Stimulation of triacylglycerol synthesis by Z protein in rat liver and intestinal mucosa

Rat liver microsomal membranes have been shown to contain an UDP-glucose binding protein. Its mol. wt was estimated to be 120 000 by gel filtration and by ultracentrifugation in a sucrose gradient. The receptor activity was purified by gel filtration on Sephadex G200 and analysed by gel-electrofocusing.     

Protein-catalyzed exchange of phosphatidylinositol between rat brain microsomes and myelin.

Exchange of phosphatidylinositol and phosphatidylcholine between microsomal and myelin membranes has been demonstrated. This exchange is reversible and catalyzed by soluble proteins from the brain homogenate precipitated at pH 5.1. The extent of exchange of phosphatidylinositol from microsomal membrane to myelin is dependent upon pH and temperature, with an optimum around pH 7 and at 50 degrees C. Maximum exchange was observed at approximately equal amounts of microsomal, myelin, and supernatant proteins. The extent of the catalyzed exchange increases 4- to 8-fold upon using sonicated or heat-treated myelin as an acceptor membrane. Heating of microsomal membranes results in no change. The extent of catalyzed exchange of phosphatidylcholine is less than that of the phosphatidylinositol. The exchange of other phospholipids and glycolipids between microsomal and myelin membranes cannot be demonstrated. The catalytic activity of the pH 5.1 supernatant proteins in rat brain for the exchange of phosphatidylinositol increases with age after birth and reaches a maximum around 21 days of age analogous to the process of myelination. The pH 5.1 supernatant proteins from quaking and jimpy mutant mice has normal catalytic activity.     

Subcellular distribution of a rat hepatic binding protein specific for uridine diphosphoglucose

A binding protein, specific for UDP-glucose, is present in microsomal membranes of rat liver. Evidence is given for the localization of this protein in a Golgi-rich fraction. The whole membranous pathway for the biosynthesis of UDP-glucose is located in the same fraction and the relationship of the binding protein with the pathway is discussed, as well as its physiological significance.     

The influence of ganglioside insertion into brain membranes on the rate of ganglioside degradation by membrane-bound sialidase

Microsomal membranes isolated from calf brain contain a sialidase which cleaves ganglioside substrates naturally occurring within these membranes as well as exogenously added [3H]ganglioside GD1a. Micelles of [3H]ganglioside GD1a bind to the microsomal membranes in two steps. The first step, called adsorption, is fast and reversible by treatment with trypsin; the second step, called uptake, is slower and not reversible. The product of the enzymic degradation, [3H]ganglioside GM1, is exclusively located in the ganglioside pool taken up by the sialidase-bearing membranes, and not in the trypsin-releasable pool. Electron spin resonance (ESR) studies using a spin-labelled analogue of ganglioside GD1a indicate that the ganglioside uptake by microsomal membranes is accompanied by the disappearance of the micellar structure and by the 'dilution' of the probe molecules with membrane lipids. These findings suggest that exogenously added ganglioside substrate inserts into the microsomal membrane before it is recognized as substrate by the membrane-bound sialidase. Therefore, the influence of pH, ionic strength and membrane-fluidizing agents on the degradation rate measured with exogenous ganglioside GD1a does not only reflect kinetic parameters of the enzymic reaction itself but also the velocity of ganglioside insertion. Increasing ionic strength reduces the degradation rate. The acceleration of insertion with falling pH values shifts the measured pH optimum of the ganglioside degradation to lower values (pH 3.6) and masks the substantial residual sialidase activity at pH 5-7. The membrane-fluidizing alcohol n-hexanol greatly accelerates ganglioside insertion as well as ganglioside degradation. The latter was clearly demonstrated by studying the hydrolysis of endogenous ganglioside substrates, and is due to a decrease of the apparent Km value and an increase in the Vmax value. The Vmax value was also enhanced by freezing and thawing of the microsomal membranes.     

