Further characterization of a novel phospholipase B (phospholipase A2--lysophospholipase) from intestinal brush-border membranes.

The intestinal brush-border membranes of rats and guinea pigs possess a high molecular weight, calcium-independent phospholipase B (phospholipase A2 - lysophospholipase activities) with the characteristics of a digestive ectoenzyme. A combination of subcellular fractionation, Triton X-114 phase partitioning, chromatofocusing, and preparative sodium dodecyl sulphate - polyacrylamide gel electrophoresis was used to purify a full-length, although denatured, form of this enzyme from the rat. Renaturation of the gel-purified fraction confirmed that both enzyme activities were associated with this protein. Gel slices containing the purified phospholipase B were used to generate a polyclonal antiserum in rabbits that could be used for immunoblotting. The relative mobility of the phospholipase B during electrophoresis in sodium dodecyl sulphate gels was dramatically affected by the percentage of acrylamide and the presence or absence of reducing agents in the gels. This was true for both the purified protein visualized by silver-staining and following electrophoresis of the total proteins of the membrane, with the phospholipase visualized by immunoblotting. Estimates for the molecular mass of the enzyme varied from 130 to 170 kDa in 7.5% gels and from 120 to 130 kDa in 5-10% gradient gels (with a best estimate of 120 kDa). Upon solubilization from the brush-border membrane by papain digestion, the major immunoreactive band migrated with an apparent mass of 80 kDa in both the 7.5% and 5-10% gradient gels. A major cross-reactive band was detected at 97 kDa following immunoblotting of the papain-solubilized proteins from guinea pig brush-border membranes, in agreement with the size of the purified fragment reported in the literature and at 140 kDa following immunoblotting of the intact proteins. Similar immunoblotting produced reaction with a 135-kDa protein from the rabbit brush-border membrane, as well as 95-kDa protein following papain solubilization. These results suggest that while there are species-specific apparent molecular weights, the intestinal brush-border membrane phospholipase B is conserved among species.     

Horse kidney neutral alpha-D-glucosidase: purification of the detergent-solubilized enzyme; comparison with the proteinase-solubilized forms

Neutral alpha-D-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from horse kidney brush-border membranes was solubilized using Emulphogene BC 720 and purified by an affinity chromatography technique. The enzyme preparation (390-fold purified), which was free of other known microvillus hydrolases, exhibited one precipitate line in crossed immunoelectrophoresis and migrated as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Several criteria (charge-shift crossed immunoelectrophoresis and hydrophobic chromatography) revealed the purified detergent form of the enzyme to be an amphipathic molecule. The papain treatment of either brush-border membrane vesicles or the purified detergent form of neutral alpha-D-glucosidase released an enzymatic form devoid of these amphipathic properties. Conversely, after trypsin treatment of the "d' form of the enzyme, two enzymatic forms were obtained: the first and major form retained these amphipathic properties; the second form exhibiting the same properties as the papain-released form. Furthermore, only a very small amount of neutral alpha-D-glucosidase can be released after trypsin solubilization of brush-border membrane vesicles, and the released enzyme did not exhibit amphipathic properties. These results were interpreted as meaning that the trypsin attack site on the detergent form of the enzyme had either poor affinity for, or obstructed access to, the proteinase when the enzyme was integrated in native membrane or in Triton X-100 micelles, whereas the proteolytic site of the papain was always accessible.     

Inhibition of ileal brush-border chloride conductance by specific antibody

Summary Antibody raised in mice was used in attempting to identify proteins responsible for the conductive chloride transport that can be measured in porcine ileal brush border membrane vesicles. Ileal brush-border membrane vesicle protein from pig was separated into five different molecular mass fractions by preparative SDS polyacrylamide disc gel electrophoresis. Separated protein fractions were used to immunize mice. Antibody was screened for reactivity with antigen by Western blotting, and for effects on conductive chloride transport in ileal brush border membrane vesicles. Immunization with brush-border protein from fraction I proteins (>110 kDa) produced polyclonal antisera which specifically inhibited the conductive component of chloride uptake by ileal brush border vesicle preparations. Western blotting of the antigen showed the presence of several protein species of molecular mass >100 kDa that were recognized by immune serum. Spleen cells from a mouse producing antiserum that inhibited conductive chloride transport were fused with a myeloma cell line. The resulting hybridoma colonies produced antibody that reacted with at least seven distinct protein bands by Western blot assay and inhibited chloride conductance in brush-border membrane vesicles.     

