Biochemical and enzymatic characterization of thymic and splenic lymphocyte plasma membranes from inbred rats.

Purified splenic and thymic lymphocytes from the ACI and F344 strains of inbred rats were disrupted by controlled hypotonic treatment, and their plasma membranes were prepared by sucrose density gradient centrifugation. The plasma membrane preparations were highly purified as judged by the structural appearance of the smooth membrane vesicles, by the 10- to 15-fold enrichment of 5'-nucleotidase, which cytochemically localized exclusively in the plasma membranes of intact lymphocytes, by the high cholesterol to phospholipid molar ratio (0.7-1.0), and by the very low specific activities of the enzymes associated predominantly with mitochondria, lysosomes, and endoplasmic reticulum. The protein and the lipid contents of the membranes were 48-55 and 37-48%, respectively. The total lipid content of plasma membranes was characteristically higher in thymic than splenic lymphocytes from both ACI and F344 strains. The specific activity of 5'-nucleotidase was similar in splenic lymphocyte membranes of the ACI strain, and in both the thymic and splenic lymphocyte membranes of the F344 strain. In contrast, the thymic lymphocyte membranes in the ACI strain showed half as much 5'-nucleotidase specific activity. Cytochemical results indicated that the 5'-nucleotidase is located on the outside surface of the lymphocyte plasma membranes.     

Localization of 3,5,3'-L-triiodothyronine receptor in rat kidney mitochondrial membranes

Binding of triiodothyronine(T₃) to submitochondrial fractions from rat kidney was studied. Both inner and outer mitochondrial membranes were purified by sucrose gradient centrifugation. Both membranes had specific binding sites for T₃. Scatchard analysis of T₃ binding by membranes gave different affinity constants between inner and outer membranes. In studies with gel filtration of soluble T₃ receptors, four main T₃ binding activities in outer membranes and two main T₃ binding activities in inner membranes were isolated. The results indicate that both inner and outer mitochondrial membranes have specific binding sites for T₃ and that each membrane has a specific structure in T₃ receptor.     

Evidence for the association of prolyl hydroxylase from chick embryos with membranes through membrane-bound procollagen.

Proline hydroxylase of membrane pellets from tissues of chick embryo was solubilized by several extraction mediums. Three kinds of enzyme linkage with membranes were pointed out: a labile and non specific adsorption, a specific linkage on endogenous procollagen which is linked with the membranes, and a part of membranes themselves. Our results suggest that, in the liver, these three kinds of linkage exist. Whereas, in tibia bones only labile linkages appear by non specific adsorption or through the link with membrane procollagen.     

动物细胞膜的分离纯化及其在病毒受体研究中的应用

细胞膜是动物细胞与胞外环境之间的屏障。病毒只有与细胞膜上的病毒受体特异性结合 ,才能进入细胞 ,进而启动其增殖周期。因此 ,病毒受体是病毒学研究的重要组成部分。分离纯化病毒受体所在的细胞膜作为病毒受体研究的实验材料 ,已经在许多病毒的研究中得到应用 ,并取得了很好的效果。现就动物细胞膜的分离纯化及其在病毒受体研究中的应用作一综述。     

Chicken erythrocyte membranes: Comparison of nuclear and plasma membranes from adults and embryos

Nuclear membranes and plasma membranes of chicken erythrocytes were tested for some enzyme activities and polypeptide content. Both membranes show ATPase and NADH cytochrome c reductase activities, which were higher for nuclear membrane preparations; besides the nuclear membrane ATPase is not stimulated by Na + K + ions. SDS solubilization of these membranes followed by separation using SDS polyacrylamide gel electrophoresis yields about 25 bands. Nuclear membranes and plasma membranes possess specific bands; some differences were seen when adult and embryonic membranes were compared.     