UDPglucosyltransferase and its kinetic fluorimetric assay

A rapid, kinetic assay for UDPglucosyltransferase has been developed using 1-naphthol as substrate. It is based on the continuous fluorimetric monitoring of 1-naphthyl glucoside formation during the reaction at physiological pH. The conjugate is easily distinguished from aglycone, since their fluorimetric properties differ. Glucoside biosynthesis in vitro by microsomal preparations isolated from the gut and fat body of cockroaches Periplaneta americana and Leucophaea maderae, and from the green gland and hepatopancreas of the crayfish Astacus astacus, has been demonstrated. The effects of buffer, pH, MgCl2, UDP-glucuronic acid, UDP-N-acetylglucosamine, sodium cholate and sonication on the enzyme activity have been assessed. The kinetic parameters of 1-naphthol and UDP-glucose have also been determined.     

Solubilization and characterization of a lactogenic receptor from human placental chorion membranes

Prolactin has a wide range of actions, including osmoregulation and the control of mammary gland development and lactation. These effects are mediated through a high-affinity cell surface receptor, which has been well characterized in a number of animal tissues. The molecular characteristics of the human receptor are unknown, however. The present studies were initiated, therefore, to determine the binding and molecular characteristics of the lactogenic receptor of human placental chorion membranes. Subcellular fractionation studies showed that the bulk of the receptor sedimented in the microsomal fraction at 45,000gav. Endogenous ligand was dissociated from the receptor with 3.5 M MgCl2 or 0.05 M acetate buffer (pH 4.8) with preservation of binding activity. The microsomal receptor bound human growth hormone (hGH), human prolactin (hPRL), ovine prolactin (oPRL), and human placental lactogen (hPL) but not non-primate growth hormones, indicating a narrow specificity for lactogenic hormones. The binding was only partially reversible in agreement with the known binding kinetics of animal lactogenic receptors. The receptor was solubilized with 45% yield from the microsomes using 16 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) detergent-250 mM NaCl, and the binding activity was fully restored by a two-fold dilution in the binding reaction to reveal a KD of 0.8 nM for hGH and a binding capacity of 200 fmol of specifically bound hGH per mg of microsomal protein. Gel filtration chromatography indicated the minimum molecular weight of the ligand-receptor complex was approximately 60,000 daltons, and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of covalently cross-linked 125I-hGH-receptor complexes revealed a molecular size of 58,000 daltons. When account was taken of the contribution of the ligand, a molecular weight of 36,000 for the receptor's binding domain was obtained. These data indicate that the chorion lactogenic receptor has very similar binding and molecular characteristics to the lactogenic receptors from other mammalian species. Chorion membranes are thus a convenient source of material for the further purification and characterization of the human lactogenic receptor.     

Identification and partial characterization of a retinal pigment epithelial membrane receptor for plasma retinol-binding protein.

We have developed a membrane binding assay by which we have been able to characterize the interaction between 125I-labeled retinol-binding protein and its receptor in microsome fractions derived from retinal pigment epithelial cells. The binding of retinol-binding protein to the membranes was fast, with a dissociation constant in the range of 31-72 nM, and maximum binding occurred at neutral pH. Receptor binding sites were also found in microsome fractions of liver and kidney, whereas lung and muscle contained few, if any. Chemical cross-linking of retinol-binding protein to the microsomal membranes yielded a major molecular complex of Mr 86,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein responsible for binding of retinol-binding protein was identified as a Mr 63,000 protein using a label transfer cross-linking technique. Further characterization demonstrated that the receptor for retinol-binding protein is a terminally glycosylated membrane protein noncovalently associated with a high molecular weight complex.     