Purification and Characterization of Neuron-Specific Surface Antigen Defined by Monoclonal Antibody BM88

Monoclonal antibody BM88 recognizes a neurospecific surface antigen in the CNS and the PNS. In the present study, the antigen recognized by BM88 was immunopurified from pig brain and shown to be a 22-kDa polypeptide by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nonreducing conditions a protein of 40 kDa was obtained, a result indicating that the antigen is composed of two polypeptide chains of equal molecular weight linked by disulfide bridges. Gel filtration of the purified antigen in the presence of Emulphogene suggested that it may be either a monomeric or a dimeric protein. However, in the presence of Triton X-100 a monomeric structure was implied. N-Glycanase digestion indicated that the protein is probably not glycosylated. The purified antigen was characterized as an integral membrane protein by hydrophobic chromatography and phase-separation experiments with Triton X-114. The antigen, or at least the antibody binding region of the molecule, is very susceptible to protease attack, as judged by protease digestion experiments on brain membranes. By using very low concentrations of papain combined with short incubation times, the antigen was converted to a 16.3-kDa membrane-associated polypeptide as assessed by immunoblotting. This polypeptide contained the BM88 binding epitope. Soluble BM88 immunoreactive polypeptides were not obtained. Bacillus cereus phospholipase C was also unable to solubilize the antigen from the membrane. Our results suggest that the molecule, possessing at least one small extramembranous domain, is attached to the membrane via a polypeptide chain.     

Candidate proteins for conductive chloride transport in porcine ileal brush-border membrane

Conductive transport of chloride ion is important in controlling ion and fluid secretion by exocrine tissues. The current study was directed at identifying proteins in the intestinal brush-border membrane that may be involved with conductive chloride transport. Reaction of total brush-border membrane protein with phenyl-isothiocyanate inhibited conductive chloride transport into brush-border membrane vesicles. The conductive transport process was protected from this inhibition by including the ligands Cl- and alpha-phenylcinnamate in the reaction mixture. Brush-border membrane protein protected by this procedure and labeled with fluorescein had an apparent molecular mass in the region of 130 and 23 kDa on separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphorylation of brush-border membrane protein with [gamma-32P] ATP and endogenous protein kinase under conditions causing activation of chloride conductance in membrane vesicles caused the transfer of 32P to several proteins, including ones in the same molecular size range (130 and 23 kDa) as those identified by the fluorescein labeling procedure. Conductive chloride transport in porcine intestinal brush-border vesicles may occur via proteins identified by this differential labeling procedure.     

Characteristics of tripeptide transport in human jejunal brush-border membrane vesicles

These studies are aimed at characterizing the transport of the tripeptide, glycylglycyl-L-proline (GlyGlyPro) across human jejunal brush-border membrane vesicles. GlyGlyPro (0.65 mM) was hydrolyzed by brush-border membrane vesicles with the extent of hydrolysis per mg protein being 23% at 0.5 min, 57% at 1 min and complete hydrolysis at 60 min. Treatment of the membrane vesicles with gel-complexed papain (to remove membrane peptidases) resulted in minimal hydrolysis of GlyGlyPro up to 10 min of incubation. Measurement of GlyGlyPro influx with papain-treated vesicles in the presence of increasing medium osmolarity showed that uptake occurred into an osmotically reactive intravesicular space. Transport of GlyGlyPro with normal and papain-treated membrane vesicles was similar in the presence of an inward Na+ or K+ gradient. No overshoot phenomenon was observed in the presence of an inward proton gradient (extravesicular pH 5.5; intravesicular pH 7.5). An interior negative membrane potential induced by a K+ diffusion potential in the presence of valinomycin stimulated the uptake of the peptide. The effect of increasing concentrations on initial rates of GlyGlyPro uptake revealed the presence of a saturable component as well as a diffusional component. Preloading the membrane vesicles with 20 mM glycylsarcosylsarcosine stimulated uptake by 4-fold. Uptake of GlyGlyPro was inhibited greater than 50% by dipeptides and tripeptides and less than 15% by free amino acids. These results indicate that GlyGlyPro uptake in jejunal brush-border membrane vesicles is not energized by a Na+ or proton gradient and that transport occurs by carrier-mediated and diffusional processes.     

