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Biomedical subjects

S Azhar

Publications and source records attributed to S Azhar.

160 records · Page 9Linked to original sources
PubMed1975

Adenosine-3':5'-monophosphate-dependent and plasma-membrane-associated protein kinase from bovine corpus luteum. Solubilization and properties of solubilized enzyme.

The solubilization of plasma membrane fractions FI and FII associated protein kinases has been attempted using monovalent salts of high ionic strength and various detergent treatments. Extraction of FI and FII plasma membranes with high ionic strength salt solutions did not release more than 20% of the protein kinase activity. Similarly, monovalent salts released little adenosine 3':5'-monophosphate (cyclic AMP) binding activity, but after extraction binding capacity of cyclic [3H]AMP to plasma membranes was increased about 150-200%. Triton X-100 was a better solubilizing agent that Lubrol WX or deoxycholate. In addition to solubilization, 0.1% Triton X-100 also stimulated the protein kinase activity 150-200%. The properties of Triton X-100 solubilized FI and FII and purified cytosol KII were characterized with respect to protein substrate specificity, effect of cyclic AMP, cyclic nucleotide specificity, effects of divalent metal ion and gonadotropins. Upon sucrose density gradient centrifugation, FI solubilized protein kinase and cyclic AMP binding activities co-sedimented with a sedimentation coefficient of 6.3 S. The FII solubilized protein kinase sedimented as two components with sedimentation coefficients of 7.7 S and 5.5 S. The cyclic AMP binding activity also sedimented as two components with sedimentation coefficient 6.7 S and 5.5 S. Cyclic AMP caused dissociation of solubilized protein kinase from FI into a single catalytic (4.8 S) and two cyclic AMP binding subunits (8.1 S and 6.7 S). FII solubilized enzyme was dissociated into one catalytic (4.8 S) and one cyclic AMP binding subunit (6.3 S). Fractionation of FI and FII solubilized enzymes on DEAE-cellulose column chromatography resolved them each into two peaks Ia, Ib and IIa, IIb, respectively. Peaks Ib and IIb were more sensitive to cyclic AMP STIMULATION THAN Ia and IIa peaks. From these studies it is concluded that the plasma-membrane associated and cytosol protein kinases have similar catalytic properties but differ in some of their physical properties.

Animals↗
PubMed1975

Solubilization and characterization of phosphoprotein phosphatase(s) from bovine corpus-luteum plasma membranes.

Plasma membrane fractions I and II isolated from bovine corpus luteum contain phosphoprotein phosphatases. Enzyme activities associated with both membrane fractions showed pH optima in the neutral range and were most active with phosphoprotamine as the exogenous substrate. The enzyme activity was partially inhibited by Co2+, Zn2+ and Fe2+. Dithioerythritol, glutathione (reduced) and 2-mercaptoethanol stimulated the enzyme activity, whereas N-ethylmaleimide and N-phenylmaleimide were inhibitory. Similarly, various cyclic nucleotides and nuclsoside triphosphates also inhibited phosphoprotein phosphatase activities. The phosphatase activity was also observed with endogenous phosphorylated membrane proteins as substrate. The endogenous phosphorylation of membranes was rapid and attained a maximal level after 15--20 min of incubation. Initially endogenous dephosphorylation was also very rapid, but did not reach completion. In addition to phosphoprotein phosphatase, membrane preparations also possessed very active cyclic-AMP-dependent protein kinase activity. Phosphoprotein phosphatase activity from plasma membranes was solubilized by ionic and nonionic detergents. Optimal solubilization was achieved with 0.1% sodium deoxycholate. Sucrose density gradient centrifugation of deoxycholate-solubilized fraction I and fraction II membranes resolved phosphoprotein phosphatase activity into two species with apparent sedimentation coefficients of 6.7 S (Mr 130000) and 4.8 S (Mr 90000). Cyclic-AMPstimulated protein kinase activity sedimented as a broad peak with a sedimentation coefficient of 5.5 S (Mr 110000).

Animals↗
PubMed1975

Adenosine 3'5'-m onophosphate dependent phosphorylation of ribosomes and ribosomal subunits from bovine corpus luteum.

In a previous publication the purification and properties of two protein kinases (KI and KII) from a soluble fraction of bovine corpus luteum and the stimulation of the latter fol. Chem. 248,494-501). We have now studied the effects oc cyclic AMP and luteinizing hormone on ribosomal protein phosphorylation of corpus luteum by protein kinase II. Protein kinase II catalyzed the phosphorylation of ribosomes by transfer of terminal phosphate of ATP to ribosomal proteinsmextraction with hot trichloroacetic acid and non-aqueous solvent revealed that about 80% of total radioactivity incorporated remain associated with the protein residue. Radioactivity was identified in the phosphoserine and phosphothreonine residues of polypeptides by high voltage paper electrophoresis; The extent of phosphorylation was stimulated by cyclic AMP but not by luteinizing hormonemat least 9 proteins of 80-S ribosomes and 12 proteins of the 60-S ribosomal subunit were phosphorylated in the presence of cyclic AMP as resolved by urea polyacrylamide gel electrophoresis. However, only one major and four minor bands were phosphorylated in the ase of 40-S ribosomal subunit under the influence of cyclic AMP. The ribosomal protein phosphorylation catalyzed by protein kinase II is regulated by cyclic AMP wherease luteinizing hormone has no effect on ribosome phosphorylation.

Animals↗
PubMed1975

Adenosine 3':5'-cyclic monophosphate-dependent and plasma-membrane-associated protein kinase(s) from bovine corpus luteum.

