Biomedical subjects

C Kasinathan

Publications and source records attributed to C Kasinathan.

30 records · Page 2Linked to original sources

PubMed1991

Identification of gastric mucosal mucus glycoprotein sulfotransferase.

Sulfation of mucus glycoproteins, reaction catalyzed by Golgi resident sulfotransferase, is an important event in posttranslational processing of gastric mucins. Here we report the purification of mucus glycoprotein sulfotransferase enzyme from the microsomal fraction of rat gastric mucosa. The enzyme was released from the membrane with 0.5% Triton X-100 and precipitated from the 100,000xg supernatant with 90% ice-cold acetone. The enzyme activity (44.7 pmol/mg/45 min) in the precipitate was enriched nearly 10-fold compared to Triton X-100 extract of microsomal membrane (4.2 pmol/mg/45 min). On SDS-PAGE, the enzyme gave a single 43 kDa protein band, which was active towards mucin, but did not catalyze the sulfation of galactosylceramide. The study is the first to report the characteristics of a sulfotransferase enzyme specific for gastric mucin.

Animals↗

PubMed1990

Purification of protein fatty acyltransferase and determination of its distribution and topology.

Studies reported from this laboratory have demonstrated that O-glycosidic glycoproteins of salivary, pulmonary, and gastrointestinal origin are acylated by fatty acyltransferase residing in Golgi and microsome-enriched fraction (Slomiany, A., Liau, Y.H., Takagi, A., Laszewicz, W., and Slomiany, B.L. (1984) J. Biol. Chem. 259, 13304-13308). Here we report on the successful purification of this enzyme from rough microsomal membranes of rat gastric mucosa and its identification in a number of diverse tissues and organs, such as heart, liver, pancreas, lung, kidney, salivary glands, and lymphoblasts. The enzymatic activity has been released from the stripped and salt-extracted microsomes with 0.5% Triton X-100 and recovered from 100,000 x g supernatant by affinity chromatography on Cibacron blue F3GA column. The retained fatty acyltransferase protein was selectively displaced from the column with 50 microM palmitoyl-CoA. On nonreducing polyacrylamide gel electrophoresis, the enzymatic activity was associated with a 234-kDa complex, and on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the complex afforded 65- and 67-kDa protein bands. Incubation of microsomes with trypsin prior to enzyme extraction resulted in a 50% inactivation of the fatty acyltransferase and generation of 53- and 55-kDa protein bands, which also had affinity to Cibacron blue F3GA and were displaced from the column together with the active (intact) enzyme. We suggest that the fatty acyltransferase is an integral rough microsomal protein partially exposed to cytosol, which catalyzes the fatty acyl-CoA-protein reaction on the cytosolic site of the rough endoplasmic reticulum and that this enzyme is responsible for processing of the group of protein which are entering rough endoplasmic reticulum-Golgi secretory pathway.

Acyl Coenzyme A↗

PubMed1989

Polyphosphoinositide formation in isolated cardiac plasma membranes.

Phosphatidylinositol (PtdIns) and phosphatidylinositol 4-phosphate (PtdIns4P) kinase activities in plasma membranes isolated from canine left ventricle were partially characterized, and their sensitivity to a number of intracellular variables was established. PtdIns and PtdIns4P kinase activities were estimated by the formation of [32P]PtdIns4P and [32P]phosphatidylinositol 4,5-bisphosphate ([32P]PtdIns(4,5)P2), respectively, when membranes were incubated with [gamma-32P]ATP and 0.1% Triton X-100. Unlike [32P]PtdIns4P formation [32P]PtdIns(4,5)P2 formation required exogenous (PtdIns4P) substrate. [32P]PtdIns4P and [32P]PtdIns(4,5)P2 formation were insensitive to Ca2+ at concentrations ranging from 0.1-30 microM. The hydrolysis of [32P]PtdIns4P was less than 15% under standard assay conditions for measuring its formation, and was unaffected by any of the variables tested. The apparent Km of the PtdIns kinase for ATP was 53 +/- 13 (S.E.M.) microM (N = 3). ADP inhibited [32P]PtdIns4P formation competitively with respect to ATP, the Ki being 0.4 mM. The data indicate that ADP is a poor competitive inhibitor of PtdIns kinase at the concentrations which are believed to be present intracellularly normally or which may be attained during mild hypoxia provided ATP levels are maintained in the millimolar range. Hence, any response of the myocardium to alpha-adrenergic hormones during mild hypoxia would be largely unimpaired by effects of Ca2+ on PtdIns and PtdIns(4,5)P2, or of ADP on PtdIns kinase activity.

