Acid and alkaline phosphatases activities in vascular smooth muscle: species differences and subcellular distribution

1. Acid and alkaline phosphatase activities were studied in rat and dog aortic muscle using p-nitrophenyl phosphate (p-NPP) as the substrate. Alkaline phosphatase activity was quite comparable to acid phosphatase activity in rat aortic microsomes as well as further purified plasma membranes, but considerably lower than acid phosphatase activity in dog aortic membranes. 2. Subcellular distribution of acid and alkaline phosphatase activities in these vascular muscles indicated that alkaline phosphatases and a large portion of acid phosphatase activities were primarily associated with plasma membranes and the distribution of acid phosphatase showed little resemblance to that of N-acetyl-beta-glucosaminidase, a lysosomal marker enzyme. 3. The rat aortic plasmalemmal acid and alkaline phosphatase activities responded very differently to magnesium, fluoride, vanadate and EDTA. The alkaline phosphatase was more susceptible to heat inactivation than acid phosphatase. 4. These results suggest that these two phosphatases are likely to be two different enzymes in the smooth muscle plasma membranes. The implication of the present findings is discussed in relation to the alteration of these phosphatases in hypertensive vascular diseases.     

Highly purified plasma membranes from rat hepatocytes following rate-isopycnic zonal centrifugation

Plasma membranes from liver parenchymal cells were isolated by rate-isopycnic zonal centrifugation. A method is described for the Beckman size 15 zonal rotor. It involved preparation from a perfused liver of a parenchymal cell-enriched homogenate in isoosmotic sucrose. The nuclear fraction containing membranes was recovered by centrifugation. The resuspended pellet was applied on the gradient of the zonal rotor. The isolated membranes had the same isopycnic banding density as 37% sucrose (w/w). The specific activity of 5′-nucleotidase, a widely used plasma membrane marker, was 105 μmoles·(mg protein)^?1·h^?1 being enriched by a factor of 50 as compared with parenchymal cell homogenate. The plasma membrane fraction was free of the mitochondrial and lysosomal enzymes, succinate dehydrogenase and acid phosphatase. No DNA and 10 μg RNA per mg plasma membrane protein were found. The purity of the membranes and their morphological appearance were controlled by electron microscopy. The preparation consisting of large membrane sheets showed a considerable purification away from other cellular components. A comparison with similar methods indicates that plasma membranes of a higher degree of purity can be obtained from parenchymal cells.     

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.     

Isolation and characterization of subcellular membranes of Entamoeba invadens

A method is described for the isolation of subcellular membranes of Entamoeba invadens. Plasma membranes were obtained by rate centrifugation followed by isopycnic centrifugation on a sucrose gradient. Intact phagolysosomes floated in a 10% sucrose solution providing a simple technique for isolation. Phagolysosomal membranes were collected by isopycnic centrifugation, after lysis of the phagolysosomes. Microsomes were obtained by differential centrifugation. Membrane fractions were examined by electron microscopy, and the contamination of each fraction was determined with marker enzymes. Mg2+-ATPase is associated with the plasma membrane. Acid phosphatase (beta-glycerophosphate) was associated mainly with phagolysosmal membranes. Plasma membranes also contained acid phosphatase activity which hydrolyzes p-nitrophenylphosphate but not beta-glycerophosphate. The localization of the two phosphatases was confirmed cytochemically. Isolated plasma membranes were contaminated with phagolysosomal membranes (15%) and with microsomes (25%). No more than 5% of the phagolysosomal membrane fraction consisted of plasma membranes. Contamination of the microsomes by plasma and phagolysosomal membranes was 10% and 7%, respectively. Plasma membranes and phagolysosomal membranes had a high ratio of cholesterol to phospholipid (0.93 and 1.05 mumol/mumol, respectively). Microsomes were relatively poor in cholesterol (0.39 mumol/mumol). Microsomes, plasma, and phagolysosomal membranes contained increasing amounts of spingolipids (12%, 17%, and 28%). Phagolysosomal membranes had a high percentage of phosphatidylserine but little phosphatidylcholine. Microsomes were rich in phosphatidylcholine (45%). Differences in phospholipid composition between plasma and phagolysosomal membranes are discussed in view of the phagocytic process.     

