Kinetics of the purified plasma membrane H+-ATPase from red beet (Beta vulgaris)

The plasma membrane H⁺-ATPase (EC 3.6.1.35) was purified by washing red beet ( Beta vulgaris L.) plasma membranes with sodium deoxycholate and separating the ATPase, solubilized with lysophosphatidylcholine, by centrifugation in a glycerol gradient. The purified H⁺-ATPase had a sedimentation coefficient of about 8S. In the absence of exogenous protein substrates, the purified ATPase preparation did not present protein kinase activity. Compared with the H⁺-ATPase in the plasma membrane, the purified ATPase presented a higher affinity for adenosine 5'-triphosphate (ATP) and a lower sensitivity to the inhibitors vanadate and inorganic phosphate. These changes in the kinetics of the ATPase could also be observed by treating the membranes with lysophosphatidylcholine, without purifying the enzyme. These results can be explained assuming that lysophosphatidylcholine interacts with the ATPase altering its kinetics probably by stimulating the transformation from the inhibitor-binding conformation E2 into the ATP-binding conformation E1.     

Discrepancies between the affinities of binding and action of the novel beta-adrenergic agonist BRL 37344 in rat brown adipose tissue

The novel brown adipose tissue (BAT) selective beta-adrenergic agonist, BRL 37344, is 31-fold more potent than (-)-isoproterenol in stimulating the respiratory rate of interscapular BAT fragments. BRL 37344 is also more potent (9-fold) than (-)-isoproterenol in stimulating adenylate cyclase activity of IBAT purified plasma membranes whereas, in the same preparation, it is 81-fold less potent than (-)-isoproterenol in competition displacement studies with the beta-adrenergic ligand, [125I]cyanopindolol. We have previously demonstrated that the photoaffinity reagent [125I]cyanopindolol-diazirine selectively labels a 62 kDa protein in IBAT plasma membranes that displays pharmacological properties of a beta 1-adrenergic subtype. Relatively high concentrations of BRL 37344 (10 microM) are required to displace [125I]cyanopindolol-diazirine binding to the 62 kDa protein. Taken together, the results suggest that two different populations of beta-adrenergic receptors may co-exist in BAT plasma membranes: a small population (about 15%) of atypical beta-receptors and a large population of beta 1-receptors that exhibit high and low affinities for BRL 37344, respectively.     

Differential acid stabilities of citraconylated amino groups of glucagon. Preparation of N alpha-citraconyl glucagon and evaluation of its biological properties

Acylation of the alpha- and epsilon-amino groups of histidine-1 and lysine-12 in glucagon with citraconic anhydride resulted in the formation of amide bonds which displayed different stabilities to hydrolysis under mild acid conditions. Treatment of N alpha,epsilon-dicitraconyl glucagon at pH 4.0 and room temperature regenerated the free epsilon-amino group within 16 h, while the citraconyl-alpha-amino group was stable. N alpha-Citraconyl glucagon was purified by anion-exchange chromatography and was a weak partial agonist in stimulating adenylate cyclase in rat liver plasma membranes. The derivative exhibited 1% of the biological potency and 35-40% of the maximal stimulation of glucagon. Binding affinity to plasma membranes was also reduced, but not to as great an extent as adenylate cyclase activity. Removal of the alpha-citraconyl group by treatment with 10 mM HCl at 40 degrees C restored full potency and stimulation to glucagon. These results suggest that the N-terminal histidine of glucagon is involved in both binding to plasma membranes and transduction of the signal to adenylate cyclase.     

Isolation and identification of prostaglandin I 2 stimulating factor from human plasma

To isolate and identify the plasma factor which stimulates prostaglandin I 2 production by rat aortic ring, a human plasma fraction which showed a major stimulating activity on prostaglandin I 2 production was purified by ultrafiltrate, Sephadex G-10 gel filtration and QAE-Sephadex column chromatography. The purified plasma factor was identified as uric acid by its ultraviolet and infrared absorption spectroscopy, and 1H nmr and 13C nmr spectroscopy. The stimulating activity of the purified plasma factor and that of authentic uric acid coincided with each other. The stimulating potency of uric acid at its physiological concentration in human plasma (about 50 micrograms/ml) was half of the deproteinized human plasma, and was about 30 fold stronger than that of L-tryptophan, a cofactor of prostaglandin hydroperoxidase.     