1-Phosphatidylinositol 4-Kinase↗

PubMed1989

Lipolytic activity of Campylobacter pylori: effect of colloidal bismuth subcitrate (De-Nol)

Infection by Campylobacter pylori appears to play a major role in the etiology of gastric disease, but the nature of impairment evoked by this pathogen in gastric mucosal defense is not well understood. We present here evidence that the extracellular material elaborated by this microorganism exhibits lipolytic activity capable of gastric mucosal lipid degradation. The colonies of bacteria, cultured from antral mucosal biopsy specimens of patients undergoing endoscopy, were washed with saline, passed through a sterilization filter, and the filtrate was examined for lipase and phospholipase activities. By following the degradation of glycerol trioleate and dipalmitoyl phosphatidylcholine, we established the presence of lipase and phospholipase A enzymes. The major product of the triglyceride degradation was glycerol monooleate, while lysophosphatidylcholine resulted from the degradation of phosphatidylcholine. The lipolytic activity of C. pylori filtrate was inhibited by an antiulcer drug, De-Nol, which at 150 micrograms/ml caused a 21% reduction in lipase activity and a 60% reduction in the activity of phospholipase A. The results suggest that De-Nol is capable of preventing degradation of mucosal lipids by C. pylori lipases and, hence, helps to preserve the mucosal integrity.

Anti-Ulcer Agents↗

PubMed1988

Differences in calcium uptake in native canine cardiac microsomes are correlated with the ratio of unphosphorylated to phosphorylated phospholamban as determined by Western blot analysis.

Phospholamban (PLM) is detectable by Western blot analysis of canine cardiac microsomes using rabbit antiserum against a peptide containing the 2 to 30 amino acid sequence of PLM. Phosphorylated PLM is distinguishable from the unphosphorylated form by virtue of a reduced electrophoretic mobility. Utilizing digital image analysis to determine relative band densities, it was found that the ratio of unphosphorylated to phosphorylated PLM is correlated with the rate of calcium uptake in 5 preparations of native microsomes (r = 0.94, p less than 0.01). The present analysis may be useful for determining the phosphorylation state of PLM in microsomes obtained from animals in physiological states characterized by impaired sarcoplasmic reticulum calcium pump activity.

Adenosine Triphosphate↗

PubMed1988

Presence of a Ca2+-sensitive CDPdiglyceride-inositol transferase in canine cardiac sarcoplasmic reticulum.