Purification of rabbit endometrial plasma membranes from receptive and non-receptive uteri

We have developed a method for isolation of plasma membranes from rabbit endometrium, with high yield and purification. Endometrial homogenates are precipitated with calcium chloride and the resulting supernatant is fractionated by centrifugation in a self-forming gradient of 20% Percoll. Before fractionation, the intact luminal epithelial surface was labelled with 125I-labelled soyabean agglutinin. Between buoyant densities of 1.015 and 1.017 g/ml, a discrete peak of surface label was obtained, which coincided with activities for 5'-nucleotidase and alkaline phosphatase, enzyme markers for the plasma membrane. This peak was well separated from the majority of cellular protein, and from marker enzyme activities for mitochondria and microsomes (NADH cytochrome C reductase) and lysosomes (acid phosphatase). Electron microscopy of the purified membranes showed membrane sheets and vesicles free from other cellular organelles. Analysis of detergent-soluble membrane proteins, fractionated by concanavalin A-affinity chromatography, revealed differences in the protein pattern of membranes from uteri of rabbits receptive (Day 6 of pregnancy) and non-receptive (Day 3) for implantation. The method will be useful for generation of immunological and affinity probes for surface antigens involved in ovoimplantation.     

Isolation of plasma membranes from ram spermatozoa by a two-phase polymer system

The plasma membranes of ram spermatozoa were disrupted in a hypotonic EDTA medium and isolated by using a two-phase polymer system of dextran--polyethyleneglycol. The plasma membranes obtained were of a relatively high degree of purity (approximately 70%) as judged by electron microscopy observations and measurements of the marker enzymes alkaline phosphatase, ATPase and AMPase. The activity of succinate cytochrome C reductase, a marker of mitochondrial membranes, was very low.     

Some properties of acid and alkaline phosphates from boar sperm plasma membranes

Boar sperm plasma membranes were purified by differential and sucrose density equilibrium centrifugation and were found to yield a single band at a density of 1.14 g/cm3. Both alkaline and acid phosphatase activities were enriched in this fraction. The alkaline phosphatase activity was optimal in 100 mM tris (hydroxymethyl) methylamine (Tris)-NaHCO3 at pH 9.9 with 0.05% Triton X-100 and 1 mM MgCl2. This activity was inhibited by ethylenediaminetetraacetic acid (EDTA), cadmium, zinc or heating at 60 degrees C for 30 min. Also, L-homoarginine caused approximately 70% inhibition and L-phenylalanine or L-leucine caused about 10 to 20% inhibition. Acid phosphatase activity was optimal in 100 mM sodium acetate at pH 5.1 with 0.05% Triton. Sodium dodecyl sulfate, potassium fluoride (KF) or sulfhydryl reagents inhibited the activity, while EDTA or heating at 60 degrees C had no effect. These data for enzymes from boar sperm plasma membranes can be used for future work on the quantitation of the enzymes, distinguishing these two phosphatases from other phosphohydrolases, purification of the enzymes and for comparison to phosphatases in other tissues.     

Acid phosphatases from beet root (Beta vulgaris) plasma membranes

Several acid phosphatases (EC 3.1.3.2) were found in beet root ( Beta vulgaris L.) plasma membranes. Two of them were partially purified by an extraction of plasma membranes with octylglucoside and successive gel-filtration and anion-exchange chromatographies. With p -nitrophenyl-phosphate (pNPP) as substrate, most of the phosphatase activity was found in a fraction containing an 82-kDa protein. This phosphatase showed an optimum pH of 5.4 and was inhibited by Cu²⁺, Zn²⁺, molybdate or vanadate. The other phosphatase had a lower specific activity with p NPP, but was able to dephosphorylate phospho-myelin basic protein (phospho-MBP). This phosphatase presented two polypeptides with molecular masses of 36 and 65 kDa and was 83% inhibited by 2 n M okadaic acid, which suggests it is a PP2A protein phosphatase. As the phosphatase activity was high in soluble (non-membrane) fractions, the possibility that phosphatases in plasma membranes were soluble contaminants was assessed. Following the method of Bérczi and Møller (Plant Physiol. 116:1029, 1998), it was found that about 45% of both acid and protein phosphatase activities could be due to soluble enzymes trapped inside membrane vesicles.     

Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae

Subcellular membranes of Saccharomyces cerevisiae, including mitochondria, microsomes, plasma membranes, secretory vesicles, vacuoles, nuclear membranes, peroxisomes, and lipid particles, were isolated by improved procedures and analyzed for their lipid composition and their capacity to synthesize phospholipids and to catalyze sterol delta 24-methylation. The microsomal fraction is heterogeneous in terms of density and classical microsomal marker proteins and also with respect to the distribution of phospholipid-synthesizing enzymes. The specific activity of phosphatidylserine synthase was highest in a microsomal subfraction which was distinct from heavier microsomes harboring phosphatidylinositol synthase and the phospholipid N-methyltransferases. The exclusive location of phosphatidylserine decarboxylase in mitochondria was confirmed. CDO-diacylglycerol synthase activity was found both in mitochondria and in microsomal membranes. Highest specific activities of glycerol-3-phosphate acyltransferase and sterol delta 24-methyltransferase were observed in the lipid particle fraction. Nuclear and plasma membranes, vacuoles, and peroxisomes contain only marginal activities of the lipid-synthesizing enzymes analyzed. The plasma membrane and secretory vesicles are enriched in ergosterol and in phosphatidylserine. Lipid particles are characterized by their high content of ergosteryl esters. The rigidity of the plasma membrane and of secretory vesicles, determined by measuring fluorescence anisotropy by using trimethylammonium diphenylhexatriene as a probe, can be attributed to the high content of ergosterol.     

Isolation and characterization of Golgi membranes from bovine liver

Zonal centrifugation has been used to isolate a fraction from bovine liver which appears to be derived from the Golgi apparatus. Morphologically, the fraction consists mainly of sacs and tubular elements. Spherical inclusions, probably lipoproteins, are occasionally seen in negative stains of this material. The preparation is biochemically unique. UDP-galactose:N-acetyl glucosamine, galactosyl transferase activity is concentrated about 40-fold in this fraction compared to the homogenate. Rotenone- or antimycin-insensitive DPNH- or TPNH- cytochrome c reductase activities are 60–80% of the level of activities found in microsomes. Purified organelles from bovine liver such as plasma membranes, rough microsomes, mitochondria and nuclei have negligible levels of galactosyl transferase. Some activity is present in smooth microsomes but at a level compatible with the possible presence of Golgi membranes in this fraction. The Golgi fraction does not contain appreciable amounts of enzymes such as ATPase, 5'-nucleotidase, glycosidase, glucose-6-phosphatase, acid phosphatase, or succinate-cytochrome c reductase. Similar fractions isolated from bovine epididymis also have very high levels of galactosyl transferase. The fraction is heavily osmicated when incubated for long periods of time at elevated temperatures, a characteristic property of Golgi membranes.     

Spontaneous and detergent-induced vesiculation of thymocyte plasma membranes

An analog of lysophosphatidylcholine (1-dodecyl-propanediol-3-phosphocholine) which does not impair membrane-bound enzymes was used for the induction of shedding of membrane vesicles from intact calf thymocytes. Without liberation of intracellular enzymes such as lactate dehydrogenase (EC 1.1.1.27) the shedded membranes contained 15--25% of the total activity of the plasma membrane enzymes alkaline phosphatase (EC 3.1.3.1), nucleotide pyrophosphatase (EC 3.1.4.1) and gamma-glutamyl transferase (EC 2.3.2.2). Membrane-free supernatants only exhibited trace activities of these enzymes. Without further purification, the specific enzyme activities in shedded membranes were of the same order of magnitude as in purified plasma membranes prepared after nitrogen cavitation of thymocytes. Small amounts of membrane vesicles which showed a different composition could be removed without detergent. These membranes exhibited a 3-fold lower specific activity of the gamma-glutamyl transferase while that of the alkaline phosphatase and nucleotide pyrophosphatase was similar as in detergent induced membrane vesicles. Distinct differences also were found in the protein pattern. The content of total cholesterol and phospholipid in vesicles shed spontaneously or after detergent treatment was nearly identical, however, significant differences were found in the fatty acid composition of the main phospholipids. The content of polyunsaturated fatty acids (linoleic and arachidonic acid) increased in the order: spontaneously shedded membranes, detergent induced vesicles, conventional purified plasma membranes. These results are discussed in terms of the heterogeneous composition of areas of the thymocyte plasma membrane.     