Isolation and identification of prostaglandin I 2 stimulating factor from human plasma

To isolate and identify the plasma factor which stimulates prostaglandin I 2 production by rat aortic ring, a human plasma fraction which showed a major stimulating activity on prostaglandin I 2 production was purified by ultrafiltrate, Sephadex G-10 gel filtration and QAE-Sephadex column chromatography. The purified plasma factor was identified as acid by its ultraviolet and infrared absorption spectroscopy, and ¹H nmr and ¹³C nmr spectroscopy. The stimulating activity of the purified plasma factor and that of authentic uric acid coincided with each other. The stimulating potency of uric acid at its physiological concentration in human plasma (about 50 μg/ml) was half of the deproteinized human plasma, and was about 30 fold stronger than that of L-tryptophan, a cofactor of prostaglandin hyperoxidase.     

Studies of (Na+ + K+)-sensitive ATPase activity in pig lymphocytes. Effects of concanavalin A.

(Na+ + K+)-ATPase activity is demonstrated in plasma membranes from pig mesenteric lymph nodes. After dodecyl sulfate treatment plasma membranes have an 18-fold higher (Na+ + K+)-ATPase activity, while their ouabain-insensitive Mg2+-ATPase is markedly lowered. A solubilized (Na+ +K+)-ATPase fraction, obtained by Lubrol WX treatment of the membranes, has very high specific activity (21 mumol Pi/h per mg protein). Concanavalin A has no effect on these partially purified (Na+ + K+)-ATPase, while inhibits (40%) this activity in less purified fractions which still contain Mg2+-ATPase activity.     

Purification and Identification of a Plasma Membrane Associated Electron Transport Protein from Maize (Zea mays L.) Roots

Plasma membranes isolated from three-day-old maize (Zea mays L.) roots by aqueous two-phase partitioning were used as starting material for the purification of a novel electron transport enzyme. The detergent-solubilized enzyme was purified by dyeligand affinity chromatography on Cibacron blue 3G-A-agarose. Elution was achieved with a gradient of 0 to 30 micromolar NADH. The purified protein fraction exhibited a single 27 kilodalton silver nitrate-stained band on sodium dodecyl sulfate polyacrylamide gel electrophoretograms. Staining intensity correlated with the enzyme activity profile when analyzed in affinity chromatography column fractions. The enzyme was capable of accepting electrons from NADPH or NADH to reduce either ferricyanide, juglone, duroquinone, or cytochrome c, but did not transfer electrons to ascorbate free-radical or nitrate. The high degree of purity of plasma membranes used as starting material as well as the demonstrated insensitivity to mitochondrial electron transport inhibitors confirmed the plasma membrane origin of this enzyme. The purified reductase was stimulated upon prolonged incubation with flavin mononucleotide suggesting that the enzyme may be a flavoprotein. Established effectors of plasma membrane electron transport systems had little effect on the purified enzyme, with the exception of the sulfhydryl inhibitor p-chloromercuriphenyl-sulfonate, which was a strong inhibitor of ferricyanide reducing activity.     

Preparation of target antigens specifically recognized by human natural killer cells

In an attempt to identify target cell membrane molecules recognized by natural killer (NK) cells, artificial membranes were prepared from detergent-solubilized plasma membranes of NK target cells and synthetic lipids. Such reconstituted membranes from human and rat NK target cells were shown to inhibit both human and rat NK-target cell conjugates in a species-specific fashion; these reconstituted membranes failed to inhibit NK cytotoxicity. The detergent-solubilized material from the human NK target K562 was subjected to various procedures prior to reconstitution and the conjugate inhibition assay. Conjugate inhibitory activity was lost upon trypsin digestion and incubation at 65 degrees C. This inhibition activity was absorbed to concanavalin A agarose and could subsequently be eluted with alpha-methylmannoside, resulting in approximately 20-fold purification. Gel filtration of this material on an AcA-34 column in detergent gave a broad activity peak with maximal activity in the molecular weight range of 30,000-165,000. Gel electrophoresis of purified membranes demonstrated multiple molecular weight bands in lipid membranes. The K562 membrane material, purified by concanavalin A agarose and gel filtration, inhibited conjugates between human NK cells and any of four human target cells, but not of conjugates with (1) human large granular lymphocytes and antibody-coated mouse tumor cells nor (2) rat NK cells and their target cells. Thus the purified glycoproteins from K562 retain the property of specific inhibition of human NK-target conjugates.     

Megakaryocyte colony growth-supporting activities in human plasma: modification by platelets and platelet membranes

Human megakaryocyte colonies are grown in methylcellulose with platelet-poor plasma and medium conditioned by phytohemagglutinin-stimulated leukocytes (PHA-LCM) as a source of megakaryocyte colony stimulating factor (MEG-CSF). The megakaryocyte colony growth-supporting activity in human plasma can be absorbed by intact platelets or degranulated platelet membranes. It was possible to recover the activity by solubilizing platelet membranes with cholic acid. Filtration of the solubilized platelet membrane preparations through a Sephadex G-100 column yielded at least two activity peaks. The molecular weight of these two activities differs from that of the growth-promoting activity in PHA-LCM.     

