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A K Grover

Publications and source records attributed to A K Grover.

At least 91 records · Page 5Linked to original sources
PubMed1986

Pig coronary artery smooth muscle: substrate and pH dependence of the two calcium pumps.

The properties of the pig coronary artery smooth muscle Ca transport mechanisms and the effects of pH were examined. The Ca pump in the plasma membrane requires less than 50 microM Mg-ATP2- for half-maximal activity, but the pump in the endoplasmic reticulum requires greater than 700 microM Mg-ATP2-. The Ca uptake by the various subcellular organelles occurs with the following affinity characteristics towards Ca2+: plasma membrane, Km = 0.91 +/- 0.06 microM, and Hill coefficient = 1.31 +/- 0.09; endoplasmic reticulum, Km = 0.58 +/- 0.05 microM, and Hill coefficient = 2.48 +/- 0.34; and mitochondria, Km = 7.1 +/- 1.16, and Hill coefficient = 0.93 +/- 0.10. The active Ca transport systems showed similar pH dependence in that the Ca uptake at pH 6.8 was greater than at 7.6. However, the pH effects were much larger on the Ca uptake by the endoplasmic reticulum and mitochondrial membranes than by the plasma membrane. The plasma membrane also showed a small amount of pH-dependent, high-affinity Ca binding. Ca efflux from passively loaded plasma membrane vesicles was similar at pH 6.4 and 7.4. From these data, it is concluded that the endoplasmic reticulum Ca pump may play a larger role in the vasodilation of large coronary artery, but the role of the plasma membrane may be more in maintenance of relaxation mechanism.

Adenosine Triphosphate↗
PubMed1986

Muscarinic receptor size on smooth muscle cells and membranes.

The loss of [3H]quinuclidinyl benzilate ([3H]QNB) binding following high-energy radiation was used to compare the muscarinic receptor size on single smooth muscle cells isolated by collagenase digestion from the canine stomach and on plasma membranes derived from intact gastric smooth muscle without exposure to exogenous proteolysis. Radiation inactivation of galactose oxidase (68 kdaltons), yeast alcohol dehydrogenase (160 kdaltons), and pyruvate kinase (224 kdaltons) activities were used as molecular-weight standards. Radiation inactivation of [3H]QNB binding to rat brain membranes, which gave a target size of 86 kdaltons, served as an additional control. In isolated smooth muscle cells, the calculated size of the muscarinic receptor was 80 +/- 8 kdaltons. In contrast, in a smooth muscle enriched plasma membrane preparation, muscarinic receptor size was significantly smaller at 45 +/- 3 kdaltons. Larger molecular sizes were obtained either in the presence of protease inhibitors (62 +/- 4 kdaltons) or by using a crude membrane preparation of gastric smooth muscle 86 +/- 7 kdaltons).

Animals↗
PubMed1986

Calmodulin stimulation of plasmalemmal Ca2+-pump of canine aortic smooth muscle.

Plasma-membrane-enriched fractions of canine aortic smooth muscle possess an ATP-supported Ca2+ accumulation which has an absolute requirement for Mg2+ and a high affinity for Ca2+ (Km approximately 0.5 microM). The rate of ATP-supported Ca2+ transport is not affected by several calmodulin antagonists, but is stimulated by exogenously added calmodulin. The maximal effect of calmodulin on the rate of ATP-dependent Ca2+ transport (at 5.0 microM Ca2+) occurs at 10 micrograms/ml calmodulin and represents an approximately 3-fold stimulation. This calmodulin stimulation of Ca2+ transport does not require pretreatment of the membranes by EGTA and is an intrinsic property of the plasma membranes. A high-affinity Ca2+-ATPase (Km for Ca2+ approximately 0.5 microM) is also present in the aortic smooth muscle plasma membrane. This high-affinity Ca2+-ATPase does not require Mg2+ for catalytic activity, but is in fact inhibited by increasing Mg2+ concentrations. Calmodulin at concentrations effective for the stimulation of the ATP-dependent Ca2+ transport has no effect on the high-affinity Ca2+-ATPase activity or on the basal ATPase activity stimulated by 5 mM Mg2+ or Ca2+. Our results indicate that isolated plasma membranes of canine aortic smooth muscle contain no endogenous calmodulin. The ability of exogenously added calmodulin to stimulate the rate of ATP-dependent Ca2+ transport by vascular smooth muscle plasma membranes suggests that calmodulin may play a role in lowering the cytoplasmic concentration of ionized calcium during vasodilatation. An Mg2+-independent, but not an Mg2+-dependent high-affinity Ca2+-ATPase, was identified in the plasma membranes. This may be separate from the plasmalemmal Ca2+-pump.