Characterization of an integral membrane glycoprotein associated with the microfilaments of pig intestinal microvilli

An integral membrane glycoprotein of pig intestinal microvilli which exists in two polypeptide forms [mol. wt. 140 K and 200 K as measured by SDS-polyacrylamide gel electrophoresis (SDS-PAGE)] was purified to homogeneity and characterized. The 200-K form is probably a precursor of the 140-K species. We have localized the glycoprotein by electron microscope immunochemistry using specific antibodies and determined its topological organization with respect to the membrane bilayer. Triton X-100 treatments which solubilize most other microvillar membrane glycoproteins from purified, closed, right-side out vesicles do not efficiently extract this protein. The protein can be partially solubilized from the detergent-insoluble residue, either by treatment with proteases (trypsin or papain) or by exposure to low ionic strength buffer in the presence of chelating agents and detergents. Once solubilized by papain or trypsin, the protein co-migrates on SDS-PAGE with the protein obtained by low ionic strength extraction. However, the form of the protein released by papain does not bind detergents and exhibits hydrophilic properties. Our observations are consistent with the 140-K protein having a small hydrophobic domain that anchors it to the microvillar membrane. The 140-K glycoprotein binds in vitro to a 110-K protein of the core cytoskeleton residue. These observations suggest that the 140-K glycoprotein may be a transmembrane protein which may in vivo provide attachment sites for direct or indirect association with polypeptides of the microvillus cytoskeleton.     

Topological studies on the hydrolases bound to the intestinal brush border membrane. I. Solubilization by papain and triton X-100

Papain digestion of closed, right side out vesicles from pig, rat and rabbit jejunum brush border induces the release of the hydrolases bound to the membrane without grossly affecting the lipid bilayer limiting the vesicles. This observation definitely proves that intestinal hydrolases are surface components attached to the external side of the membrane. All proteins released by papain could be identified by electrophoresis and immunoelectrophoresis to already known intestinal hydrolases, with the exception of an unidentified substance strongly stained by the Schiff's reagent.The early observation that the aminopeptidase form released from pig brush border by Triton X-100 is different from that released by papain was extended to other hydrolases from pig, rat and rabbit. In some cases, the Triton-released form could be converted by further proteolytic digestion into a new form similar to that liberated by papain. These facts may be related to the existence of hydrophobic anchors retaining the intestinal hydrolases to the membrane surface.     

The B cell-associated CD37 antigen (gp40-52). Structure and subcellular expression of an extensively glycosylated glycoprotein

The human B lymphocyte-associated CD37 antigen (gp40-52) has been characterized by the monoclonal antibody HD28. The CD37 antigen is strongly expressed on surface immunoglobulin positive B lymphocytes and weakly on a subpopulation of T lymphocytes and myeloid cells. The total molecular mass of the antigen ranges from approximately 40 to 52 kDa in B cell-derived leukemias and malignant lymphomas as well as in normal and anti-mu/B cell growth factor-activated tonsillar B cells. The polydisperse nature of the electrophoretic pattern of the CD37 antigen was found to be due to a microheterogeneity in its carbohydrate moiety. Biochemical analysis showed that the CD37 antigen derived from B cell-lines BJAB and LICR-LON-HMy2 consists of a single chain protein core of approximately 25 kDa to which two N-linked, complex carbohydrate antennae of various length are bound. The glycosylation of the molecule comprises about 50% of the total molecular mass. The molecule does not contain O-linked carbohydrate chains. In contrast, the non-Hodgkin's lymphoma cell line, OCI.LY1, which is growth-dependent on human serum, carries a CD37 antigen with an additional carbohydrate chain resulting in a total molecular mass of approximately 40 to 64 kDa. At the electron microscopy level, this cell surface-expressed antigen was found to be associated with intracellular vesicles. The subcellular distribution of the CD37 antigen may reflect a function of this antigen both at the cell surface and in the cytoplasm. We found that, both due to its peculiar biochemical structure and its ultrastructural distribution, the CD37 antigen closely resembles the 46-kDa species of the mannose 6-phosphate receptor. The implications of this possible congruence for the function of the CD37 antigen are discussed.     

