Simultaneous preparation of basolateral and brush-border membrane vesicles from sea bass intestinal epithelium

A method is described for simultaneous preparation of brush-border and basolateral sea bass enterocyte membranes using simple differential centrifugation and discontinuous sucrose gradient density centrifugation techniques. Basolateral membranes were purified with a Na+/K(+)-ATPase yield of about 11% of the original activity, with an enrichment factor of 12. The yield of maltase-glucoamylase, a specific marker of brush-border membranes, was also about 11% of the original activity, with 15-fold enrichment. The characteristics of these membrane preparations were determined. Electron microscopy analysis showed that these two membrane preparations were uniform in size and vesicular in nature. Orientation studies revealed that the luminal membrane vesicles were right-side out and 43% of the antiluminal membrane vesicles were sealed inside out. Investigation of D-glucose and L-leucine uptake showed that these two plasma membrane preparations retained their transport properties.     

Isolation of a highly enriched sarcolemma membrane fraction from canine heart.

A highly enriched sarcolemma preparation was isolated by differential centrifugation of a canine ventricular homogenate followed by centrifugation of a membrane fraction layered over 22% (w/v) sucrose. Ouabain binding, ouabain-sensitive potassium phosphatase activity and 5'-nucleotidase activity were enriched 19--27 fold over the homogenate whereas Ca2+-ATPase and succinate dehydrogenase activities were 0.75 and 0.36, respectively, of that for the homogenate. The isolation procedure was relatively rapid and yielded about 2.0 mg protein/100 g of ventricular muscle. The highest salt concentration used in the procedure was 0.6 M KCl and no detergents were employed. Initial characterization studies suggested that the sarcolemma-enriched fraction consists predominantly if not totally of freely permeable membrane vesicles and that the sarcolemma does not manifest a Ca2+-ATPase activity, at least within the limits of the assay procedures employed. This preparation was concluded to be about 1.5- to 4-fold more highly enriched with sarcolemmal markers than preparations obtained by previously published procedures. Accordingly, the preparation provides an improved basis for the probe of calcium movements that occur across the sarcolemma in association with the excitation-contraction-relaxation sequence of the mammalian myocardial cell.     

Comparison of brush border membrane vesicles prepared by three methods from larval Manduca sexta midgut

Brush border membrane vesicles (BBMV) were prepared from freshly isolated posterior larval Manduca sexta midguts by differential calcium precipitation, differential magnesium precipitation and differential ultrasonication. BBMV were also prepared from frozen posterior larval M. sexta midguts by differential calcium precipitaion and differential magnesium precipitation. The yield of BBMV by both differential precipitation methods was 5–6 times greater than that by the differential ultrasonification method. Enrichments of the brush border membrane marker enzymes alkaline phosphatase, γ-glutamyl transferase, and aminopeptidase were similar in all preparations. The polypeptide composition of all preparations was also similar. The specific activity of mitochondrial and microsomal marker enzymes was higher in BBMV prepared from freshly isolated midguts by the differential precipitation methods than in BBMV prepared by the ultrasonication method. The specific activity of cytochrome-c oxidase was 2.5–7 times higher in BBMV prepared from frozen midguts than in BBMV prepared from fresh tissue.     

Studies of pyridine nucleotide oxidizing enzymes from human neutrophils

An NADH cytochrome c reductase has been identified in plasma membrane fractions from neutrophils in addition to the superoxide producing NADPH oxidase which has been extensively studied by other investigators. Activation of neutrophils resulted in increased enzyme activities but to different degrees; the NADH cytochrome c reductase increased 2 fold in specific activity and the NADPH oxidase 30 fold. Treatment of the plasma membrane fraction with sonication and differential centrifugation yielded a particulate fraction (R2) with a 2 fold increase in specific activities of both enzymes and concentrations of cytochrome b and FAD. The cytochrome b in the preparation was not reduced under anaerobic conditions by either NADH or NADPH. Treatment of preparations of R2 with deoxycholate or potassium thiocyanate separated the two enzymes yielding particulate preparations with only NADPH oxidase or NADH cytochrome c reductase activity, respectively.     

Uridine phosphorylase activity of isolated plasma membranes of rat liver.