5'-Nucleotidase↗
PubMed1986

Determination of molecular size of alpha-1 and alpha-2 adrenoceptors in rat mesenteric artery by radiation inactivation.

Radiation inactivation of alpha-1 and alpha-2 adrenoceptors in the purified plasma membranes of rat mesenteric artery has been performed with high energy electrons at -45 to -55 degrees C. Alpha-1 and alpha-2 adrenoceptor inactivation was monitored with [3H] prazosin and [3H]yohimbine binding, respectively. Internal endogenous and external standards of known molecular weight were used in these studies to determine the molecular size. The average value of D37 for the [3H]prazosin binding site was 6.75 +/- 0.62 Mrad (n = 4) with an estimated molecular size of 122,921 +/- 11,329 Daltons. However, the average value of D37 for the [3H] yohimbine binding site was higher (D37 = 10.05 +/- 0.91 Mrad) and accordingly the molecular size of this binding site was less than the [3H]prazosin binding sites (molecular weight = 82,540 +/- 7478 Daltons; n = 4). Irradiation did not change the dissociation constant of either radioligand, suggesting that the loss of the radioligand binding sites after radiation is due to receptor protein inactivation. These results confirm our earlier finding that [3H]prazosin and [3H]yohimbine bind to two distinct sites in the plasma membranes of rat mesenteric artery. Whether both of these sites are the subunits of a common macromolecule of alpha adrenoceptor on vascular smooth muscle in rat mesenteric artery cannot be concluded from these results. This report is the first one in the literature on the molecular size of alpha-1 and alpha-2 binding sites in vascular smooth muscle.

Animals↗
PubMed1986

Determination of the molecular weight of neuronectin, a conditioned medium-derived, substrate-binding neurite-extension factor: comparison with laminin using radiation-inactivation analysis.

Neurite outgrowth from a wide variety of peripheral neurons is stimulated and may be directed in culture by a substrate-binding factor(s) derived from medium conditioned over numerous types of cells. This factor, or family of factors, which we shall call neuronectin by reason of its ability to serve as an attachment molecule for neurons, has been studied by target-size analysis using radiation inactivation. The radiation-inactivation method has the unique advantage of providing a means for determining the actual functional size of a biologically active molecule irrespective of its state of purification. By this method, the functional size of the major neurite outgrowth-promoting activity (neuronectin) from mouse heart cell conditioned medium has been found to be 350,000 Da. While neuronectin has not yet been purified, determination of the actual functional size provides a framework within which possible models must fit. Thus, although neurite outgrowth-promoting activity in this system is found to be associated with a complex containing laminin, fibronectin, heparan sulfate proteoglycan, and other extracellular matrix molecules, the total size of the functional molecule or molecular complex serving as the major source of activity is limited to 350,000 Da. Consequently, our results suggest that neuronectin from mouse heart cell-conditioned medium is different from laminin (Mr approximately 900,000), a molecule that also exhibits neurite-promoting activity. In addition to the difference in molecular size, neuronectin and laminin differ in that laminin, unlike neuronectin, gives rise to toxic or inhibitory products when exposed to high-energy radiation.

Animals↗
PubMed1985

Calcium channel antagonist binding and pharmacology in rat uterine smooth muscle.

The calcium channel antagonists altered Ca-dependence of high K+-contractions of the estrogen dominant rat myometrium with the following pA2 values: PN-200-110, 10.63; nitrendipine, 9.56; nifedipine, 9.41; D-600, 9.05; and diltiazem, 7.57. Specific binding of 3H-nitrendipine occurred to the isolated plasma membrane vesicles with Kd of 0.1 to 0.3 nM and was inhibited by PN-200-110, nitrendipine, nifedipine and D-600, and slightly activated by diltiazem. The binding studies and the contractility studies were in excellent agreement for the three dihydropyridines, but not for D-600 and diltiazem.

Animals↗
PubMed1985

Target size of Ca-pumps in pig coronary artery smooth muscle.

The target sizes of the oxalate-independent Ca uptake by the plasma membrane enriched fraction F2, and the oxalate-stimulated Ca uptake by a fraction F3 slightly enriched in the endoplasmic reticulum were determined by radiation inactivation. The oxalate-independent Ca uptake was inactivated with a D37 value of 1.96 +/- 0.30 Mrad but the oxalate-stimulated Ca uptake had a D37 value of 0.45 +/- 0.07 Mrad. Thus, in the smooth muscle the oxalate-stimulated Ca uptake appeared to be due to a structure 3 to 6 times larger than was the oxalate-independent Ca uptake. The subcellular site of the ATP-dependent azide insensitive Ca uptake in the smooth muscle has been disputed in the past. It has been suggested to be plasma membrane (PM) by several workers, and endoplasmic reticulum (ER) by others. Recently, however, there has been substantial evidence to support the hypothesis that one Ca uptake system, unaffected by oxalate, resides in the PM and another, stimulated by oxalate, is located in the ER of the smooth muscle. The evidence has been reviewed recently. Here, we show that the two modes of Ca uptake differ in their target sizes as well. To our knowledge, this is the first report on the use of radiation inactivation to distinguish between the two modes of Ca uptake in any tissue.

