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Biomedical subjects

V K Kalra

Publications and source records attributed to V K Kalra.

At least 55 records · Page 3Linked to original sources
PubMed1982

Phospholipid methylation of kidney cortex brush border membranes. Effect on fluidity and transport.

Exposure of intact brush border membrane vesicles of hog kidney cortex to cholesterol oxidase resulted in 24% oxidation of membrane cholesterol compared with more than 95% oxidation of cholesterol in lipids isolated from membranes, showing that cholesterol is asymmetrically distributed in membranes. Phospholipase C, hydrolyzed 76% of phosphatidylcholine and 10-12% phosphatidylethanolamine while phosphatidylserine was not hydrolyzed, thus indicating that majority of phosphatidylcholine is present on the outer surface of these vesicles while phosphatidylethanolamine and phosphatidylserine are present on the inner surface. Methylation of phospholipids in brush border membrane with S-adenosyl-[methyl-3H]methionine resulted in the formation of phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N]dimethylethanolamine and phosphatidylcholine from endogenous phosphatidylethanolamine. The Km for S-adenosylmethionine was 1.10(-4) M with an optimum pH 9.0 for the formation of all three methyl derivatives. Mg2+ was without any effect between pH 5 to 10. Addition of exogenous mono- and dimethylphosphatidylethanolamine derivatives enhanced methyl group incorporation by 4-5-fold as compared to the addition of phosphatidylethanolamine. The conversion of endogenous phosphatidylethanolamine to phosphatidyl-N-monomethylethanolamine or addition of exogenous phosphatidylmonomethylethanolamine to brush border membrane did not result in a change in bulk membrane fluidity as determined by fluorescence polarization of diphenylhexatriene. Methylation of phosphatidylethanolamine in brush border membrane did not affect the Na+-dependent uptake of either D-glucose or phosphate, although the accessibility of cholesterol in membrane to cholesterol oxidase was diminished by 21%, presumably due to altered flip-flop movement of cholesterol in the membrane.

Animals↗
PubMed1982

Proteoliposome interaction with human erythrocyte membranes. Functional implantation of gamma-glutamyl transpeptidase.

The transfer of detergent solubilized and purified gamma-glutamyl transpeptidase (gamma-GTase), of hog kidney cortex, from proteoliposomes into human erythrocyte ghost membranes has been studied. The transfer of gamma-glutamyl transpeptidase was observed upon incubation of gamma-GTase incorporated dipalmitoylphosphatidylcholine vesicles with erythrocyte ghost membranes at 37 degrees C for 12 h. The extent of transfer was dependent upon the fluidity of donor proteoliposomes, being more when dipalmitoylphosphatidylcholine proteoliposomes were used compared to dimyristoylphosphatidylcholine, and intermediate values were observed when binary mixtures of DMPC and DPPC were used. Moreover, the transfer of gamma-GTase was facilitated when rigid basic phospholipid proteoliposomes were used as donor. The transfer of gamma-GTase has been observed to be associated with the removal of intrinsic membrane proteins and lipids from erythrocytes, mainly acetylcholinesterase, sphingomyelin, and cholesterol. An enhancement in the fluorescence due to resonance energy transfer was observed when ghost membranes containing fluorescent donor probe were incubated with proteoliposomes containing fluorescent acceptor probe, indicating that fusion but not adsorption of vesicles occurs during the transfer process. However, the inability of entrapped [14C]-sucrose delivery from proteoliposomes into ghost membrane vesicle suggest that fusion per se is not primarily involved in the transfer process. It appears that the transfer of gamma-glutamyl transpeptidase occurs by a collisional transfer process resulting in intermembrane protein transfer. The gamma-glutamyl transpeptidase implanted ghost membranes exhibited the uptake of L-glutamate which was inhibited by serine-borate, an inhibitor of transpeptidase activity. In addition, the uptake of L-glutamate was inhibited by the dipeptide gamma-glutamyl-L-glutamate, thus supporting the proposed role of gamma-glutamyl transpeptidase in the uptake of amino acids in biological membranes.