Plasma membranes were isolated from rat liver homogenates either by differential centrifugation or by fractionation in discontinuous sucrose density gradients. Both membrane preparations contained about 17% of the total uridine phosphorylase (EC 2.4.2.3) activity and 44% of the total 5'-nucleotidase (EC 3.1.3.5). The enrichment factor for uridine phosphorylase in the fractions prepared by differential centrifugation was about 2.8 and by the gradient method, as much as 11.0; the respective enrichment factors for 5'-nucleotidase were 1.8 and 9.5. Uridine phosphorylase activity of isolated plasma membrane fractions was stimulated 2.5-fold by 0.1% Triton X-100. Unlike the cytosol enzyme, uridine phosphorylase of plasma membranes showed little or no deoxyuridine-cleaving activity. Contamination of the membrane fractions by thymidine phosphorylase (EC 2.4.2.4) of the cytosol was negligible. The other subcellular organelles obtained by either procedure and characterized by marker enzyme activities were found not to contain significant uridine phosphorylase activity; the cytosol fractions contained just over 70% of the total uridine phosphorylase activity with an enrichment of only about 2.8-fold. The activity of the cytosol enzyme was not stimulated by Triton X-100.     

Purification of plasma membranes from roots of barley: specificity of the phosphotungstic Acid-chromic Acid stain

Plasma membrane vesicles from roots of barley (Hordeum vulgare L., var. Arivat) had an equilibrium density in sucrose of about 1.16 grams per cubic centimeter, but could not be purified satisfactorily with the procedure developed for roots of other plant species. The reported procedure involving differential centrifugation to remove mitochondria (peak density of 1.18 grams per cubic centimeter) and subsequent density gradient centrifugation to purify plasma membrane vesicles was modified to include a narrower differential centrifugation fraction (13,000 to 40,000g instead of 13,000 to 80,000g) and a narrower density range in the sucrose gradient (1.15 to 1.18 grams per cubic centimeter instead of 1.15 to 1.20 grams per cubic centimeter). The fraction obtained by the modified procedure was between 60 and 70% pure as determined by staining with the phosphotungstic acid-chromic acid procedure, which was judged to be reliable for identifying plasma membrane vesicles in subcellular fractions from barley roots. The plasma membrane fraction was enriched in K⁺-stimulated ATPase activity at pH 6.5. The presence of nonspecific ATP-hydrolyzing activity in the plasma membrane fraction made it difficult to determine if the ATPase had properties in common with those reported for cation absorption in barley roots.     

Isolation of ribosomes from Escherichia coli by chromatography on methyl-albumin-kieselgel.

The present communication shows that ribosomes isolated by differential centrifugation retain their composition and biological activity during subsequent methyl-albumin-kieselgel (MAK)-chromatography. The data obtained were used to develop a novel chromatographic procedure for the isolation of ribosomes from E. coli lysates. It could be demonstrated that purified preparations of active ribosomes can be obtained by MAK-chromatography avoiding ultracentrifugation.     

A differential scanning calorimetry study of acetylcholine receptor-rich membranes from Torpedo californica

Various acetylcholine receptor-rich membrane preparations from Torpedo californica electroplax tissue were examined using the techniques of differential scanning calorimetry coupled with gel electrophoretic analysis of heat-denaturing material and functional assays following passage through discrete transitions. In unfractionated membranes, four irreversible calorimetric transitions were observed, one of which (Td = 59 degrees C) could be assigned to a complete loss of acetylcholine receptor function. A second lower temperature transition apparently corresponds to loss of certain peripheral membrane proteins including the Mr = 43,000 polypeptide and the acetylcholinesterase activity. Membrane preparations highly enriched in acetylcholine receptor polypeptides contained a major transition at 59 degrees C which could be shown to be sensitive to the presence of added ligands of the acetylcholine receptor, supporting its assignment to structural alterations of the receptor protein or its arrangement in the membrane.     

A comparison of the intrinsic protein kinase activities of membrane preparations from various tissues.