Characteristics of specific bindings of nitrendipine and PN200-110 to various crude membranes: induction of irreversible bindings by UV irradiation

The characteristics of the specific bindings of [3H]nitrendipine (Nit) and [3H](+)PN200-110 (PN) to crude membranes from rat skeletal, cardiac, and uterine muscle and whole brain were investigated, with special interest in the effect of UV irradiation on these bindings. The specific bindings of [3H]Nit and [3H](+)PN to these crude membranes were saturable and reversible. The specific bindings of [3H]Nit to all these membranes except crude skeletal membranes was maximum in the presence of 0.15 M NaCl plus 1 mM CaCl2 and minimal in the absence of these ions, but the specific bindings of [3H](+)PN to these crude membranes was not affected significantly by these ions. A calcium agonist and antagonists inhibited the specific bindings of [3H]Nit and [3H](+)PN to these crude membranes, the order of their inhibitory effects on specific [3H]Nit bindings being roughly Nit greater than or equal to (+)PN greater than or equal to (-)PN much greater than Bay K 8644 (Bay) greater than verapamil (Ver) greater than diltiazem (Dil). In crude skeletal membranes only, PN caused significant stereospecific inhibition. The order of inhibitions of specific [3H](+)PN bindings to these crude membranes was generally (+)PN greater than Nit greater than or equal to (-)PN greater than Bay much greater than Ver greater than or equal to Dil. In all these crude membranes, UV irradiation completely prevented decrease in the amount of specific binding of [3H](+)PN binding on addition of excess unlabeled (+)PN. These findings suggested that [3H]Nit and [3H](+)PN bind to voltage-sensitive calcium channels in crude membranes from rat skeletal, cardiac, and uterine muscle and whole brain, and that UV irradiation changes the specific bindings of [3H]Nit and [3H](+)PN from reversible to irreversible bindings.     

Glycoprotein synthesis in the adult rat pancreas: II. Characterization of Golgi-rich fractions

Golgi-rich fractions were prepared from homogenates of adult rat pancreas by discontinuous gradient centrifugation. These fractions were characterized by stacks of cisternae associated with large, irregular vesicles and were relatively free of rough microsomes, mitochondria, and zymogen granules. The Golgi-rich fractions contained 50% of the UDP-galactose: glycoprotein galactosyltransferase activity; the specific activity was 12-fold greater than the homogenate. Such fractions represented < 19% of thiamine pyrophosphatase, uridine diphosphatase, adenosine diphosphatase, and Mg²⁺-adenosine triphosphatase. Zymogen granules and the Golgi-rich fractions were extracted with 0.2 m NaHCO3, pH 8.2, and the membranes were isolated by centrifugation. The glycoprotein galactosyltransferase could not be detected in granule membranes, while the specific activity in Golgi membranes was 25-fold greater than the homogenate.At least 35 polypeptide species were detected in Golgi membranes by polyacrylamide gel electrophoresis in 1% sodium dodecylsulfate. These ranged in molecular weight from 12,000 to <160,000. There were only minor differences between Golgi membranes and smooth microsomal membrane. In contrast, zymogen granule membranes contained fewer polypeptides. A major polypeptide, which represented 30–40% of the granule membrane profile, accounted for less than 3% of the polypeptides of Golgi membranes or smooth microsomal membranes.     

Isolation of plasma and nuclear membranes of thymocytes. II. Biochemical composition

Thymocyte plasma and nuclear membranes obtained by the procedure described in the accompanying paper were analyzed for their biochemical composition. Plasma membranes were very rich in phospholipid, cholesterol, sialic aicd; they did not contain nucleic acids. In comparison, nuclear membranes had a lower phospholipid to protein ratio and contained much less sialic acid and cholesterol. 50% of the cellular cholesterol and of the membrane-bound sialic acid were found in the plasma membranes, 14% in the nuclear membranes. Live cells were labeled with 131I, and the acid-insoluble radioactivity was followed in the subfractions. A good correlation with the distribution and enrichment of plasma membrane market-enzymes was obtained. Label enrichment was about 50-fold in the two lightest of the three plasma membrane fractions. 60% of the label was contained in the plasma membranes, only 4% in the nuclear membranes. Cross-contamination of these two types of membranes was thus negligible. Sodium dodecyl sulfate-gel electrophoresis revealed three different patterns specific for, respectively, plasma membranes, the microsomal-mitochondrial fraction, and nuclear membranes. Each pattern was characterized by a set of proteins and glycoproteins, among which high molecular weight glycoproteins could be considered as marker-proteins of, respectively, 280,000, 260,000, and 230,000 daltons. 131I-labeling of live cells tagged with a very high specific activity three glycoproteins of mol wt 280,000, 200,000, and 135,000 daltons. Nuclear membranes prepared from labeled isolated nuclei had a set of labeled proteins completely different from plasma membranes.     