Dephosphorylation of phosphoproteins of human liver plasma membranes by endogenous and purified liver alkaline phosphatases

Purified alkaline phosphatase and plasma membranes from human liver were shown to dephosphorylate phosphohistones and plasma membrane phosphoproteins. The protein phosphatase activity of the liver plasma membranes was inhibited by levamisole, a specific inhibitor of alkaline phosphatase, and by phenyl phosphonate and orthovanadate, but was relatively insensitive to fluoride (50 mM). Endogenous membrane protein phosphatase activity was optimal at pH 8.0, compared to pH 7.8 for purified liver alkaline phosphatase. Plasma membranes also exhibited protein kinase activity using exogenous histone or endogenous membrane proteins (autophosphorylation) as substrates; this activity was cAMP-dependent. Autophosphorylation of plasma membrane proteins was apparently enhanced by phenyl phosphonate, levamisole, or orthovanadate. The dephosphorylation of phosphohistones by protein phosphatase 1 was not inhibited by levamisole but was inhibited by fluoride. Inhibition of endogenous protein phosphatase activity by orthovanadate during autophosphorylation of plasma membranes could be reversed by complexation of the inhibitor with (R)-(-)-epinephrine, and the dephosphorylation that followed was levamisole-sensitive. Neither plasma membranes nor purified liver alkaline phosphatase dephosphorylated glycogen phosphorylase a. These results suggest that the increased [32P]phosphate incorporation by endogenous protein kinases into the membrane proteins is due to inhibition of alkaline phosphatase and that the major protein phosphatase of these plasma membranes is alkaline phosphatase.     

RGS1 is expressed in monocytes and acts as a GTPase-activating protein for G-protein-coupled chemoattractant receptors.

The leukocyte response to chemoattractants is transduced by the interaction of transmembrane receptors with GTP-binding regulatory proteins (G-proteins). RGS1 is a member of a protein family constituting a newly appreciated and large group of proteins that act as deactivators of G-protein signaling pathways by accelerating the GTPase activity of G-protein alpha subunits. We demonstrate here that RGS1 is expressed in human monocytes; by immunofluorescence and subcellular fractionation RGS1 was localized to the plasma membrane. By using a mixture of RGS1 and plasma membranes, we were able to demonstrate GAP activity of RGS1 on receptor-activated G-proteins; RGS1 did not affect ligand-stimulated GDP-GTP exchange. We found that RGS1 desensitizes a variety of chemotactic receptors including receptors for N-formyl-methionyl-leucyl-phenylalanine, leukotriene B4, and C5a. Interaction of RGS proteins and ligand-induced G-protein signaling can be demonstrated by determining GTPase activity using purified RGS proteins and plasma membranes.     

High affinity antibody from hen's eggs directed against the human insulin receptor and the human IGF-I receptor

Large quantities of high affinity antibodies directed against the human insulin receptor and the human insulin-like growth factor-I (IGF-I) receptor were obtained from hen's eggs. Hens were immunized with human placental membranes and human liver membranes by intramuscular injections. Specific antibodies to the receptors were demonstrated in serum and egg yolks at 5 weeks and these antibodies presisted for at least 6 months. Antibodies from egg yolks were purified by the polyethylene glycol precipitation technique of Polson et al. The eggs provided the equivalent of about 450 ml of immunized serum per month per bird. The purified antibodies were approximately equally reactive with receptors for insulin or IGF-I. Antibodies immunoprecipitated affinity-labeled receptors, inhibited binding of each ligand, and were capable of stimulating 2-deoxyglucose uptake in rat adipocytes and thymidine incorporation in cultured fibroblasts. The presence of antibodies directed against the IGF-I receptor in those hens immunized with human liver membranes was unexpected, since liver membranes possess little or no IGF-I binding activity. We conclude that antibodies against human antigens may be relatively easily obtained by immunization of hens and purification of those antibodies from eggs.     

Diacylglycerol kinase in plasma membranes from wheat.