The ability of membrane preparations from different tissues to catalyse the phosphorylation of their endogenous protein (intrinsic protein kinase activity) was determined. It was found that membrane fragments prepared from a large variety of tissues contain this activity although the actual level varies quite widely. Preparations from vas deferens and brain have nearly ten times more activity than preparations from heart, kidney, or erythrocytes. Plasma membranes from skeletal muscle have no detectable activity. The intrinsic protein kinase activity of membrane fragments from most tissues is stimulated by cyclic AMP although the phosphorylation of proteins in preparations of kidney microsomes or heart plasma membranes, is not affected. cyclic GMP (10 micronM) has no effect on the intrinsic protein kinase activity of any membrane preparation examined. A specific inhibitor of soluble, cyclic AMP-stimulated, protein kinase has no effect on the intrinsic protein kinase activity of any of the membrane preparations examined. This suggests that the intrinsic protein kinase activity of membrane preparations may be due to the presence of a specific protein kinase. It is suggested that an examination of the distribution of membrane-bound intrinsic protein kinase activity among different tissues may be helpful in determining the function of the reaction.     

Isolation of adenylate cyclase-enriched membranes from mammalian cells using concanavalin A.

Plasma membrane vesicles containing adenylate cyclase and beta-adrenergic receptors were prepared from 1321N1 human astrocytoma cells by a procedure involving the use of concanavalin A to stabilize the plasma membrane to fragmentation and vesiculation upon cell lysis. Treatment of cells with concanavalin A causes these plasma membrane markers to sediment to a higher density of sucrose and in a narrower band than observed with untreated cells. Upon treatment of the heavy membrane fragments with alpha-methylmannoside to remove bound concanavalin A, the enzyme markers again sediment a lower densities of sucrose. This reversible change in sedimentation behavior has been used to obtain preparations of plasma membranes enriched 14- to 21-fold (recovery 25%) in adenylate cyclase activity and about 12-fold (recovery 16%) in beta-adrenergic receptor density, as compared to lysates. The adenylate cyclase of purified membranes responded normally to isoproterenol and prostaglandin E1. Experiments with S49 and YAC mouse lymphoma cells and human skin fibroblasts indicate that this procedure may be adaptable to the isolation of plasma membranes from a variety of cultured cell lines.     

Sarcoplasmic reticulum from flounder muscle having improved lipid peroxidative activity

A procedure for the isolation of sarcoplasmic reticulum from winter flounder (Pseudopleuronectes americanus) resulted in a fraction with a specific activity of lipid peroxidation two to three times that of previous preparations. In addition, good stability of the NADH-dependent lipid peroxidative activity was achieved. There appeared to be minimal contamination of the preparation with lysosomes and mitochondria. The flounder sarcoplasmic reticulum was highly active with respect to ATPase and calcium uptake. The membrane fraction contained 43% lipid and 57% protein; 60% of the lipids were phospholipids. Phosphatidylcholine was the major phospholipid present.     

Protein synthesis by microsomal particles from regenerating rat liver

1. Washed microsome particles from regenerating liver were shown to incorporate [(14)C]leucine into protein more actively than similar preparations from normal liver. 2. The total incorporation in the preparations from regenerating liver increased linearly with the amount of protein incubated, whereas this was not so with preparations from normal liver. 3. The greater activity of regenerating-liver microsomes appeared to be associated with the bound polysomes. 4. The size distribution of polysomes obtained after removal of membrane with deoxycholate was the same in normal and regenerating liver. 5. In general the activity of polysome preparations from normal and regenerating liver was similar. 6. It is concluded that the greater activity of the particles in the microsome fraction from regenerating liver is to be attributed to the ribosomes bound to membrane and that their activity is controlled by factors present in the membrane.     

Staphylococcus aureus adenosine triphosphatase: inhibitor sensitivity and release from membrane.

Homogeneous preparations of cytoplasmic membrane isolated from Staphylococcus aureus 6538P exhibited membrane-associated adenosine triphosphatase (ATPase) activity. Membrane ATPase activity was activated by divalent cations (4.0 mM: Mg2+ greater than Mn2+ greater than Co2+ greater than Zn2+), and ATP was hydrolyzed more readily than other nucleoside triphosphates and phosphorylated substrates. The pH optimum for the membrane ATPase was 6.5. The ATPase could not be released from the membrane by differential osmotic treatments, but detergent treatment effectively solubilized active enzyme. The nonionic detergent Triton X-100 (1%) released a protein with ATPase activity, after substrate-dependent staining in polyacrylamide gels, that differed slightly in electrophoretic migration when compared to the active enzyme solubilized with sodium dodecyl sulfate (0.1%). Membrane-associated ATPase activity was inhibited by N,N'-dicyclohexylcarbodiimide (0.001 to 1 mM) and NaF (50% inhibition at 5 mM NaF). Azide and trypsin inhibited activity, whereas ouabain had a slight inhibitory effect. Diethylstilbestrol showed appreciable activation of the membrane ATPase over the range employed (0.001 to 1 mM).     