Some properties of a Ca2+- and (or) Mg2+-requiring nucleoside di- and tri-phosphatase(s) associated with the membranes of rat pancreatic zymogen granules.

Membranes of rat pancreatic zymogen granules have been purified and found to be essentially free of contamination by mitochondria, microsomes, and plasma membranes. They possessed an acid phosphatase activity which derived probably from lysed lysosomes contaminating the purified zymogen granules from which the membranes were prepared. These membranes were found to contain a strong Ca2+- and (or) Mg2+-requiring activity toward all nucleoside di- and tri-phosphates. Various data support the tentative conclusion that a single protein catalyzes the hydrolysis of the nucleoside di- and tri-phosphates. This protein appears to be intrinsic with its active site localized on the internal face of the membranes.     

Functional Expression of p64, an Intracellular Chloride Channel Protein

p64 is a protein identified as a chloride channel by biochemical purification from kidney microsomes. We expressed p64 in HeLa cells using a recombinant vaccinia virus/T7 RNA polymerase driven system. Total cell membranes were prepared from infected/transfected cells and fused to a planar lipid bilayer. A novel chloride channel activity was found in cells expressing p64 and not in control cells. The p64-associated activity shows strong anion over cation selectivity. Single channels show prominent outward rectification with single channel conductance at positive potentials of 42 pS. The chloride channel activity is activated by treatment of the membranes with alkaline phosphatase and inhibited by DNDS and by TS-TM calix(4)arene. Whole membrane anion permeability was determined by a chloride efflux assay, revealing that membranes from cells expressing p64 showed a small but highly significant increase in chloride permeability, consistent with expression of a novel chloride channel activity. Received: 17 November 1997/Revised: 9 February 1998     

Localization of particulate guanylate cyclase in plasma membranes and microsomes of rat liver.

The subcellular localization of guanylate cyclase was examined in rat liver. About 80% of the enzyme activity of homogenates was found in the soluble fraction. Particulate guanylate cyclase was localized in plasma membranes and microsomes. Crude nuclear and microsomal fractions were applied to discontinuous sucrose gradients, and the resulting fractions were examined for guanylate cyclase, various enzyme markers of cell components, and electron microscopy. Purified plasma membrane fractions obtained from either preparation had the highest specific activity of guanylate cyclase, 30 to 80 pmol/min/mg of protein, and the recovery and relative specific activity of guanylate cyclase paralleled that of 5'-nucleotidase and adenylate cyclase in these fractions. Significant amounts of guanylate cyclase, adenylate cyclase, 5'-nucleotidase, and glucose-6-phosphatase were recovered in purified preparation of microsomes. We cannot exclude the presence of guanylate cyclase in other cell components such as Golgi. The electron microscopic studies of fractions supported the biochemical studies with enzyme markers. Soluble guanylate cyclase had typical Michaelis-Menten kinetics with respect to GTP and had an apparent Km for GTP of 35 muM. Ca-2+ stimulated the soluble activity in the presence of low concentrations of Mn-2+. The properties of guanylate cyclase in plasma membranes and microsomes were similar except that Ca-2+ inhibited the activity associated with plasma membranes and had no effect on that of microsomes. Both particulate enzymes were allosteric in nature; double reciprocal plots of velocity versus GTP were not linear, and Hill coefficients for preparations of plasma membranes and microsomes were calculated to be 1.60 and 1.58, respectively. The soluble and particulate enzymes were inhibited by ATP, and inhibition of the soluble enzyme was slightly greater. While Mg-2+ was less effective than Mn-2+ as a sole cation, all enzyme fractions were markedly stimulated with Mg-2+ in the presence of a low concentration of Mn-2+. Triton X-100 increased the activity of particulate fractions about 3- to 10-fold and increased the soluble activity 50 to 100%.     