Plasma-membrane fractions FI and FII isolated from bovine corpus luteum by discontinuous sucrose-density-gradient centrifugation, at sucrose-density interfaces of 1.14/1.16 and 1.16/1.18 respectively, contained membrane-associated protein kinases that phosphorylated both the structural proteins of membranes as well as exogenously added protein substrates. Both fractions were characterized with respect to endogenous and exogenous protein substrate specificity, pH-dependence, effect of bivalent metal ions and sensitivity toward cyclic nucleotides. These membrane-associated kinases showed an optimum pH of 6.0 and had an absolute requirement for bivalent metal ions such as Mg2+, Mn2+, or Co2+ that cannot be replaced by Ca2+. Both the activities were stimulated two- to four-fold by cyclic AMP in vitro with an apparent Km of 83 and 50 nM for fractions FI and FII respectively. Other cyclic 3':5'-nucleotides were effective only at higher concentrations, but even the most effective, cyclic IMP, showed a stimulation nearly an order of magnitude lower than that of cyclic AMP. In contrast, stimulation by cyclic dTMP and cyclic dAMP was very weak. Cyclic AMP showed no significant effect on the apparent Km value of both enzymes for histone and MgCl2 but it somewhat decreased the Km value for ATP. Nucleoside triphosphates like GTP, CTP and UTP inhibited the transfer of [32P]Pi from [gamma-32P]ATP into mixed histone catalysed by membrane-associated kinases either in the presence or in the absence of cyclic AMP. In addition to protein kinases, these membrane fractions also possessed cyclic AMP-binding activities. The apparent association constant (Kalpha) for cyclic AMP binding was 1.0 X 10(10) and 2.6 X 10(10) M for FI and FII membrane fractions respectively.

Adenosine Triphosphate↗
PubMed1975

Tryptophan metabolism in Cicer arietinum.

Crude extracts of Cicer arietinum seedlings contain enzymes that mediate the oxidation of tryptophan. The most active is formylase followed by tryptophan oxygenase and kynureninase. Tryptophan is also utilized for the synthesis of indolyl-3-acetic acid. The stimulation of tryptophan oxygenase in seedlings incubated in presnece of indolyl-3-acetic acid has been shown by specific labelling of haem moeity and the apoenzyme not to involve de novo synthesis.

Kynurenine↗
PubMed1975

Loss of epinephrine stimulated synthesis of cyclic adenosine 3':5' monophosphate during maturation of rabbit and human reticulocytes.

The adenylate cyclase activity of rabbit reticulocytes is localized in the stroma. Rabbit reticulocytes are four times more active than mature erythrocytes in transforming in situ formed [14C] adenosine triphosphate into [14C] cyclic adenosine 3':5'-monophosphate. Similarly, anaemic human red blood cells show higher activity in effecting this transformation. The adenylate cyclase activity is stimulated by epinephrine, 40% in rabbit reticulocytes, and 70-80% in anaemic human RBC but not in erythrocytes. Both the reticulocytes and erythrocytes can bind [14C] epinephrine, but the reticulocytes bind a relatively large amount. A glycogen phosphorylase that can be activated by cyclic adenosine 3':5'-monophosphate is detected only in reticulocytes. Both reticulocytes and erythrocytes contain a phosphodiesterase which can hydrolyse cyclic 3':5'-adenosine monophosphate and which is activated by imidazole and inhibited by methyl xanthines.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1975

Ribonuclease activity of Entamoeba histolytica.

Ribonuclease activity of Entamoeba histolytica was purified 65-fold by calcium phosphate gel, ammonium sulphate and acetone treatments. The enzyme was inhibited by metal ions, like Mg-2+, Mn-2+, Co-2+ and Ca-2+. Metal chelating and sulphydryl agents showed no effect. EDTA and alpha, a'-dipyridyl reversed the inhibition produced by Mg-2+, Mn-2+ and Fe-2+. Various sodium salts had negligible effect on the enzyme; however, sodium chloride activated the enzyme slightly. Amoebicidal drugs like enterovioform and chloroquine phosphate inhibited the enzyme completely, while emetine showed marked inhibitory effect. Neomycin exhibited a slight stimulatory action on this enzyme.

Amebicides↗
PubMedDate not supplied

Evidence for surface entrapment of cholesterol-rich lipoproteins in luteinized ovary.

Previous studies showed that perfused 125I-labeled low and high density lipoproteins (LDL, HDL) have affinity for specialized microvillar regions of luteal cells in hormone-primed, luteinized rat ovaries. In the current report, we re-examined the interaction of cholesterol-rich lipoproteins with these specialized plasma membrane regions using native lipoproteins visualized as discrete particles by standard electron microscopic techniques. In ovaries perfused with the various lipoproteins, spherical particles (varying in size from 12 to 28 nm depending on the particle used) were found over the surfaces of all luteal cells and filling up extensive "channel" space formed by the apposed plasma membranes of adjacent microvilli or cytoplasmic surfaces. Only 30% of these tissue-associated particles were removable after prolonged washing with perfused media or heparin. Few intact particles were found inside the cells, despite the fact that the lipoproteins induced a substantial hormone response by the ovary. To determine the total protein internalized by cells during the course of the experiments, parallel biochemical experiments were carried out with nonreleasable (14C-sucrose-coupled) human LDL. Of the total bound 14C-sucrose LDL, only 8.5% was degraded (trichloroacetic acid-soluble) and presumed internalized by the cells. Thus, while large numbers of cholesterol-rich lipoprotein particles interact with the luteal cell surface in specialized microvillar channels and elicit a progesterone response, relatively few intact lipoprotein particles appear to enter the cells to be degraded. We speculate that in the luteinized ovary, a large majority of the lipoprotein-cholesterol transfer occurs at the surface of the luteal cells, and that the membranes of the microvillar channels are involved in this process.

Animals↗