Amino Acids↗
PubMed1981

Transport of amino acids in gamma-glutamyl transpeptidase-implanted human erythrocytes.

gamma-Glutamyl transpeptidase purified from hog kidney cortex was implanted in the human erythrocyte membrane by incubation of erythrocytes at 37 degrees c with gamma-glutamyl transpeptidase-incorporated dipalmitoyl phosphatidylcholine vesicles. Membranes prepared from these implanted cells exhibited 4- to 5-fold increase in gamma-glutamyl transpeptidase activity. The association/insertion of gamma-glutamyl transpeptidase into erythrocyte membrane was further demonstrated by antibody to gamma-glutamyl transpeptidase. Implantation of gamma-glutamyl transpeptidase into erythrocyte membrane led to stimulation of uptake of glutamate and alanine, which are normally transported at a slow rate in human erythrocytes. The uptake of these amino acids in the implanted system was inhibited by inhibitors (serine-borate and azaserine) of transpeptidase activity as well as by antibody to gamma-glutamyl transpeptidase. These results in the implanted human erythrocytes demonstrate that gamma-glutamyl transpeptidase enzyme can mediate the translocation of amino acids and provide further evidence in support of its postulated role in the transport of amino acids in natural membranes.

Alanine↗
PubMed1980

Gamma-glutamyl transpeptidase-mediated transport of amino acid in lecithin vesicles.

Gamma-Glutamyl transpeptidase, a membrane-bound enzyme purified from hog kidney cortex following solubilization with detergent, was incorporated by sonication procedure into large phospholipid vesicles using a 1:2 molar ratio of deoxycholate to phospholipid. tthese vesicles, when containing entrapped glutathione, exhibited the uptake of L-[14C]glutamate, but not of L-proline. The glutamate uptake did not occur to a significant extent in these vesicles when glutathione was added externally. The uptake of glutamate in the reconstituted system was sensitive to the temperature of incubation and to the inhibitors (serine-borate, azaserine, and 6-diazo-5-oxo-L-norleucine) of gamma-glutamyl transpeptidase activity. The accumulated product during the transport of glutamate in the gamma-glutamyl transpeptidase reconstituted system containing intravesicular glutathione was identified as gamma-glutamyl-glutamate. These results indicate that gamma-glutamyl transpeptidase mediates the translocation of glutamate in vitro and provides evidence in support of its role in the transport of amino acids in natural membranes.

Animals↗
PubMed1980

Fusion of human erythrocytes induced by uranyl acetate and rare earth metals.

Incubation of human erythrocytes with either uranyl ions (UO22+) or rare earth metals (La3+, Nd3+, Sm3+, Eu3+, Tb3+, Dy3+ and Yb3+) at 37 degrees C for 30-45 min resulted in the fusion of erythrocytes. Redistribution of membrane-associated particles was observed using colloidal-iron charge labelling and freeze-fracture electron microscopy. The fusion of erythrocytes induced by these agents, unlike Ca2+, did not exhibit the absolute requirement for phosphate. Moreover, agglutination and fusion by these agents was observed in neuraminidase-treated erythrocytes in contrast to Ca2+- and phosphate-induced fusion. Inhibitors of intrinsic transglutaminase activity partially inhibited (35-45%) the fusion induced by UO22+ suggesting that cross-linking of membrane proteins results in protein-free areas of lipid where fusion may be initiated.

Calcium↗
PubMed1979

Resolution and reconstitution of active transport of calcium by a protein(s) from Mycobacterium phlei.

Membrane protein(s) responsible for the active transport of calcium in membrane vesicles from Mycobacterium phlei have been solubilized from membranes by sodium cholate treatment and partially purified using a hydrophobic resin. Reconstitution of calcium transport was demonstrated by reconstitution of detergent extracted membranes with the partially purified protein. The uptake of calcium in the reconstituted system was sensitive to proton-conducting uncouplers. Liposomes prepared with partially purified calcium translocating protein were capable of accumulating calcium. The uptake of calcium in this system occurred as a result of an artificial proton gradient generated by the reduction of entrapped ferricyanide with ascorbate-benzoquinone serving as a hydrogen carrier. The addition of the ionophore A23187 caused efflux of accumulated calcium in both native and proteoliposomal-reconstituted system.