Comparison of plasma membranes and endoplasmic reticulum fractions obtained from whole white adipose tissue and isolated adipocytes.

The influence of the mode of preparation upon some of the characteristics of white adipose tissue plasma membranes and microsomes has been reported. Plasma membrane fractions prepared from mitochondrial pellet were shown to have higher specific activities of (Mg2+ + Na+ + K+)-ATPase than plasma membranes originating in crude microsomes. Isolation of fat cells by collagenase treatment was found to result in a decrease in specific activity of the plasma membrane enzymes; in plasma membranes prepared from isolated fat cells, the specific activity values obtained for (Mg2+ + Na+ +k+)-ATPase and 5'-nucleotidase were only 42% and 6.3% respectively of those obtained in plasma membranes prepared from whole adipose tissue. Purification of whole adipose tissue crude microsomes by hypotonic treatment caused extensive solubilization of the endoplasmic reticulum marker enzymes, NADH oxidase and NADPH cytochrome c reductase. The lability of endoplasmic reticulum marker enzymes, however, was found to be greatly diminished in the preparations from isolated fat cells. The possibility that NADH oxidase and NADPH cytochrome c reductase activities found in the plasma membranes are microsomal enzymes adsorbed by the plasma membranes is discussed. The peptide patterns as well as the NADH oxidase and NADPH cytochrome c reductase activity patterns of plasma membranes and purified microsomes were compared by means of sodium dodecyl sulfate or Triton X-100 polyacrylamide gel electrophoresis.     

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.     

Localization of the ecto-ATPase (ecto-nucleotidase) in the rat hepatocyte plasma membrane. Implications for the functions of the ecto-ATPase

The surface distribution of the plasma membrane Ca2+ (Mg2+)-ATPase (ecto-ATPase) in rat hepatocytes was determined by several methods. 1) Two polyclonal antibodies specific for the ecto-ATPase were used to examine the distribution of the enzyme in frozen sections of rat liver by immunofluorescence. Fluorescent staining was observed at the bile canalicular region of hepatocytes. 2) Plasma membranes were isolated from the canalicular and sinusoidal regions of rat liver. The specific activity of ecto-ATPase in the canalicular membranes was 22 times higher than that of sinusoidal membranes. The enrichment of the ecto-ATPase activity in the canalicular membrane is closely parallel to that of two other canalicular membrane markers, gamma-glutamyltranspeptidase and leucine aminopeptidase. 3) By immunoblots with polyclonal antibodies against the ecto-ATPase and the Na+,K+-ATPase, it was found that the ecto-ATPase protein was only detected in canalicular membranes and not in sinusoidal membranes, while the Na+,K+-ATPase protein was only detected in sinusoidal membranes and not in canalicular membranes. These results indicate that the ecto-ATPase is enriched in the canalicular membranes of rat hepatocytes.     

A simple and rapid method for the preparation of plasma membranes

A simple and rapid method for preparing plasma membranes from isolated cells or tissues is described. The membranes were characterised (a) biochemically by an analysis of specific marker enzymes, (b) by quantitation of cell surface receptors, and (c) immunologically by their ability to elicit specific allogeneic responses from cytotoxic T cells in secondary in vitro stimulations. Based on both biochemical and immunologic criteria, plasma membranes prepared by the method described here are of equal or greater 'purity' compared to those prepared by two other methods that are most widely used to date and the yields are several-fold higher.     

Subcellular distribution and characterization of GTP-binding proteins in human neutrophils

The subcellular distribution of GTP binding proteins in human neutrophils and their functional coupling to the N-formylmethionylleucylphenylalanine (FMLP) receptor was characterized to provide insight into mechanisms of cellular activation. Human neutrophils were nitrogen cavitated and fractionated on discontinuous Percoll gradients. Four subcellular fractions were obtained: cytosol, light membranes enriched for plasma membranes, specific granules and azurophilic granules. ADP-ribosylation catalyzed by pertussis toxin (PT) revealed a major substrate of 40 kDa only in plasma membrane and cytosol, and antiserum specific for Gi alpha confirmed the presence of neutrophil Gi alpha in plasma membrane and cytosol and its absence from specific granules. The cytosolic PT substrate was shown to be mostly in monomeric form by molecular sieve chromatography. The rate of the ribosyltransferase reaction was several-fold lower in cytosol compared to plasma membranes, and the extent of ADP-ribosylation was greatly augmented by supplementation with beta gamma subunits in cytosol. ADP-ribosylation catalyzed by cholera toxin (CT) revealed substrates of 52, 43 and 40 kDa in plasma membrane alone. FMLP receptors in plasma membrane were shown to be coupled to the 40 kDa substrate for CT by ligand-modulation of ADP-ribosylation, while FMLP added to specific granules did not induce ribosylation of this substrate even though FMLP receptors were found in high density in this compartment. Both 24 and 26 kDa [32P]GTP binding proteins were found to codistribute with FMLP receptors in specific granules and plasma membranes. Functional evidence for the coupling of GTP binding proteins to the FMLP receptor in specific granules was obtained by modulating [3H]FMLP binding with GTP gamma S, and by accelerating [35S]GTP gamma S binding with FMLP.     

