermine, while CTS-catalyzed phosphorylation is enhanced by this polycation concentration
Kinetic behaviour of human erythrocyte casein kinases. Effect of 2, 3-bisphosphoglycerate and heparin.
No full textThe predominant cAMP-independent casein kinase CS isolated from human erythrocyte hemolysate and membrane casein kinase MS are inhibited to the same extent by 2,3-bisphosphoglycerate (2,3-DPG). However, under some conditions, both enzymes may appear to be unaffected or even stimulated by this prominent red cell metabolite, depending on casein and monovalent salt (NaCl, KCl) concentrations employed in the assay. In addition, both casein kinases CS and MS exhibit identical response to heparin, being unaffected by increasing concentrations of this mucopolysaccharide up to 400 ng/ml. On the contrary, the minor cytosolic casein kinase CTS, is inhibited by both 2,3-DPG and heparin under all conditions tested in the assay
Comparative Characterization of Membrane-associated and Cytosolic Tyr-protein Kinases In Human-erythrocytes
Full text linkIn recent years, two protein-tyrosine kinase activities, phosphorylating tyrosine residues on the transmembrane band-3 protein, have been isolated from human erythrocyte membranes and partially characterized by different laboratories, i.e. one extracted by non-ionic detergent (Triton X-100 or Nonidet P-40), the other solubilized by 0.25 M NaCl from the detergent-insoluble residue. The present paper shows that these two membrane-associated Tyr-protein kinases purified, in the presence of bovine serum albumin, by phosphocellulose chromatography followed by heparin-Sepharose chromatography, have the same apparent molecular mass (36 kDa) determined by Ultrogel Ac44 filtration. Moreover, both Tyr-protein kinases exhibit several identical properties, including Km values for band 3, the random acidic copolymer poly(Glu,Tyr)4:1 and angiotensin II, pH dependence, response to Mn2+ and Mg2+, response to NaCl and 2,3-bisphosphoglycerate. All these properties are identical or very similar to those exhibited by the Tyr-protein kinase previously isolated by us from human erythrocyte cytosol. These results suggest that the two membrane-associated and the cytosolic Tyr-protein kinase activities are mediated by the same enzyme, distributed between the cytosol and the membrane structures
Comparative study of mitochondrial and cytosol protein kinase activities.
No full textBoth cytosol and mitochondria of rat liver display protein kinase activity, cyclic AMP-independent, which is resolved by Sepharose 6B filtration and P-cellulose chromatography into multiple forms phosphorylating, besides endogenous mitochondrial membrane-bound proteins, also exogenous phosphoproteins such as casein and phosvitin. However, the forms by far predominant in the cytosol phosphorylate both phosphorylserine and phosphorylthreonine residues of casein, while most of the activity associated to mitochondrial structures is due to the forms phosphorylating only phosphorylserine residues
Partial-purification and Characterization of Cytosolic Tyr-protein Kinase(s) From Human-erythrocytes
Full text linkyrosine-protein kinase, phosphorylating tyrosine residues of transmembrane band 3 protein, has been partially purified from human erythrocyte cytosol by DEAE-Sepharose chromatography followed by heparin-Sepharose chromatography. Such a Tyr-protein kinase (36 kDa), as distinct from the Ser/Thre-protein kinases (casein kinase S and TS), appears to display a broader site specificity than does the previously described human erythrocyte P-Tyr-protein phosphatase, dephosphorylating band 3 protein. That is, it is able to phosphorylate not only the highly acidic copolymer poly(Glu-Tyr) but also angiotensin II, lacking an acidic amino acid sequence around the target Tyr residue. Moreover, the phosphorylation of these two substrates exhibits a different pH dependence and a different response to NaCl and 2,3-bisphosphoglycerate. These results suggest that in intact erythrocytes the cytosolic Tyr-protein kinase might phosphorylate band 3 not only on Tyr-8, surrounded by several acidic side-chains (as demonstrated preferentially to occur in isolated ghosts), but also on other Tyr residues surrounded by other amino acid sequences
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