An electron microscope comparison of plasma membrane vesicles from meristematic and mature soybean root tissue

Plasma membrane vesicles were isolated from homogenates of meristematic and mature soybean root tissue by differential sucrose gradient centrifugation. Vesicles were positively identified by the phosphotungstic acid-chromic acid procedure (PACP). The two preparations were comparable in size class distribution, mitochondrial contamination, and per cent plasma membrane vesicles present. Purity levels were estimated to be greater than 75%. The specificity of PACP was observed for a variety of cell types from both regions. Some variability in PACP staining was offset by careful modulation of the stain protocol and was found to be independent of developmental stage in subcellular fractions. Patchy or discontinuous staining, observed in both intact tissue and in subcellular fractions from both regions, was found to be a function of stain time.     

A comparison between adenylate cyclase solubilized from normal and Rous sarcoma virus-transformed chicken embryo fibroblasts

The specific activity of adenylate cyclase in membrane preparations obtained from Rous Sarcoma virus-transformed chicken embryo fibroblasts is two to four times lower than that found in untransformed membranes. Adenylate cyclase was solubilized from normal and transformed membranes in order to evaluate the influence of the membrane phase on the properties of the enzyme. Adenylate cyclase in normal and transformed membranes differed in specific activity, V for ATP, activation entropies, sensitivity to Ca2+, and stability at 37 degrees C. Solubilization with Brij 96 abolished or greatly reduced these differences. These data suggest that the differences between adenylate cyclase activities in normal and transformed chicken embryo fibroblasts are due either to differential modulation of enzyme activity by an effector which requires intact membranes for its effects, or indirect effects due to altered membrane properties.     

Kinetic characterization of plasma membrane atpase from Saccharomyces cerevisiae

1. Plasma membrane preparations have been isolated from spheroplasts of Saccharomyces cerevisiae, strain R XII, via lysis and subsequent differential centrifugation. These preparations are almost devoid of mitochondrial contamination. 2. The plasma membrane ATPase is fairly stable when refrigerated, but loses activity at 8 degrees C and above. Below pH 5.6 the ATPase is irreversibly inactivated. The enzyme also splits GTP and ITP, although to a lesser extent. 3. Mg2+-ions are essential as part of the reactive substrate, MgATP, and furthermore they activate the ATPase. Optimal conditions depend on substrate concentration. When the concentration of free Mg2+ ions exceeds about 0.1 mM, competitive inhibition occurs. 4. In the range of pH 5.6-9.2 two functional groups dissociate. One, with pKb = 8.1 +/- 0.1 participated in substrate binding and another one with pKb' = 8.1 +/- 0.1 is involved in substrate splitting. 5. The experiments with group-specific inhibitors suggest that an alpha-amino group and a sulfhydryl residue are involved in substrate binding and conversion. Furthermore, imidazole, tryptophan and carboxyl residues may be important for the catalytic process.     

Mitochondrial hexokinase from small-intestinal mucosa and brain

1. The submitochondrial localization of hexokinase activity in preparations of mitochondria from the small intestine of the guinea pig was studied by conventional methods. 2. Hexokinase activity in this tissue was predominantly associated with the outer mitochondrial membrane. 3. The inactivation of mitochondrial enzymes by trypsin in iso-osmotic and hypo-osmotic conditions was also used to determine the submitochondrial localization of hexokinase activity. 4. Hexokinase activity was found to be on the outside of the outer mitochondrial membrane. 5. It was shown that both type I and type II hexokinase activities are bound to the outside of the outer mitochondrial membrane. The types are present in the same ratio as that in which they occur in the cytosol of the cell. 6. Mitochondrial hexokinase from the small intestine did not show the latency phenomenon demonstrated by mitochondrial hexokinase from brain when subjected to a variety of treatments. However, hexokinase activity was solubilized from preparations of mitochondria from the small intestine by the same treatments as for mitochondrial hexokinase from brain. 7. The submitochondrial distribution of hexokinase activity in mitochondrial preparations from rat brain was determined by the trypsin inactivation method. 8. Hexokinase activity in preparations of mitochondria from rat brain was found on the outside of the outer membrane, between the mitochondrial membranes, and within the inner mitochondrial membrane. 9. Hexokinase from rat brain showed latency properties irrespective of its submitochondrial location.     