Isolation and identification of Methylomonas methanica membranes

The crude membrane preparation of Methylomonas methanica was fractionated by sucrose density gradient centrifugation and in an aqueous dextran -- polyethylene glycol two-phase system. Fractions of a higher purity were prepared by sucrose density gradient centrifugation. Two subcellular fractions were isolated and characterized. One of them enriched in lipopolysaccharides was represented by the cell wall debris; the other possessing greater specific activities of the enzymes contained mainly intracytoplasmic membranes. The effect of various factors on the separation of membranes and on the specific enzyme activities was investigated.     

Prostaglandin receptor-adenylate cyclase system in plasma membranes of rat liver and ascites hepatomas, and the effect of GTP upon it.

1. Adenylate cyclase in plasma membranes from rat liver was stimulated by prostaglandin E1, and to a lesser extent by prostaglandin E2. Prostaglandin F1alpha and A1 did not stimulate the cyclase. The prostaglandin E1-mediated activation was found to require GTP when the substrate ATP concentration was reduced from 3 mM to 0.3 mM in the reaction mixture. Adenylate cyclase of the plasma membranes from rat ascites hepatomas AH-130 and AH-7974 was not stimulated by prostaglandin E1 in the presence or the absence of GTP, although the basal activity of adenylate cyclase as well as its stimulation by GTP alone were similar to normal liver plasma membranes. 2. Liver plasma membranes were found to have two specific binders for [3H] prostaglandin E1 with dissociation constants of 17.6-10(-9) M and 13.6-10(8) M (37 degrees C) and one specific binder for [3H]prostaglandin F2alpha with a dissociation constant of 2.31-10(8) M (37 degrees C). The specific binders for prostaglandin E1 could not be detected in the hepatoma plasma membranes. 3. Binding of [3H] prostaglandin E1 to the liver plasma membranes was exchange by, GTP dGPT, GDP, ATP and GMP-P(N)P, but not by GMP, CGMP, DTTP, UTP or CTP. The increase in the binding of [3H] prostaglandin E1 was found to be due to the increased affinity of the specific binders to prostaglandin F2alpha was not affected by GTP. 4. GTP alone was found to increase V of adenylate cyclase of liver plasma membranes, while GTP plus prostaglandin E1 was found to decrease Km of adenylate cyclase in addition to the increase of V to a further extent.     

Nick translation of DNA immobilized on nylon membranes

Nick translation of DNA bound to nylon membranes is described. Phage lambda DNA was digested with restriction endonuclease HindIII. The fragments were separated by agarose electrophoresis and electrophoretically transferred to Zeta-Probe nylon membranes. After being air-dried, the areas with DNA fragments attached were cut out and subjected to nick translation. The labeled fragments, removed from the membranes by a single wash step, can be used as specific hybridization probes. Currently used methods require time-consuming electroelution and often additional purification procedures if a specific DNA fragment, separated by gel electrophoresis, is to be labeled by nick translation. With the procedure described it is possible to label many DNA fragments in parallel in a time- and cost-saving manner.     