Proteolytic fragmentation of brain myosin and localisation of the heavy-chain phosphorylation site

The heavy chains and the 19-kDa and 20-kDa light chains of bovine brain myosin can by phosphorylated. To localise the site of heavy-chain phosphorylation, the myosin was initially subjected to digestion with chymotrypsin and papain under a variety of conditions and the fragments thus produced were identified. Irrespective of the ionic strength, i.e. whether the myosin was monomeric or filamentous, chymotryptic digestion produced two major fragments of 68 kDa and 140 kDa; the 140-kDa fragment was further digested by papain to yield a 120-kDa and a 23-kDa fragment. These fragments were characterised by (a) a gel overlay technique using 125I-labelled light chains, which showed that the 140-kDa and 23-kDa polypeptides contain the light-chain-binding sites; (b) using myosin photoaffinity labelled at the active site with [3H]UTP, which showed that the 68-kDa fragment contained the catalytic site, and (c) electron microscopy, using rotary shadowing and negative-staining techniques, which demonstrated that after chymotryptic digestion the myosin head remains attached to the tail whereas on papain digestion isolated heads and tails were observed. Thus the 120-kDa polypeptide derived from the 140-kDa fragment is the tail of the myosin, and the 68-kDa fragment containing the catalytic site and the 23-kDa fragment, with the light-chain-binding sites, form the head (S1) portion of the myosin. When [32P]-phosphorylated brain myosin was digested with chymotrypsin and papain it was shown that the heavy-chain phosphorylation site is located in a 5-kDa peptide at the C-terminal end of the heavy chain, i.e. the end of the myosin tail. Using hydrodynamic and electron microscopic techniques, no significant effect of either light-chain or heavy-chain phosphorylation on the stability of brain myosin filaments was observed, even in the presence of MgATP. Brain myosin filaments appear to be more stable than those of other non-muscle myosins. Light-chain phosphorylation did, however, have an effect on the conformation of brain myosin, for example in the presence of MgATP non-phosphorylated myosin molecules were induced to fold into a very compact folded state.     

Identification of Na+,Pi-binding protein in kidney and intestinal brush-border membranes.

An Na+, Pi-binding protein has been extracted from kidney and intestinal brush-border membranes with an organic solvent and has been purified by Kieselghur and Sephadex LH-60 chromatography. The molecular mass of this protein has been estimated to be about 155 kDa as determined by gel-filtration chromatography on Sepharose 2B. Under denaturing conditions, polyacrylamide-gel electrophoresis revealed a monomer of molecular mass about 70 kDa. The protein has high specificity and high affinity for Pi [K0.5 (concentration at which half-maximal binding is observed) near 10 microM]. Na2+ binding also exhibits saturation behaviour, with a K0.5 near 7.5 mM. Pi binding is inhibited by known inhibitors of Pi transport in brush-border membrane vesicles. It appears that this protein could be involved in Na+/Pi co-transport across the renal and intestinal brush-border membranes.     

Specific binding of the endocytosis tracer horseradish peroxidase to intestinal fatty acid-binding protein (I-FABP) in apical membranes of carp enterocytes

In a previous study we had demonstrated that a 15-kDa protein present in carp intestinal brush-border membrane vesicles (BBMV) was able to bind the endocytosis tracer horseradish peroxidase (HRP) with high specificity. Here we show that this protein corresponds to a peripheral membrane protein, identified by partial amino acid sequence analysis as the intestinal fatty acid-binding protein (I-FABP), a member of the small cytosolic fatty acid binding protein family (FABPs). The presence of I-FABP and its HRP-binding activity was demonstrated both in the cytosolic and membrane-associated fractions of intestinal mucosa by Western and ligand blot analyses, respectively. Also, both fractions displayed significant capacity to bind [(3)H]palmitic acid, a known ligand for I-FABP. Immunohistochemical analysis showed that I-FABP localizes both in the cytosol and in the brush-border membranes of epithelial cells. Taken together the unusual extra-cellular localization of I-FABP as well as its ability to interact with HRP suggests a novel function for this protein in the intestinal mucosa.     