Animals↗
PubMed1985

Subcellular fractionation of pig coronary artery smooth muscle.

A detailed procedure for subcellular fractionation of the smooth muscle from pig coronary arteries based on dissection of the proper tissue, homogenization, differential centrifugation and sucrose density gradient centrifugation is described. A number of marker enzymes and Ca2+ uptake in presence or absence of oxalate, ruthenium red and azide were studied. The ATP-dependent oxalate-independent azide- or ruthenium red-insensitive Ca2+ uptake, and the plasma membrane markers K+-activated ouabain-sensitive p-nitrophenylphosphatase, 5'-nucleotidase and Mg2+-ATPase showed maximum enrichment in the F2 fraction (15-28% sucrose) which was also contaminated with the endoplasmic reticulum marker NADPH: cytochrome c reductase, and to a small extent with the inner mitochondrial marker cytochrome c reductase, and also showed a small degree of oxalate stimulation of the Ca2+ uptake. F3 fraction (28-40% sucrose) was maximally enriched in the ATP- and oxalate-dependent azide-insensitive Ca2+ uptake and the endoplasmic reticulum marker NADPH: cytochrome c reductase but was heavily contaminated with the plasma membrane and the inner mitochondrial markers. The mitochondrial fraction was enriched in cytochrome c oxidase and azide- or ruthenium red-sensitive ATP-dependent Ca2+ uptake but was heavily contaminated with other membranes. Electron microscopy showed that F2 contained predominantly smooth surface vesicles and F3 contained smooth surface vesicles, rough endoplasmic reticulum and mitochondria. The ATP-dependent azide-insensitive oxalate-independent and oxalate-stimulated Ca2+ uptake comigrated with the plasma membrane and the endoplasmic reticulum markers, respectively, and were preferentially inhibited by digitonin and phosphatidylserine, respectively. This study establishes a basis for studies on receptor distribution and further Ca2+ uptake studies to understand the physiology of coronary artery vasodilation.

4-Nitrophenylphosphatase↗
PubMed1985

Binding and degradation of angiotensin II by mesenteric artery subcellular membranes.

Rat mesenteric artery microsomes were previously reported to degrade 125I-angiotensin II (AII). It is now shown here that washing the membranes with EDTA and including EGTA in the assay media reduces the 125I-AII degradation to very low levels without reducing the specific binding of 125I-AII. Using EDTA wash and including 5 mM MgCl2 and 0.2 mM EGTA the following characteristics of the binding were observed: microscopic association rate constant (k1) = 1.3 to 2.2 X 10(5) M-1 s-1, microscopic dissociation rate constant (k-1) = 3.8 to 6.4 X 10(-4) s-1, equilibrium dissociation constant (Kd) = 1.8 nM and number of binding sites (Bmax) = 193 fmol/mg protein. The subcellular distribution of the specific binding of 125I-AII at 0.16 nM and 1.63 nM was studied along with the distribution of the marker enzymes. The specific binding paralleled the plasma membrane marker (5'-nucleotidase), but not the putative endoplasmic reticulum marker (NADPH-cyt. c-reductase) or the inner mitochondrial marker (cyt. c-oxidase). Thus the binding to the plasma membrane-enriched fraction F2 occurred with a similar affinity (Kd = 2.2 nM) but with higher number of binding sites (420 fmol/mg protein). This study establishes the 125I-AII binding method suitable for determining the changes in the angiotensin receptor characteristics in the pathophysiology of the vascular smooth muscle.

Angiotensin II↗
PubMed1985

Solubilization of a high affinity CA-ATPase from dog antrum smooth muscle.

A Mg-independent high affinity Ca-ATPase has recently been reported to be present in the plasma membranes of smooth muscle. This enzyme has now been solubilized using deoxycholate. The membrane-bound and the solubilized enzymes resemble each other in Km for Ca2+, and inhibition by fluphenazine. The solubilized enzyme is, however, more sensitive to inhibition by Mg2+ than the membrane bound enzyme. Radiation inactivation analysis shows that whereas the membrane-bound enzyme had a target size of 98,000 +/- 4,000 Daltons, the solubilized enzyme was only 70,000, +/- 7,000 Daltons.