Sarcoplasmic reticulum (SR) and plasma membranes from canine left ventricle were used to evaluate the presence of the enzyme CDPdiglyceride-inositol transferase in these membranes. (K+,-Ca2+)-ATPase activity, a marker for SR, was 79.2 +/- 5.0 (SE) and 11.2 +/- 2.0 mumol.mg-1.h-1 in SR and plasma membrane preparations, respectively, and (Na+,K+)-ATPase activity, a marker for plasma membranes, was 5.6 +/- 1.2 and 99.2 +/- 8.0 mumol.mg-1.h-1, respectively. Contamination of SR and plasma membrane preparations by mitochondria was estimated to be 2% and 8%, respectively, and by Golgi membranes, 0.9% and 1.8%, respectively. Transferase activity, measured at pH 6.8, was 1.32 +/- 0.04 (SE) and 0.28 +/- 0.04 nmol of [3H]phosphatidylinositol ([3H]PtdIns).mg-1.min-1 in three SR and plasma membrane preparations, respectively. The transferase activity detected in the plasma membrane preparation could be accounted for largely, but not entirely, by contaminating SR membranes. The pH optimum for the SR transferase activity was between 8.0 and 9.0; little or no activity was detectable at pH 6.3 and 5.5, the lowest pH tested. Ca2+ inhibited the enzyme, half-maximal inhibition occurring at about 10 microM Ca2+; removal of the Ca2+ by addition of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid restored activity. No loss of [3H]PtdIns could be detected when membranes were incubated in the presence or absence of Ca2+. The Ca2+ inhibition of the transferase was noncompetitive with respect to CDP-dipalmitin while that with respect to myo-inositol was slightly noncompetitive at low [Ca2+] and became uncompetitive at higher [Ca2+].(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗

PubMed1986

Proteolytic activation of the canine cardiac sarcoplasmic reticulum calcium pump.

Mild trypsin treatment of canine cardiac microsomes consisting largely of sarcoplasmic reticulum vesicles produced a severalfold activation of oxalate-facilitated calcium uptake. The increase in calcium uptake was associated with an increase in ATP hydrolysis. Proteases other than trypsin were also effective although to a lesser degree. Trypsin produced a shift of the Ca2+ concentration dependency curve for calcium uptake toward lower Ca2+ concentrations, which was almost identical with that produced by phosphorylation of microsomes by cyclic AMP dependent protein kinase when the trypsin and the protein kinase were present at maximally activating concentrations. The Hill numbers (+/- SD) of the Ca2+ dependency after treatment of microsomes with trypsin (1.5 +/- 0.1) or protein kinase (1.7 +/- 0.1) were similar and were not significantly different from those for untreated control microsomes (1.6 +/- 0.1 and 1.8 +/- 0.1, respectively). Autoradiograms of sodium dodecyl sulfate-polyacrylamide electrophoretic gels indicate that 32P incorporation into phospholamban (Mr 27.3K) or its presumed monomeric subunit (Mr 5.5K) was markedly reduced when trypsin-treated microsomes were incubated in the presence of cyclic AMP dependent protein kinase and [gamma-32P]ATP compared to control microsomes incubated similarly but pretreated with trypsin inhibitor inactivated trypsin. The activation of calcium uptake by increasing concentrations of trypsin was paralleled by the reduction of phosphorylation of phospholamban. Trypsin treatment of microsomes previously thiophosphorylated in the presence of cyclic AMP dependent protein kinase and [gamma-35S]thio-ATP did not result in a loss of 35S label from phospholamban, which suggests that phosphorylation of phospholamban protects against trypsin attack.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1984

Phospholipid synthesizing enzymes of dermatophytes. III. Glycerol kinase of dermatophytes.

Glycerol kinase, the key enzyme for glycerol use in phospholipid synthesis, was identified in cytosolic fractions of 2 dermatophytes, Microsporum gypseum and Epidermophyton floccosum. Ammonium sulfate was observed to activate and stabilize this enzyme in both dermatophytes. Two pH optima, 8.0 and 10.5, were observed for both dermatophyte enzymes. Glycerol kinase from M. gypseum was purified up to 33-fold with a 225% recovery by ammonium sulfate precipitation and gel filtration. The molecular weight of the enzyme was ca. 4.5 X 10(5). It had 2 pH optima of 8.0 and 10.5. The enzyme had Km values of 0.35 mM and 2.3 mM for glycerol and ATP. Reactivity of the enzyme for various nucleotides was ATP greater than TTP greater than GTP greater than ITP = CTP = UTP. Kinetic studies showed the enzyme to catalyze the reaction by the ping-pong mechanism. Fructose 1,6-bisphosphate and glucose-6-phosphate inhibited the enzyme competitively, whereas glucose was not inhibitory.

Ammonium Sulfate↗

PubMed1983