Bile salt-binding polypeptides in brush-border membrane vesicles from rat small intestine revealed by photoaffinity labeling

Photoaffinity labeling of small intestinal brush-border membrane vesicles with photolabile bile salt derivatives was performed to identify bile salt-binding polypeptides in these membranes. The derivatives used in this study were the sodium salts of 7,7-azo-3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 3 beta-azido-7 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acid, their respective taurine conjugates, and (11 xi-azido-12-oxo-3 alpha, 7 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonic acid. With ileal brush-border membrane vesicles, photoaffinity labeling resulted in the identification of 5 polypeptides with apparent molecular weights of 125,000, 99,000, 83,000, 67,000, and 43,000. The extent of labeling depended on the photolabile derivative employed. In jejunal brush-border membrane vesicles, polypeptides with apparent molecular weights of 125,000, 94,000, 83,000, 67,000, and 43,000 were labeled. The results indicate that the binding polypeptides involved in bile salt transport in ileal brush-border membrane vesicles are 1) similar with one exception to those concerned with bile salt transport in jejunal brush-border membranes, and 2) markedly different from those previously shown to be concerned with bile salt transport in plasma membranes of hepatocytes.     

Purification of a new, calcium-independent, high molecular weight phospholipase A2/lysophospholipase (phospholipase B) from guinea pig intestinal brush-border membrane

A phospholipase A2 activity directed against phosphatidylcholine was previously described in brush-border membrane from guinea pig intestine (Diagne, A., Mitjavila, S., Fauvel, J., Chap, H., and Douste-Blazy, L. (1987) Lipids 22, 33-40). In the present study, this enzyme was solubilized either with Triton X-100 or upon papain treatment, suggesting a structural similarity with other intestinal hydrolases such as leucine aminopeptidase, sucrase, or trehalase. The papain-solubilized form, which is thought to lack the short hydrophobic tail responsible for membrane anchoring, was purified 1800-fold to about 90% purity by ion exchange chromatography on DEAE-Sephacel, gel filtration on Ultrogel AcA44, and hydrophobic chromatography on phenyl-Sepharose. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a main band with an apparent molecular mass of 97 kDa was detected under reducing and nonreducing conditions. In the latter case, phospholipase A2 activity could be recovered from the gel and was shown to coincide with the 97-kDa protein detected by silver staining. The enzyme activity was unaffected by EGTA and slightly inhibited by CaCl2. The purified enzyme displayed a similar activity against phosphatidylcholine and phosphatidylethanolamine, whereas 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine hydrolysis was reduced by 50% compared to diacylglycerophospholipids. Using phosphatidylcholine labeled with either [3H]palmitic acid or [14C]linoleic acid in the 1- or 2-positions, respectively, the purified enzyme catalyzed the removal of [3H]palmitic acid, although at a lower rate compared to [14C]linoleic acid. This resulted in the formation of sn-glycero-3-phosphocholine, but only 1-[3H]palmitoyl-sn-glycero-3-phosphocholine was detected as an intermediary product. In agreement with this, 1-acyl-2-lyso-sn-[14C]glycero-3-phosphocholine was deacylated at almost the same rate as the sn-2-position of phosphatidylcholine. Since upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the two hydrolytic activities were detected at the same position as 97-kDa protein, the enzyme is thus considered as a phospholipase A2 with lysophospholipase activity (phospholipase B), which might be involved in phospholipid digestion.     