Activation Analysis↗
PubMed1985

Ca-pumps in smooth muscle: one in plasma membrane and another in endoplasmic reticulum.

For several years it has been debated whether the Ca-pump in smooth muscle is located in the plasma membrane or in the endoplasmic reticulum (alias sarcoplasmic reticulum). Experimental evidence using skinned smooth muscle cells and subcellular membrane fractions isolated from a number of smooth muscles is reviewed here to hopefully resolve this issue. The inescapable conclusion is that there are two modes of nonmitochondrial ATP-dependent Ca-transport. The first one, unaffected by oxalate, is localized in the plasma membranes and the second, potentiated by oxalate, is localized in the endoplasmic reticulum. Clear experiments to delineate the roles of the two pumps in the excitation-contraction cycle of the smooth muscle remain to be conducted.

Animals↗
PubMed1985

Target size of 5'-nucleotidase in smooth muscle.

Target size of the 5'-nucleotidase in six different smooth muscles was determined by radiation inactivation. The enzyme in the soluble fraction of rat myometrium and vas deferens gave a target size of approximately 80,000 daltons. The plasma membrane bound 5'-nucleotidase however, gave target size of 80,000 to 110,000 daltons in rat gastric fundus and vas deferens and dog stomach and ileum, 135,000 daltons in rat mesenteric artery and 210,000 daltons in rat myometrium.

5'-Nucleotidase↗
PubMed1985

Ion movements in membrane vesicles: a new fluorescence method and application to smooth muscle.

A method is described for studying ion permeabilities of membrane vesicles based on the principle that when membrane permeability to H+ is very high, the H+ movement is determined by the membrane potential generated by the H+ movement. The rate of H+ movement under these conditions thus gives a measure of the rate of dissipation of this membrane potential by comovement of anions or countermovement of cations present. Thus, by studying the H+ efflux using an impermeant cation and different anions, the membrane permeability to the anions can be assessed. Similarly, the use of an impermeant anion allows the study of the permeation of various cations. H+ movement was followed across the membranes by monitoring a change in the fluorescence intensity of the pH-sensitive dye pyranine trapped inside the membranes. This method when tested using phosphatidylcholine liposomes yielded the expected results, i.e., permeability of the liposomal membrane was: Cl- greater than SO2-4 and K+ greater than Na+. A plasma membrane-enriched fraction loaded with pyranine was isolated from estrogen-dominant rat myometrium. The anion permeability characteristics of this membrane were studied using tetramethylammonium (TMA+) as the poorly permeant cation, and the cation permeability was studied using L-glutamate- as the poorly permeant anion. The anion permeabilities were D-glutamate- less than L-glutamate- less than glutarate2- less than Cl- less than or equal to SO2-4, and the cation permeabilities were TMA+ less than K+ less than Na+. It is hypothesized that the observed anomalously higher Na+ and SO2-4 movements may involve special mechanisms.

Animals↗
PubMed1985

Calcium pump, high-affinity Ca2+-ATPase, and other ATPases in dog antrum smooth muscle plasma membrane.

The plasma membrane-enriched fraction from dog antrum smooth muscle is enriched in ATP-dependent azide-insensitive Ca2+ uptake (0.3-0.4 microM Ca2+ required for half-maximal activity), a high-affinity Ca2+-ATPase (Km of 0.3-0.8 microM for Ca2+), a low-affinity Ca2+-ATPase (Km for 250-400 microM for Ca2+), and a Mg2+-ATPase. Studies using membranes washed with EDTA and assay media treated with Chelex 100 showed that the high-affinity Ca2+-ATPase did not depend on contaminating Mg2+. Thus, whereas the ATP-dependent Ca2+ uptake had an absolute requirement for Mg2+, the Ca2+-ATPases did not. Studies using gamma-irradiation showed that the protein responsible for the ATP-dependent Ca2+ uptake was inactivated at significantly lower doses of radiation than the three ATPases. The Ca2+ uptake and the high-affinity Ca2+-ATPase also differed in their inhibition by calmodulin antagonists and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Thus it is unlikely that the high-affinity Ca2+-ATPase by itself is responsible for the ATP-dependent Ca2+ uptake.

Adenosine Triphosphatases↗
PubMed1985

Effect of gamma-thio-ATP on dog stomach smooth muscle membranes.

The effect of gamma-Thio-ATP on high affinity ATPase and on the ATP-dependent Ca-uptake of a plasma membrane fraction prepared from the dog stomach smooth muscle was examined. gamma-Thio-ATP did not act as a substrate for the ATP-dependent Ca-uptake and was only a poor substrate for the high affinity Ca-ATPase reaction. The compound, however, reversibly inhibited the uptake and the ATPase.

Adenosine Triphosphate↗