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R K Puri

Publications and source records attributed to R K Puri.

At least 181 records · Page 10Linked to original sources
PubMed1986

Peptide mapping analysis of the avian progesterone receptor.

Progesterone receptor from the chicken oviduct has been shown to exist as two 8 S forms (I and II). Form I contains a protein of Mr = 75,000 and form II contains a protein of Mr = 110,000. In addition to these hormone-binding proteins, both receptor forms contain a protein with Mr = 90,000 that does not bind steroid. To investigate the possibility that these proteins are structurally related, they were isolated by preparative sodium dodecyl sulfate gel electrophoresis and subjected to peptide mapping analyses after digestion with Staphylococcus aureus V-8 protease, papain, or alpha-chymotrypsin. Receptor proteins labeled with [32P]orthophosphate in tissue minces were also subjected to peptide mapping analysis. The electrophoretic patterns of peptide fragments of the 90-kDa protein from receptor forms I and II were identical but were different from the peptide patterns obtained from the 75- and 110-kDa proteins which generated similar peptide patterns, indicating that these are structurally related. However, some differences were evident, indicating that these latter two proteins are not identical substrates for proteases. A one-dimensional comparison of the phosphopeptide patterns from the 75- and 110-kDa proteins also showed them to be similar, but not identical. Two-dimensional maps of phosphopeptides generated from the 75- and 110-kDa protein after complete tryptic digestion revealed multiple sites of phosphorylation which were identical except for one phosphopeptide that was unique to the 110-kDa protein. These results show the two progesterone-binding proteins to be very similar in structure, but to differ considerably from the 90-kDa protein.

Animals↗
PubMed1985

Isolation of steroid receptor binding protein from chicken oviduct and production of monoclonal antibodies.

Previous studies have shown that the molybdate-stabilized progesterone receptor from the chick oviduct contains a nonhormone binding component with a molecular weight of 90 000. This protein has also been shown to be associated with some other molybdate-stabilized steroid receptors of the oviduct. In order to access this larger pool of the receptor binding protein, we have developed an isolation procedure based on the observation that the protein is selectively shed from proteins adsorbed to heparin-agarose when molybdate is removed. The protein obtained by this procedure is shown to be the same as that isolated from affinity-purified progesterone receptor as compared by protease digestion and one-dimensional peptide mapping. Four immunoglobulin G secreting hybridoma cell lines were generated against the 90 000-dalton antigen. All of the antibodies recognize the 90 000-dalton protein obtained by electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels. In addition, two of the antibodies complex the molybdate-stabilized progesterone receptor as demonstrated by sedimentation analysis on sucrose gradients. One of these antibodies was used to show the presence of the 90 000-dalton component in molybdate-stabilized glucocorticoid and androgen receptors and also to show its presence in brain, liver, and skeletal muscle, but not in serum.

Animals↗
PubMed1985

Local anesthetic-phospholipid interactions. The pH dependence of the binding of dibucaine to dimyristoylphosphatidylcholine vesicles.

The interaction of the local anesthetic dibucaine with unilamellar vesicles of dimyristoylphosphatidylcholine was studied by equilibrium dialysis. Saturating binding profiles (as a function of dibucaine) were found, with apparent association constant ranging from 1.26 X 10(3)M-1 to 2.57 X 10(3)M-1 as pH is increased from 5.0 to 7.5. The number of phospholipid molecules comprising a binding site was found to be about 5 at each pH. Analysis of the data was also achieved using the Stern model, which takes into account the electrostatic effect on binding of the cationic drug due to the build up of a surface potential.

Dibucaine↗
PubMed1985

Oral submucous fibrosis--treatment with hyalase.

Oral Submucous Fibrosis (OSMF) is a disease of unknown etiology. A total of 96 patients with Oral Submucous Fibrosis have received four regimens of treatment--local dexamethasone, local hyaluronidase, local combination of dexamethasone and hyaluronidase, and local placental extract. The patients were followed up for a period varying from 3 months to 2 years. The group of patients receiving hyaluronidase alone showed quicker improvement in symptoms although its combination with dexamethasone gave somewhat better longer-term results. A new regimen for the treatment of submucous fibrosis is recommended.

Dexamethasone↗
PubMed1984

Polypeptide components of two 8 S forms of chicken oviduct progesterone receptor.

