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K R Anumula

Publications and source records attributed to K R Anumula.

26 records · Page 2Linked to original sources
PubMed1989

Quantitative determination of kinins released by trypsin using enzyme-linked immunosorbent assay (ELISA) and identification by high-performance liquid chromatography (HPLC).

Rapid ELISA and HPLC procedures were developed for quantitation and identification of various natural kinins. Antibradykinin mouse monoclonal antibodies were used to determine kinin levels in the range of 20-200 ng. Bradykinin coupled to bovine serum albumin was used to coat the plates in a 3- to 4-hr ELISA. Synthetic kinin standards isoleucine-seryl-bradykinin (Ileu-Ser-BK), methionyl-lysyl-bradykinin (Met-Lys-BK), tyrosine-bradykinin (Tyr-BK) and bradykinin (BK) yielded almost identical curves with a mixture of A5 and D9 monoclonal antibodies. [Tyr5]-BK, [Tyr8]-BK and des-arginine9 bradykinin (des-Arg9-BK) showed negligible amounts of cross-reactivity. ELISA-compatible trypsin digestion developed for release of kinins from plasma of normal humans, rats and turpentine-treated rats gave values of 3.2, 6.9 and 70 micrograms/ml plasma, respectively. High performance liquid chromatography methods were developed for complete resolution of kinins on a C-18 reversed phase mu-Bondapak column before and after derivatization with phenyl isothiocyanate (PITC). The simple PITC derivatization procedure yielded good quantitation above 20 pmol. The ELISA and HPLC methods were used in a complementary fashion to assay and identify kinins in biological fluids as well as during the course of kininogen purification.

Animals↗
PubMed1983

Release of glucose-containing polymannose oligosaccharides during glycoprotein biosynthesis. Studies with thyroid microsomal enzymes and slices.

Incubations of thyroid microsomes with radiolabeled dolichyl pyrophosphoryl oligosaccharide (Glc3Man9-GlcNAc2) under conditions optimal for the N-glycosylation of protein resulted in the release, by apparently independent enzymatic reactions, of two types of neutral glucosylated polymannose oligosaccharides which differed from each other by terminating either in an N-acetylglucosamine residue (Glc3Man9GlcNAc1) or a di-N-acetylchitobiose moiety (Glc3Man9GlcNAc2). The first mentioned oligosaccharide, which was released in a steady and slow process unaffected by the addition of EDTA, appeared to be primarily the product of endo-beta-N-acetylglucosaminidase action on newly synthesized glycoprotein and such an enzyme with a neutral pH optimum capable of hydrolyzing exogenous glycopeptides and oligosaccharides (Km = 18 microM) was found in the thyroid microsomal fraction. The Glc3Man9GlcNAc2 oligosaccharide, in contrast, appeared to originate from the oligosaccharide-lipid by a rapid hydrolysis reaction which closely paralleled the N-glycosylation step, progressing as long as oligosaccharide transfer to protein occurred and terminating when carbohydrate attachment ceased either due to limitation of lipid-saccharide donor or addition of EDTA. There was a striking similarity between oligosaccharide release and transfer to protein with lipid-linked Glc3Man9GlcNAc2 serving as a 10-fold better substrate for both reactions than lipid-linked Man9-8GlcNAc2. The coincidence of transferase and hydrolase activities suggest the possibility of the existence of one enzyme with both functions. The physiological relevance of oligosaccharide release was indicated by the formation of such molecules in thyroid slices radiolabeled with [2-3H]mannose. Large oligosaccharides predominated (12 nmol/g) and consisted of two families of components; one group terminating in N-acetylglucosamine, ranged from Glc1Man9GlcNAc1 to Man5GlcNAc1 while the other contained the di-N-acetylchitobiose sequence and included Glc3Man9GlcNAc2, Glc1Man9GlcNAc2, and Man9GlcNAc2.

Acetylglucosaminidase↗
PubMed1983

Biosynthesis of lutropin in ovine pituitary slices: incorporation of [35S]sulfate in carbohydrate units.