A comparison of brush-border membranes prepared from rabbit small intestine by procedures involving Ca2+ and Mg2+ precipitation

Brush-border membranes were isolated from rabbit small intestine by procedures involving precipitation of undesired membranes with either 10 mM MgCl2 or 10 mM CaCl2. The membranes were compared on the basis of marker enzyme content and lipid composition. Ca2+-prepared membranes displayed a greater enrichment of alkaline phosphatase and sucrase activity compared to homogenate than did the Mg2+-prepared membranes. The former also displayed an impoverishment of (Na+ + K+)-ATPase activity, the specific activity of which increased several-fold in Mg2+-prepared membranes. Membranes prepared with Ca2+ were characterized by a lower phosphoacylglycerol-protein ratio and a higher phosphatidylethanolamine-phosphatidylcholine ratio. Although lysophosphoacylglycerols accounted for about 6% of the total phospholipids in these membranes compared to 2% in Mg2+-prepared membranes, the free fatty acid content was similar in both types of membranes. It was concluded that Ca2+ prepared membranes were less contaminated by basolateral membranes than were Mg2+-prepared membranes and the use of Ca2+ did not notably enhance degradation of endogenous lipids by brush-border membrane phospholipase A.     

Subcellular site of mannosyl transfer to dolichyl phosphate in Phaseolus aureus

Cellular membranes were isolated from hypocotyls of Phaseolus aureus by continuous sucrose gradient centrifugation. Membrane fractions were used to study the subcellular localization of mannosyl transfer reactions that involved GDP-mannose as the mannosyl donor. The highest specific activity of the enzyme responsible for transfer of the mannosyl moiety to dolichyl monophosphate was found in the fraction enriched in membranes of the endoplasmic reticulum. In contrast, the highest specific activity of enzymes capable of incorporating mannose into ethanol-insoluble polymer, or possibly the highest concentration of endogenous mannosyl acceptors, was located in the fraction enriched in membranes of the Golgi apparatus.     

Identification of the Ndh (NAD(P)H-plastoquinone-oxidoreductase) complex in etioplast membranes of barley: changes during photomorphogenesis of chloroplasts

In the last few years the presence in thylakoid membranes of chloroplasts of a NAD(P)H-plastoquinone oxidoreductase complex (Ndh complex) homologous to mitochondrial complex I has been well established. Herein, we report the identification of the Ndh complex in barley etioplast membranes. Two plastid DNA-encoded polypeptides of the Ndh complex (NDH-A and NDH-F) were relatively more abundant in etioplast membranes than in thylakoids from greening chloroplasts. Conversion of etioplast into chloroplast, after light exposure of barley seedlings grown in the dark, was accompanied by a decrease in the NADH dehydrogenase activity associated to plastid membranes. Using native-PAGE and immunolabelling techniques we have determined that a NADH specific dehydrogenase activity associated with plastid membranes, which was more active in etioplasts than in greening chloroplasts, contained the NDH-A and NDH-F polypeptides. These results complemented by those obtained through blue-native-PAGE indicated that NDH-A and NDH-F polypeptides are part of a 580 kDa NADH dependent dehydrogenase complex present in etioplast membranes. This finding proves that accumulation of the Ndh complex is independent of light. The decrease in the relative levels and specific activity of this complex during the transition from etioplast to chloroplasts was accompanied by a parallel decrease in the specific activity of peroxidase associated to plastid membranes. Based on the mentioned observations it is proposed that an electron transport chain from NADH to H2O2 could be active in barley etioplasts.     

Selective enzymatic radioactive and spin labelling of phospholipids in biological membranes: application to study of temperature-induced changes of microsomal phosphatidylinositols and mitochondrial polyglycerophosphatides.

A new method for the covalent radioactive and spin labelling of lipids within isolated biological membranes has been described in detail and applied to studies of temperature-induced changes of microsomal and mitochondrial membranes. The method is based on the enzymatic use of radioactive substrates carrying covalently bound doxyl derivatives of stearic acid in the biosynthesis of phospholipids in isolated membranes. Radioactive-and spin-labelled lipids bound to the microsomal and mitochondrial membranes were then used as internal probes in monitoring temperature-induced changes of these membranes. Since the analysis of isolated radioactive-and spin-labelled lipids revealed the exact composition of membrane-bound labelled lipids, specific temperature-induced changes were correlated with specific lipids of examined membranes. Phosphatidylinositol of microsomal membranes and polyglycerophosphatides (phosphatidyl-glycerol and cardiolipin) of mitochondrial and inner mitochondrial membranes were found to be involved in the apparent formation of lipid clusters at around 20-30 degrees C. Cardiolipin was found to be involved in the fluidization of inner mitochondrial membranes. These findings are discussed in view of the present state of knowledge of the organization of lipids in biological membranes.