Two 8 S forms of progesterone receptor from the chicken oviduct were purified to near homogeneity and analyzed for peptide composition by gel electrophoresis. Form I contains two major peptides with molecular weights of 90,000 and 75,000. Form II also contains two major peptides with molecular weights of 90,000 and 110,000. In glycerol gradients containing molybdate, the 90,000, 110,000, and 75,000 molecular weight peptides co-sediment with the [3H]progesterone peak at 8 S. In high salt gradients lacking molybdate, the [3H]progesterone peak co-sediments with the 110,000 and 75,000 molecular weight peptides at 4 S, while the 90,000 molecular weight peptide sediments at 6-7 S. On photoactivation, the synthetic progestin R5020 binds covalently to the 110,000 and 75,000 molecular weight peptides. Thus, both 8 S forms of progesterone receptor contain 90,000 molecular weight peptides which do not bind progesterone, and each 8 S form contains a separate form of progesterone-binding peptide. When receptor is purified from oviduct minces incubated with [32P]orthophosphate, autoradiography indicates the presence of 32P in the 90,000 and 110,000 molecular weight peptides and in a peptide which appears to be slightly larger than 75,000 and may be a more highly phosphorylated fraction of the 75,000 molecular weight peptide. Thus, three separate peptides have been identified as components of the 8 S form of progesterone receptor, and all three appear to exist as phosphoproteins.

Animals↗
PubMed1984

The avian progesterone receptor: isolation and characterization of phosphorylated forms.

Methods have been developed for isolation of the avian progesterone receptor in the nontransformed, molybdate-stabilized state. The final step in this procedure. DEAE-Sephadex chromatography, resolves the receptor into two forms, components I and II. Analysis of these components by polyacrylamide gel electrophoresis under denaturing conditions shows that both contain a peptide with Mr = 90,000. These peptides contain phosphorylated serine residues, as shown by [32P]orthophosphate incorporation studies. When the cytosol receptor is treated with alkaline phosphatase, its steroid binding capacity is abolished. These studies show that the nontransformed progesterone receptor is a phosphoprotein and indicate that receptor phosphorylation may be important to the maintenance of steroid binding capacity.

Alkaline Phosphatase↗
PubMed1984

Microbial transformation of primaquine by Candida tropicalis.

The microbial metabolism of primaquine, a 6-methoxy-8-aminoquinoline antimalarial agent, was investigated. The yeast Candida tropicalis was found to convert primaquine to the previously reported N-acetylated derivative. On continued incubation of C. tropicalis in the presence of the N-acetylated derivative, a minor dimeric metabolite was formed. The proposed structure of the metabolite was based primarily on the analysis of its spectroscopic properties (1H and 13C nuclear magnetic resonance spectra and field-desorption mass spectrum). The structure of the metabolite was proven by direct comparison with an authentic sample of the minor dimeric metabolite prepared by treatment of the N-acetylated derivative with formaldehyde in the presence of formic acid in methanol.

Acetylation↗
PubMed1984

Production of a novel dimeric metabolite of primaquine by Streptomyces rimosus.

Primaquine, an 8-amino-6-methoxyquinoline antimalarial agent, was subjected to metabolic studies with microorganisms. Streptomyces rimosus converted primaquine to the previously reported N-acetyl derivative. Continued incubation of S. rimosus resulted in the formation of a minor dimeric metabolite. The structure of the minor dimeric metabolite was proposed based primarily on its spectral data (1H and 13C nuclear magnetic resonance spectra and mass spectrum). The proposed structure of the metabolite was confirmed by synthesis of the dimer by treatment of primaquine-N-acetate with potassium ferricyanide in a biphasic chloroform-aqueous sodium bicarbonate system with a phase-transfer catalyst. Since (+/-)-primaquine was used for both the microbial transformation and synthesis, a diastereomeric mixture of symmetrical dimers was formed in each case. The metabolite sample was identical to the synthetic sample, as shown by direct comparison (thin-layer chromatography, co-thin-layer chromatography, high-pressure liquid chromatography, co-high-pressure liquid chromatography, 1H nuclear magnetic resonance spectra, and mass spectrum).

Chromatography, High Pressure Liquid↗
PubMed1984

Transformation of highly purified avian progesterone receptor.