Diacylglycerol kinase activity was demonstrated in highly purified plasma membranes isolated from shoots and roots of dark-grown wheat (Triticum aestivum L.) by aqueous polymer two-phase partitioning. The active site of the diacylglycerol kinase was localized to the inner cytoplasmic surface of the plasma membrane using isolated inside-out and right-side-out plasma membrane vesicles from roots. The enzyme activity in plasma membrane vesicles from shoots showed a broad pH optimum around pH 7. The reaction was Mg2+ and ATP dependent, and maximal activity was observed around 0.5 mM ATP and 3 mM MgCl2. The Mg2+ requirement could be substituted only partially by Mn2+ and not at all by Ca2+. The phosphorylation of endogenous diacylglycerol was strongly inhibited by detergents indicating an extreme dependence of the lipid environment. Inositol phospholipids stimulated the activity of diacylglycerol kinase in plasma membranes from shoots and roots, whereas the activity was inhibited by R59022, a putative inhibitor of several diacylglycerol kinase isoenzymes involved in uncoupling diacylglycerol activation of mammalian protein kinase C.     

Distribution of liver plasma membrane 5' nucleotidase as indicated by its reaction with anti-plasma membrane serum

Antiserum against mouse liver plasma membranes was used to investigate the properties and distribution of the surface membrane enzyme 5′ nucleotidase.The antiserum inhibited 5′ nucleotidase but had no effect on alkaline phosphodiesterase, nucleotide pyrophosphatase, or insulin-binding activity.5′ Nucleotidase was purified from mouse liver plasma membranes and the purified enzyme was shown to be inhibited by the antiserum. The membrane-bound and the purified enzyme were both inhibited in a noncompetitive manner.The reaction of the antiserum with 5′ nucleotidase activity of mouse liver plasma membrane “light” and “heavy” subfractions, and of rat liver and pig lymphocyte surface-membrane fractions was investigated. In each case the enzyme was inhibited by the antiserum.Since a protein must be partially exposed on the membrane surface in order to react with its antibody, the results are discussed in terms of the disposition of 5′ nucleotidase within the membrane.     

Purification and characterization of a doxorubicin-inhibited NADH-quinone (NADH-ferricyanide) reductase from rat liver plasma membranes

Plasma membrane-associated redox systems play important roles in regulation of cell growth, internal pH, signal transduction, apoptosis, and defense against pathogens. Stimulation of cell growth and stimulation of the redox system of plasma membranes are correlated. When cell growth is inhibited by antitumor agents such as doxorubicin, capsaicin, and antitumor sulfonylureas, redox activities of the plasma membrane also are inhibited. A doxorubicin-inhibited NADH-quinone reductase was characterized and purified from plasma membranes of rat liver. First, an NADH-cytochrome b(5) reductase, which was doxorubicin-insensitive, was removed from the plasma membranes by the lysosomal protease, cathepsin D. After removal of the NADH-cytochrome b(5) reductase, the plasma membranes retained a doxorubicin-inhibited NADH-quinone reductase activity. The enzyme, with an apparent molecular mass of 57 kDa, was purified 200-fold over the cathepsin D-treated plasma membranes. The purified enzyme had also an NADH-coenzyme Q(0) reductase (NADH: external acceptor (quinone) reductase; EC 1.6.5.) activity. Partial amino acid sequence of the enzyme showed that it was unique with no sequence homology to any known protein. Antibody against the enzyme (peptide sequence) was produced and affinity-purified. The purified antibody immunoprecipitated both the NADH-ferricyanide reductase activity and NADH-coenzyme Q(0) reductase activity of plasma membranes and cross-reacted with human chronic myelogenous leukemia K562 cells and doxorubicin-resistant human chronic myelogenous leukemia K562R cells. Localization by fluorescence microscopy showed that the reaction was with the external surface of the plasma membranes. The doxorubicin-inhibited NADH-quinone reductase may provide a target for the anthracycline antitumor agents and a candidate ferricyanide reductase for plasma membrane electron transport.     

Binding of parathyroid hormone to bovine kidney-cortex plasma membranes

1. Plasma membranes were purified from bovine kidney cortex, with a fourfold increase in specific activity of parathyroid hormone-sensitive adenylate cyclase over that in the crude homogenate. The membranes were characterized by enzyme studies. 2. Parathyroid hormone was labelled with (125)I by an enzymic method and the labelled hormone shown to bind to the plasma membranes and to be specifically displaced by unlabelled hormone. Parathyroid hormone labelled by the chloramine-t procedure showed no specific binding. (75)Se-labelled human parathyroid hormone, prepared in cell culture, also bound to the membranes. 3. Parathyroid hormone was shown to retain biological activity after iodination by the enzymic method, but no detectable activity remained after chloramine-t treatment. 4. High concentration of pig insulin inhibited binding of labelled parathyroid hormone to plasma membranes and partially inhibited the hormone-sensitive adenylate cyclase activity in a crude kidney-cortex preparation. 5. EDTA enhanced and Ca(2+) inhibited binding of labelled parathyroid hormone to plasma membranes. 6. Whereas rat kidney homogenates were capable of degrading labelled parathyroid hormone to trichloroacetic acid-soluble fragments, neither crude homogenates nor purified membranes from bovine kidney showed this property. 7. Binding of parathyroid hormone is discussed in relation to metabolism and initial events in hormone action.     