Properties of Brush Border Vesicles Isolated from Rat Kidney Cortex by Calcium Precipitation

Brush border membrane vesicles were isolated from rat kidney cortex by differential centrifugation in the presence of 10 mM calcium. Their properties were compared to brush border vesicles isolated by free-flow electrophoresis. By the calcium precipitation method membrane vesicles were obtained in a shorter time with a similar enrichment of brush border marker enzymes (11- to 12-fold for alkaline phosphatase and maltase), with a similarly reduced activity of the marker enzyme for basal-lateral plasma membranes and an almost identical protein composition as revealed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The transport properties of the two membrane preparations for D-glucose, L-phenylalanine, and phosphate are essentially the same; there is some indication for a lower sodium permeability of the vesicles prepared by the calcium precipitation method. The latter vesicles were also shown to exhibit sodium gradient stimulated uptake of L-glutamate.     

Bradykinin modulates the ouabain-insensitive Na+-ATPase activity from basolateral membrane of the proximal tubule.

This paper studies the modulation by bradykinin of the ouabain-insensitive Na+-ATPase activity in both renal cortex homogenate and basolateral membrane from proximal tubule. The increase in bradykinin concentration from 10-14 to 10-10 M stimulated the ouabain-insensitive Na+-ATPase activity in cortex homogenates about 2.2-fold, but inhibited the enzyme activity of basolateral membrane preparations by 60%. In both preparations, the maximal effect was obtained with 10-10 M bradykinin. Further increase in the concentration of bradykinin completely abolished these effects. The antagonist of the B2 receptor, Hyp3, completely abolished the effect of 10-10 M bradykinin on the Na+-ATPase activity in the basolateral membrane preparation in a dose-dependent manner, but had no effect on the bradykinin stimulated enzyme activity of the cortex homogenate. Furthermore, in the presence of 10-7 M Hyp3, 10-10 M bradykinin stimulated the Na+-ATPase activity by 45% in the basolateral membrane preparations. The increase in des-Arg9-bradykinin concentration from 10-12 to 10-7 M, an agonist of the B1 receptor, stimulated the Na+-ATPase activity of the cortex homogenates and of the basolateral membrane preparations by 105 and 148%, respectively. In the presence of 25 microM mergetpa, an inhibitor of kininase I, the increase in bradykinin concentration from 10-12 to 10-10 M promoted similar inhibition of the Na+-ATPase activity of both cortex homogenates and basolateral membrane preparations. These results suggest that bradykinin stimulated the Na+-ATPase activity of proximal tubule through the interaction with B1 receptors and inhibited the enzyme through the interaction with B2 receptors. Furthermore, the cortex homogenate expresses a kininase I activity that cleaves bradykinin to des-Arg9-bradykinin.     

Isolation of complex III from various mitochondria. Comparison of the structural and functional properties of the preparations from beef heart, calf liver and Neurospora crassa

Active complex III was isolated by an improved procedure from beef heart mitochondria, from Neurospora crassa mitochondria and for the first time from mitochondria originating from mammalian tissue other than heart, i.e. calf liver. The described procedure consists of differential extraction of the respective mitochondria, hydroxyapatite chromatography and, finally, either gel- or affinity chromatography. The preparations contain the well known prosthetic groups, i.e. 6-8 mumol b-type heme, 3-4 mumol c-type heme and 5-8 mumol non-heme iron per g of protein. The preparations from beef heart and from calf liver mitochondria are indistinguishable in their subunit composition by sodium dodecyl sulfate polyacrylamide gel electrophoresis, whereas the preparation from Neurospora crassa mitochondria is clearly different. The phospholipid content of all preparations is rather low, amounting to about 100 mumol/g protein. The molar catalytic activity of ubiquinol-9-cytochrome c reductase at 25 degrees C amounts to 50s-1 for the N. crassa complex III and 70-100s-1 for the bovine complexes. After reincorporation into phospholipid vesicles, all preparations how tight coupling between electron transfer from ubiquinol to cytochrome c and proton translocation across the phospholipid bilayer.