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.     

Membrane Anomalies in Huntington''s Disease Fibroblasts

Plasma membranes, microsomes, and mitochondria were isolated from paired, passage number matched, cultured human fibroblasts. The cells were obtained from skin biopsies of Huntington's disease (HD) subjects and from sex and age matched controls. All fibroblasts were cultured in identical media for three to seven passages. Enrichment of surface marker enzymes such as Na+,K+-ATPase indicated a 10-fold purification of the isolated plasma membrane. The specific activity of Na+,K+-ATPase was 62 and 82% greater in the crude homogenate and isolated plasma membrane, respectively, of HD fibroblasts than in control fibroblasts. The specific activity of plasma membrane Na+,K+-ATPase was correlated with lipid composition and with membrane structure as determined by measurement of the rotational relaxation time and limiting anisotropy of fluorescence probe molecules. Major alterations in the structure of the plasma membranes in HD fibroblasts were not noted. The rotational relaxation time and limiting anisotropy of 1,6-diphenyl-1,3,5-hexatriene and of trans-parinaric acid were not significantly different between the plasma membrane, microsomes, or mitochondria of HD versus those of control fibroblasts. trans-Parinaric acid demonstrated the coexistence of fluid and solid domains in all three subcellular membrane fractions of the normal and HD skin fibroblasts. Lastly, both trans-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene displayed characteristic breakpoints in Arrhenius plots of absorbance corrected fluorescence in plasma membranes, microsomes, and mitochondria. In all cases, similar breakpoint temperatures, indicative of phase alterations, were noted near 20 degrees and 30 degrees C. These breakpoints were unaltered in HD. In summary, the data do not support the concept of major membrane structural defects in HD.     

The characterization of rat kidney plasma membranes prepared by zonal centrifugation

1. Plasma membranes were isolated from a 10000g-min pellet prepared from a renal cortical homogenate in 20mm-NaHCO(3) by isopycnic centrifugation in a linear sucrose gradient in an ;A'-type zonal rotor. 2. The preparation was characterized by electron microscopy, and alkaline phosphatase, 5'-nucleotidase, l-leucine beta-naphthylamidase and l-leucine p-nitroanilidase activities were found to be selectively associated with the renal plasma membrane. 3. The preparation had a high degree of purity, as indicated by the presence of low activities of marker enzymes associated with subcellular organelles. A preliminary chemical analysis indicated that the chemical composition resembled that of plasma membranes of other tissues. 4. Plasma membranes were also prepared from tubular fragments and their enzyme contents were found to be similar to those of plasma membranes prepared from cortical homogenates. 5. l-Leucine beta-naphthylamidase, l-leucine p-nitroanilidase and 5'-nucleotidase were not enriched to the same extent as alkaline phosphatase in the preparation of plasma membranes from tubular fragments. A possible explanation for this finding is discussed.     

Rapid isolation of neuroblastoma plasma membranes on Percoll gradients. Characterization and lipid composition

A purified plasma membrane fraction was isolated from cultured neuroblastoma (N1E-115) cells on a discontinuous gradient of 5, 25 and 35% Percoll within 1 h of cell disruption by nitrogen cavitation. Yield of plasma membrane, banding in the 25% Percoll (d = 1.051), was high as judged by the recoveries of the marker enzymes, 5'-nucleotidase (58.0 +/- 5.4%, n = 5), alkaline phosphatase (46.0 +/- 3.0%, n = 4) and Mg2+-stimulated neutral sphingomyelinase (48.0 +/- 4.2%, n = 3); enrichment of specific activities of these enzymes relative to total cell homogenate (lysate) were 10.9 +/- 1.0-, 9.1 +/- 1.0- and 9.6 +/- 0.4-fold, respectively. Levels of marker enzymes for other organelles were less than 3% of total activity, except for microsomes (less than 9%). The plasma membrane fraction was further characterized by 2-, 5- and 6-fold higher content (nmol/mg protein) of total phospholipids, free cholesterol and sphingomyelin, respectively, compared to lysate. Ratios of free cholesterol to phospholipids and of sphingomyelin to phosphatidylcholine in the plasma membrane fraction were about 2-fold greater than that of lysate. The cholesterol ester content of plasma membrane (36 +/- 8 nmol/mg protein) was 2-3-fold higher than that of lysate. Sphingomyelin of the plasma membrane fraction had a higher concentration of long-chain fatty acids (more than 18 carbon atoms) relative to lysate or microsomes. Significant differences also were observed in the fatty acyl composition of diphosphatidylglycerol, cholesterol esters and triacylglycerol of plasma membrane. Thus, we have devised a rapid and reliable method for isolation of highly purified plasma membranes of cultured neuroblastoma cells that is suitable for comparison of metabolic relationships between the plasma membrane and other cellular organelles.     