Developmental distribution of a cell surface glycoprotein in the sea urchin Strongylocentrotus purpuratus

Recent studies from this laboratory have shown that an antigen recognized by a monoclonal antibody (MAb 1223) displays a bimodal distribution of expression in development of the embryo of Strongylocentrotus purpuratus. This molecule is specifically localized to the primary mesenchyme cells of the embryo, but is also found within the egg. In the current study, immunoelectron microscopy was used to determine the subcellular distribution of the antigen and to determine its fate during early stages of development of the embryo. In eggs, the epitope recognized by MAb 1223 was localized to the cortical vesicles. Immunoblot analysis of an isolated cell surface complex (CSC) that contained the cortical vesicles revealed the presence of a 130-kDa protein, as well as immunoreactive components of higher molecular weight. Upon fertilization, the antigen was exocytosed from the cortical vesicles and became associated with the hyaline layer, the fertilization envelope, and the plasma membrane. Subsequently, the epitope could be detected within small vesicles and yolk platelets. By 60 min postfertilization, the amount of epitope detected intracellularly or in the perivitelline compartment was greatly reduced. At later stages of development, when formation of the embryonic skeleton occurred, the 1223 antigen was principally localized to the Golgi complex and to the syncytial cell surface of the primary mesenchyme cells. Thus, the results of this study suggest that in S. purpuratus the 1223 antigen is stored and secreted from the cortical vesicles of the egg, degraded after fertilization, and then later expressed on the surface of the primary mesenchyme cells.     

Comparison of the Molecular Forms of the Kex2/Subtilisin-Like Serine Proteases SPC2, SPC3, and Furin in Neuroendocrine Secretory Vesicles Reveals Differences in Carboxyl-Terminus Truncation and Membrane Association

Abstract: The molecular forms and membrane association of SPC2, SPC3, and furin were investigated in neuroendocrine secretory vesicles from the anterior, intermediate, and neural lobes of bovine pituitary and bovine adrenal medulla. The major immunoreactive form of SPC2 was the full-length enzyme with a molecular mass of 64 kDa. The major immunoreactive form of SPC3 was truncated at the carboxyl terminus and had a molecular mass of 64 kDa. Full-length 86-kDa SPC3 with an intact carboxyl terminus was found only in bovine chromaffin granules. Immunoreactive furin was also detected in secretory vesicles. The molecular masses of 80 and 76 kDa were consistent with carboxyl-terminal truncation of furin to remove the transmembrane domain. All three enzymes were distributed between the soluble and membrane fractions of secretory vesicles although the degree of membrane association was tissue specific and, in the case of SPC3, dependent on the molecular form of the enzyme. Significant amounts of membrane-associated and soluble forms of SPC2, SPC3, and furin were found in pituitary secretory vesicles, whereas the majority of the immunoreactivity in chromaffin granules was membrane associated. More detailed analyses of chromaffin granule membranes revealed that 86-kDa SPC3 was more tightly associated with the membrane fraction than the carboxyl terminus-truncated 64-kDa form.     

The effect of papain upon proline and sodium transport of rat renal brush-border membrane vesicles

Treatment of renal brush-border membrane vesicles with papain resulted in the removal of the activity of maltase, gamma-glutamyl transpeptidase and leucine aminopeptidase by 85, 50 and 75%, respectively. Stripping of these membrane enzyme activities constituted about 2% of the total membrane proteins and resulted in a widespread diminution in the ability of a variety of amino acids and sugars to be taken up by the membrane vesicles which remained osmotically responsive. Kinetic analysis of the uptake of proline, which was shown previously to be transported by both sodium-dependent and sodium-independent systems, revealed that the Vmax for the sodium-dependent system and Km for the sodium-independent system were halved, but other parameters were not affected indicating that the papain treatment altered sodium-gradient-stimulated entry and the affinity of the sodium-gradient-independent system for proline. Experiments on sodium entry and efflux demonstrate a marked enhancement of flux, so that equilibration of the sodium gradient occurred about 5-times more rapidly than in untreated vesicles. This occurred without any change in the osmotic properties of the vesicle with regard to sodium or amino acid uptake. Studies of fluorescence polarization suggest that incubation with papain does not alter the lipid domains of the membrane.     