Hormone regulated enzyme activities of plasma membrane of decidual endometrium of the rat

Plasma membranes were purified from deciduoma of pseudopregnant rats and rat liver. Preparations contained 80% plasma membrane-derived material as based on electron microscope morphometry and analysis of enzyme markers. Several plasma membrane enzymes were tested for direct response to hormones. NADH-ferricyanide reductase of plasma membranes from both tissues was stimulated by glucagon and inhibited by insulin but was unresponsive to steroids. For steroids, responsiveness was limited to a reduction in NaF-stimulated adenylate cyclase activity by the steroid R5020. Thus, interaction of steroid hormones with plasma membranes, unlike that of glucagon and insulin, is not reflected in an altered activity of plasma membrane-bound dehydrogenases but may be exerted directly on adenylate cyclase.     

Role of cytochrome b-559 in arachidonic acid activation of resting human neutrophils

Whether or not cytochrome b-559 is a necessary component of NADPH oxidase activity in neutrophils is still controversial. In highly purified plasma membranes isolated from resting neutrophils and lacking cytochrome b, addition of arachidonic acid induced an NADPH oxidase activity. This activity was similar to that of plasma membranes isolated from phorbol myristate acetate (PMA)-stimulated cells which possessed cytochrome b. Addition of arachidonic acid to the latter plasma membranes did not alter the oxidase activity. It can be concluded that plasma membranes isolated from resting neutrophils have, in the presence of arachidonic acid, an NADPH oxidase activity similar to that of PMA-stimulated cells, except that it is independent of cytochrome b-559.     

Kinetics of cytolytic T lymphocyte binding to target cells in suspension

Cytolytic T lymphocytes (CTL) were able to specifically bind and lyse allogeneic P815 tumor cells and LPS blast cells in suspension. An assay was developed to measure the rate of target cell binding in suspension independent of the rate of lysis. Target cell binding was found to plateau within 3 hr in suspension. The presence of free, functional CTL and targets at these plateaus was demonstrated, indicating that target cell binding was an equilibrium process. Scatchard plots were used to derive values for Kd (apparent affinity) and bmax (maximum binding). Target cell binding in suspension could not be blocked by purified plasma membranes. Target cell binding was compared for CTL generated by secondary in vitro stimulation with intact cells or with purified membranes. These 2 CTL populations yielded distinct values for Kd and bmax. Implications of this kinetic difference for CTL recognition of purified plasma membranes are discussed.     

Plasma-membrane-bound malate dehydrogenase activity in maize roots

Summary Plasma membranes were isolated and purified from 14-day-old maize roots (Zea mays L.) by two-phase partitioning at a 6.5% polymer concentration, and compared to isolated mitochondria, microsomes, and soluble fraction. Marker enzyme analysis demonstrated that the plasma membranes were devoid of cytoplasmic, mitochondrial, tonoplast, and endoplasmic-reticulum contaminations. Isolated plasma membranes exhibited malate dehydrogenase activity, catalyzing NADH-dependent reduction of oxaloacetate as well as NAD⁺-dependent malate oxidation. Malate dehydrogenase activity was resistant to osmotic shock, freeze-thaw treatment, and salt washing and stimulated by solubilization with Triton X-100, indicating that the enzyme is tightly bound to the plasma membrane. Malate dehydrogenase activity was highly specific to NAD⁺ and NADH. The enzyme exhibited a high degree of latency in both right-side-out (80%) and inside-out (70%) vesicle preparations. Kinetic and regulatory properties with ATP and P_i, as well as pH dependence of plasma-membrane-bound malate dehydrogenase were different from mitochondrial and soluble malate dehydrogenases. Starch gel electrophoresis revealed a characteristic isozyme form present in the plasma membrane isolate, but not present in the soluble, mitochondrial, and microsomal fractions. The results presented show that purified plasma membranes isolated from maize roots contain a tightly associated malate dehydrogenase, having properties different from mitochondrial and soluble malate dehydrogenases.Abbreviations FCR ferricyanide reductase - MDH malate dehydrogenase