Transformation of the avian progesterone receptor to the nuclear form was studied using highly purified receptor preparations. The progesterone receptor was purified to near homogeneity in the presence of 10 mM sodium molybdate by affinity chromatography on deoxycorticosterone-Sepharose followed by DEAE-Sephadex chromatography. This latter step resolved the receptor into two 8S forms, I and II. Receptor transformation was measured by the binding of receptor to the polyanion resins (DNA-cellulose, phosphocellulose, or ATP-Sepharose) and to isolated nuclei and by the change in sedimentation coefficient from 8S to 4S. Molybdate was removed from the purified receptor preparations by agarose gel filtration. This step resulted in transformation of a major portion of receptor, as indicated by the above criteria. The extent of transformation was enhanced slightly by further incubation of the receptor in 0.2 M KCl. Control samples, which contained 10 mM molybdate, remained nontransformed, as tested by sedimentation or binding analysis. However, receptor transformation could not be reversed by adding molybdate back to transformed receptor. Although transformation of both receptor components I and II was observed, the extent of component I transformation was generally 2- to 5-fold greater than that of component II. About 50-90% of component I could be converted to a form that bound DNA-cellulose, ATP-Sepharose, and phosphocellulose. Since the progesterone receptor is a phosphoprotein, 32P-labeled receptor was tested for any loss of phosphate during transformation or receptor inactivation by incubation at 37 C. No observable loss of 32P occurred with either treatment. Our results show that transformation of the 8S receptor components I and II can be achieved in the absence of other cytosolic factors.

Animals↗
PubMed1982

Phosphorylation in vivo of chicken oviduct progesterone receptor.

Progesterone receptor from an oviduct tissue mince incubated with (32P)orthophosphate was purified using affinity chromatography, gel filtration, and DEAE-Sephadex chromatography. Two receptor peaks were eluted from the DEAE column. Peak I contained one major protein band with a molecular weight of 90,000 and a 32P band at 90,000. Peak II also had a major 90,000-dalton protein band and a 32P band at the same position. In addition, peak II contained a major protein band at 104,000 daltons and a 32P band which appeared to be slightly larger. Peaks I and II from chickens injected with (32P)orthophosphate showed the same pattern of labeling. The 90,000-molecular weight proteins from peaks I and II and the 32P-labeled component of II with Mr greater than 104,000 all contained phosphoserine. On two-dimensional gels, the 90,000-dalton proteins from peaks I and II were indistinguishable. Both contained labeled phosphate. Two-dimensional gels separated the 104,000-dalton peptide from the slightly larger 32P-labeled protein which traveled close to it on a one-dimensional gel. The relationships of the 104,000-dalton protein and this phosphoprotein to the progesterone receptor are not known. The 90,000-dalton receptor form common to both peaks is a phosphoprotein.

Animals↗
PubMed1982

Purification of "nontransformed" avian progesterone receptor and preliminary characterization.

The nontransformed (molybdate-stabilized) avian oviduct progesterone receptor has been purified to near homogeneity by a simple four-step procedure: ammonium sulfate fractionation, affinity chromatography, gel filtration, and DEAE-Sephadex A-25 chromatography. The affinity resin (deoxycorticosterone-agarose) is very resistant to chemical breakdown and enzymatic destruction and can be used repeatedly. The material obtained after gel filtration was separated into two receptor components by DEAE-chromatography. The two components, termed I and II, are obtained in about 18% yield and are purified by about 6000-fold (I) and 4000-fold (II). They are highly purified as only a single major Coomassie blue-stained polypeptide of about Mr = 90,000 in both components I and II is seen on sodium dodecyl sulfate-gel electrophoresis plus a second band of Mr = 104,000 which is seen only in component II. Both purified receptor components have sedimentation coefficients of 8 S in glycerol gradients containing molybdate (10 mM). In the absence of molybdate, however, both receptor forms have sedimentation coefficients of about 4 S in gradients containing 300 mM KCl. Therefore, stabilization of the 8 S form by molybdate is still evident in highly purified receptor preparations. The Stokes radii, binding specificity, and steroid dissociation rate of purified receptor are similar to those found in crude cytosol. These results confirm our previous observation on the existence of two 8 S forms of the progesterone receptor and show that these molybdate-stabilized forms remain intact throughout purification.

Animals↗