Sulfate incorporation into carbohydrate of lutropin (LH) has been studied in sheep pituitary slices using H2(35)SO4. Labeled ovine LH was purified to homogeneity by Sephadex G-100 and carboxymethyl-Sephadex chromatography from both the incubation medium and tissue extract. Autoradiography of the gel showed only two protein bands which comigrated with the alpha and beta subunits of ovine LH in both the purified ovine LH and the immunoprecipitate obtained with LH-specific rabbit antiserum. Furthermore, [35S]sulfate was also incorporated into several other proteins in addition to LH. The location of 35SO2-(4) in the oligosaccharides of ovine LH was evidenced by its presence in the glycopeptides obtained by exhaustive Pronase digestion. The location and the point of attachment of sulfate in the carbohydrate unit were established by the isolation of 4-O-[35S]sulfo-N-acetylhexosaminyl-glycerols and 4-O-[35S] sulfo-N-acetylglucosaminitol from the Smith degradation products and by the release of 35SO2-(4) by chondro-4-sulfatase. Thus, the present line of experimentation indicates the presence of sulfate on both the terminal N-acetylglucosamine and N-acetylgalactosamine in the oligosaccharide chains of the labeled ovine LH.

Animals↗
PubMed1981

Isolation and properties of alpha-D-mannose:beta-1,2-N-acetylglucosaminyltransferase from trachea mucosa.

A beta-1,2-N-acetylglucosaminyltransferase which transfers N-acetylglucosamine from UDP-N-acetylglucosamine to the branched terminal mannosyl residues found in the complex type oligosaccharide units of N-linked glycoproteins was isolated from swine trachea mucosa. The enzyme was purified from microsomes after solubilization with 1% Triton X-100 and 1% Nonidet P-40, and it was also isolated from a soluble extract of this tissue. The enzyme was purified by chromatography on DEAE-cellulose, cellulose phosphate, and Sephacryl S300 columns and by affinity chromatography on a Sepharose 4B column containing covalently bound ovomucoid. The purified enzyme forms beta-1,2 bonds between N-acetylglucosamine and terminal-branched mannosyl residues of glycoproteins and glycopeptides. The enzyme has an absolute specificity for terminal branched mannosyl residues, and solute specificity for terminal branched mannosyl residues, and no activity is observed with mannose, p-nitrophenyl alpha-D-mannoside, p-nitrophenyl beta-O-mannoside, and glycopeptides which contain unbranched oligosaccharide chains terminating in free mannose. Evidence obtained by kinetic and structural analysis shows that the purified enzyme catalyzes the reactions shown in Scheme I.

Animals↗
PubMedDate not supplied

Fluorescent N-methylanthranilyl (Mantyl) tag for peptides: its application in subpicomole determination of kinins.

Highly fluorescent N-methylanthranilyl (Mantyl) peptide derivatives were prepared by a one-step reaction with N-methylisatoic anhydride (MIA) for quantitative detection in HPLC. Reactions were carried out in an organic medium of acetonitrile-triethylamine, in aqueous alkaline sodium carbonate and sodium phosphate buffers. 4-Dimethylaminopyridine (DMAP) catalyzed specific mantylation of -NH2 groups of peptides in the organic reaction medium. The DMAP had no effect in the aqueous buffered reaction systems. Proline amino-terminal peptides reacted equally well with MIA. Mantyl-bradykinin had excitation and fluorescence maxima at 350 nm and 426 nm in water and water/acetonitrile (ACN)/trifluoroacetic acid (TFA) solvent mixtures, respectively. Fluorescence intensity increased with an increase in ACN concentration and decreased with an increase in acid content. Mantyl kinins were completely resolved on a C18 reversed phase HPLC column using an ACN-0.1% TFA gradient and their behavior on the column was similar to having an extra amino acid. Di-Mantyl derivatives obtained with Lys-BK and Met-Lys-BK did not exhibit fluorescence appreciably higher than Mantyl-BK. Fluorescence detection of Mantyl kinins was about 50-100 times more sensitive (lower limits of 0.1 to 0.5 picomole) than UV detection of the phenylisothiocyanate-derivatized kinins under typical HPLC conditions.

4-Aminopyridine↗