Characterization of myosin-II binding to Golgi stacks in vitro

In addition to important roles near the actin-rich cell cortex, ample evidence indicates that multiple myosins are also involved in membrane movements in the endomembrane system. Nonmuscle myosin-II has been shown to have roles in anterograde and retrograde trafficking at the Golgi. Myosin-II is present on Golgi stacks isolated from intestinal epithelial cells and has been localized to the Golgi in several polarized and unpolarized cell lines. An understanding of roles of myosin-II in Golgi physiology will be facilitated by understanding the molecular arrangement of myosin-II at the Golgi. Salt-washing removes endogenous myosin-II from isolated Golgi and purified brush border myosin-II can bind in vitro. Brush border myosin-II binds to a tightly bound Golgi peripheral membrane protein with a K(1/2) of 75 nM and binding is saturated at 0.7 pmol myosin/microg Golgi. Binding studies using papain cleavage fragments of brush border myosin-II show that the 120-kDa rod domain, but not the head domain, of myosin heavy chain can bind directly to Golgi stacks. The 120-kDa domain does not bind to Golgi membranes when phosphorylated in vitro with casein kinase-II. These results suggest that phosphorylation in the rod domain may regulate the binding and/or release of myosin-II from the Golgi. These data support a model in which myosin-II is tethered to the Golgi membrane by its tail and actin filaments by its head. Thus, translocation along actin filaments may extend Golgi membrane tubules and/or vesicles away from the Golgi complex.     

Phospholipid topology and flip-flop in intestinal brush-border membrane

The topological distribution of the two major phospholipids of brush-border membrane, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), has been investigated using brush-border membrane vesicles from rabbit small intestine. Bee venom phospholipase A2 and phosphatidylcholine exchange protein from bovine liver were used as membrane probes. It is shown that the brush-border membrane retains its integrity under conditions of phospholipase hydrolysis and intermembrane phospholipid exchange. Kinetic analysis of the data of phospholipase hydrolysis and phospholipid exchange at temperatures under 10 degrees C shows that both PC and PE occur in two pools: a minor (about 25%) more readily accessible pool and a major one (about 75%) less readily available. The rate of PC exchange between these two pools is relatively fast. The half-time derived under conditions of phospholipase hydrolysis is of the order of 20 min. Under conditions of phospholipid exchange the exchange rates may be even faster. The difference in exchange kinetics observed with the two methods of probing is probably due to changes in membrane properties such as the bilayer fluidity induced by the probing process itself. It is proposed that the two pools represent the transverse distribution of the phospholipids. The two major phospholipids of brush-border membranes, PC and PE, would be distributed mainly on the inner (cytoplasmic) side of the brush-border membrane. The phospholipid exchange between the brush-border vesicles and unilamellar phosphatidylcholine vesicles in the presence of phosphatidylcholine exchange protein reveals that significant quantities of phospholipid are taken up by brush-border membrane independently, i.e., in a separate process independent of the exchange protein-catalyzed phosphatidylcholine exchange.     

Carrier-mediated transport systems of tetraethylammonium in rat renal brush-border and basolateral membrane vesicles

Transport of [3H]tetraethylammonium, an organic cation, has been studied in brush-border and basolateral membrane vesicles isolated from rat kidney cortex. Some characteristics of carrier-mediated transport for tetraethylammonium were demonstrated in brush-border and basolateral membrane vesicles; the uptake was saturable, was stimulated by the countertransport effect, and showed discontinuity in an Arrhenius plot. In brush-border membrane vesicles, the presence of an H+ gradient ( [H+]i greater than [H+]o) induced a marked stimulation of tetraethylammonium uptake against its concentration gradient (overshoot phenomenon), and this concentrative uptake was completely inhibited by HgCl2. In contrast, the uptake of tetraethylammonium by basolateral membrane vesicles was unaffected by an H+ gradient. Tetraethylammonium uptake by basolateral membrane vesicles was significantly stimulated by a valinomycin-induced inside-negative membrane potential, while no effect of membrane potential was observed in brush-border membrane vesicles. These results suggest that tetraethylammonium transport across brush-border membranes is driven by an H+ gradient via an electroneutral H+-tetraethylammonium antiport system, and that tetraethylammonium is transported across basolateral membranes via a carrier-mediated system and this process is stimulated by an inside-negative membrane potential.