The plasma membrane of Saccharomyces cerevisiae. Isolation and some properties.

The isolation of Saccharomyces cerevisiae plasma membrane was carried out after hypotonic lysis of yeast protoplasts treated with concanavalin A by two independent methods: a, at low speed centrifugation and b, at high speed centrifugation in a density gradient. Several techniques (electron microscopic, enzymic, tagging, etc.) were used to ascertain the degree of purification of the plasma membranes obtained. The low speed centrifugation technique as compared with the other method gave a higher yield of plasma membranes with a similar degree of purification. Analysis of the yeast plasma membrane of normally growing cells by sodium dodecyl sulphate polyacrylamide gel electrophoresis showed at least 25 polypeptide bands. Twelve glycoprotein bands were also found, and their apparent molecular weights were determined. Treatment of the protoplasts with cycloheximide resulted in a significant decrease in the carbohydrate and protein content of the plasma membrane. The electrophoretic pattern of the plasma membrane of cycloheximide-treated cells showed a redistribution of the relative amounts of each protein band and a drastic reduction in the number of Schiff-positive bands. The isoelectric point of the most abundant proteins was low (pI 4) or lower than expected from previous data. A large part of the mannosyl transferase activity found in the cell (80%) was associated with the internal membranes, the remaining activity (20%) was located in the plasma membrane preparation. Part of the mannosyl transferase activity of the cells is located at the plasma membrane surface. Invertase (an external mannoprotein) is found in both the plasma and internal membranes, and as the specific activity dropped significantly following cycloheximide treatment of the cells, it is suggested that these membranes systems are the structures for the glycosylation of a precursor invertase and its subsequent release into the periplasmic space. Other transferase found in the plasma membrane preparation transfers glucose residues from UDPglucose to a poly(alpha(1 leads to 4) polymer identified as glycogen.     

Sub cellular Fractionation of the Longitudinal Smooth Muscle/Myenteric Plexus of Dog Ileum: Dissociation of the Distribution of Two Plasma Membrane Marker Enzymes

The distribution of plasma membrane markers, the sodium pump [evaluated as ouabain-sensitive, potassium-stimulated p-nitrophenyl phosphatase (K+-pNPPase)], [3H]saxitoxin binding, and 5'-AMPase, was studied in the subcellular fractions prepared from the homogenates of the longitudinal smooth muscle/myenteric plexus of dog ileum. The K+-pNPPase activity and [3H]-saxitoxin binding were found to be predominantly associated with the synaptosomal fraction as indicated by the high level of these activities in the crude synaptosomal fraction and by the copurification of K+-pNPPase and [3H]saxitoxin binding, but not 5'-AMPase, with several synaptosomal markers during the fractionation of the crude synaptosomal fraction on density gradients. In contrast to the K+-pNPPase activity and [3H]saxitoxin binding, the 5'-AMPase activity was found to be concentrated in the microsomal pellet. Further fractionation of microsomes on density gradient resulted in copurification of 5'-AMPase but not K+-pNPPase or [3H]saxitoxin binding, with other smooth muscle plasma membrane-bound enzymes, such as high-affinity Ca2+-ATPase, Mg2+-ATPase, and Ca2+-ATPase. It was concluded that in the longitudinal smooth muscle/myenteric plexus, the sodium pump activity is present in higher density in the neuronal plasma membranes whereas 5'-AMPase activity is concentrated in the smooth muscle plasma membranes.