Biological Transport, Active↗
PubMed1979

Photoaffinity labeling of procaine-binding sites in normal and sickle cell membranes.

A photoaffinity probe, procaine azide, was employed to determine the sites of interaction of procaine in normal and sickle cell erythrocytes. Studies show that the number of binding sites and affinity of procaine to membranes derived from normal and sickled cell erythrocytes were similar, although procaine retards the in vitro formation of irreversibly sickled cells from cells, The results show that procaine azide, a photoaffinity analogue of procaine, is covalently incorporated into both protein (60--70%) and lipid (40--30%) components of the membrane. Sodium dodecyl sulfate-gel electrophoresis of the labeled ghosts show that procaine binds specifically to band 3 and periodic acid Schiff staining bands in membranes derived from labeled erythrocytes. Binding of procaine or covalent incorporation of procaine azide into membrane proteins does not affect the phosphate transport. Moreover, pre treatment of intact erythrocytes with 4,4-'diisothiocyano-2,2'-stilbene disulfonate, an anion transport inhibitor, did not affect either the binding or covalent incorporationof procaine azide into erythrocytes. These results indicate that the binding of procaine azide to Band 3 protein occurs at a locus different than that involved in anion translocation process.

Affinity Labels↗
PubMed1977

Trypsin-induced changes in the orientation of latent ATPase in protoplast ghosts from Mycobacterium phlei.

Latent ATPase, located on the inner surface of protoplast ghosts of Mycobacterium phlei, was unmasked either by trypsin or an impermeable form of trypsin, ethylene maleic anhydride-trypsin. Density gradient experiments showed that the ghost preparations remained intact following trypsin treatment. Evidence was obtained that 125I-trypsin failed to penetrate the ghost membranes. Thus, attempts were made to determine whether the ATPase molecule in the ghost membranes is accessible from the outer surface. Treatment of protoplast ghosts and trypsin-treated ghosts with 125I by the lactoperoxidase method resulted in the labeling of ATPase only in the trypsin-treated ghost preparations. The antibody to latent ATPase inhibited ATPase activity in trypsin-treated ghosts. The changes in the fluorescence polarization of diphenyl hexatriene indicated that trypsin treatment of the ghost membranes resulted in an increase in membrane fluidity. These studies suggest that the latent ATPase moiety has undergone translocation to the outer surface or it became accessible to trypsin digestion from the outer surface of the membranes as a result of removal of some proteins covering ATPase molecule in the membranes.

Adenosine Triphosphatases↗
PubMed1977

Binding of nucleotides to purified coupling factor-latent ATPase from Mycobacterium phlei.

Binding studies of various nucleotides to the purified coupling factor-latent ATPase from Mycobacterium phlei have been carried out using gel filtration, equilibrium dialysis, and ultrafiltration methods. The purified latent ATPase binds 3 mol of ADP per mol of the enzyme with an apparent dissociation constant of 68 muM. Binding of nucleotides occurred in the decreasing order: ADP, epsilon-ATP, epsilon-ADP, UDP, adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), IDP, and adenosine 5'-(alpha,beta-methylene)diphosphate (AdoP(CH2)P). AMP-P(NH)P inhibits both soluble (Ki = 77 muM) and membrane-bound latent ATPase activity. However, AMP-P(NH)P does not affect oxidative phosphorylation in membrane vesicles of M. phlei. AMP-P(NH)P exhibits one binding site per molecule of the enzyme with a dissociation constant of 71 muM. After trypsin treatment of the enzyme, the binding of ADP decreases 35%, while AMP-P(NH)P binding remains unchanged. Moreover, AMP-P(NH)P binding was not displaced by ADP. Studies with sulfhydryl agents showed that, in contrast to AMP-P(NH)P, binding of at least 1 mol of ADP requires the participation of sulfhydryl groups. The results indicate that AMP-P(NH)P and ADP do not share a common binding site and that the latent ATPase enzyme has separate sites for ATP hydrolysis and ATP synthesis.

Adenosine Diphosphate↗