UDP-glucose: ceramide glucosyltransferase of rat brain: an association with smooth microsomes and requirement of an intact membrane for enzyme activity

Subcellular distribution of rat brain UDP-glucose:ceramide glucosyltransferase, the enzyme which catalyses the first step during the sequential addition of carbohydrate moieties for ganglioside biosynthesis, was studied. The activity of the enzyme was highest in the fraction rich in microsomes. Subfractionation of crude microsomal fractions resulted in a further enrichment of the enzyme activity in the fraction which contained smooth microsomes, thus suggesting that the enzyme is associated with microsomal membranes. The enzyme does not appear to be associated with synaptosomes or myelin. Treatment of the microsomal fraction with phospholipase A and C or detergents resulted in the loss of enzyme activity. Preincubation of the microsomal fraction at 37 °C also resulted in a loss of enzyme activity. These results suggest the requirement of specific membrane structure for the activity of the enzyme UDP-glucose:ceramide glucosyltransferase of rat brain. The amount of the enzyme activity lost during preincubation was dependent on the composition of the incubation medium and the age of the rats from which microsomal fractions were obtained.     

Properties of a Solubilized Microsomal Auxin-binding Protein from Coleoptiles and Primary Leaves of Zea mays

An auxin-binding protein can be solubilized from microsomal membranes of Zea mays using either Triton X-100 extraction of the membranes or buffer extraction of the acetone-precipitated membranes. This paper describes the properties of the binding protein solubilized by these two methods. The binding is assayed by gel filtration chromatography in the presence of naphthalene [2-¹⁴C]acetic acid. Binding is rapid and reversible with an optimum at pH 5. Both preparations show similar molecular weights by gel filtration (80,000 daltons) at pH 7.6 and 0.1 molar NaCl, and both aggregate at low ionic strength. They appear to be the same active molecular species. The binding activity is destroyed by trypsin, pronase or para-chloromercuribenzoic acid, but not significantly reduced by phospholipase C, DNase, RNase, or dithioerythritol. Since saturating amounts of naphthalene acetic acid protect the molecule from inhibition by para-chloromercuribenzoic acid, it is concluded that the binding protein has a sulfhydryl group at the binding site, or protects such a group in its binding conformation. The dissociation constant of the protein for naphthalene acetic acid is 4.6 × 10⁻⁸ molar with 30 picomoles of sites per gram of tissue fresh weight. Binding constants were estimated for 13 other natural and synthetic auxins by competition with naphthalene[2-¹⁴C]acetic acid. Their dissociation constants are in general agreement with published values for their binding to intact membranes and their biological activity, although several exceptions were noted. A supernatant factor from the same tissue changes the apparent affinity of the protein for naphthalene acetic acid. This factor may be the same one as has been previously reported to alter the affinity of intact microsomes for auxin.     

A High-Affinity Binding Site for the AVR9 Peptide Elicitor of Cladosporium fulvum Is Present on Plasma Membranes of Tomato and Other Solanaceous Plants

The race-specific Cladosporium fulvum peptide elicitor AVR9, which specifically induces a hypersensitive response in tomato genotypes carrying the Cf-9 resistance gene, was labeled with iodine-125 at the N-terminal tyrosine residue and used in binding studies. 125I-AVR9 showed specific, saturable, and reversible binding to plasma membranes isolated from leaves of tomato cultivar Moneymaker without Cf resistance genes (MM-Cf0) or from a near-isogenic genotype with the Cf-9 resistance gene (MM-Cf9). The dissociation constant was found to be 0.07 nM, and the receptor concentration was 0.8 pmol/mg microsomal protein. Binding was highly influenced by pH and the ionic strength of the binding buffer and by temperature, indicating the involvement of both electrostatic and hydrophobic interactions. Binding kinetics and binding capacity were similar for membranes of the MM-Cf0 and MM-Cf9 genotypes. In all solanaceous plant species tested, an AVR9 binding site was present, whereas in the nonsolanaceous species that were analyzed, such a binding site could not be identified. The ability of membranes isolated from different solanaceous plant species to bind AVR9 seems to correlate with the presence of members of the Cf-9 gene family, but whether this correlation is functional remains to be determined.     

Transfer of glucose from UDP-glucose in microsomal membranes of rat hepatocytes (author's transl)]

Microsomal preparations from rat liver mediate transfer of glucosyl units from UDP-glucose to three different kinds of acceptors: an endogenous glycoprotein, exogenous glycogen and collagen. Both glucosyl transferases work at acidic pH, 6.5 for transfer on endogeneous acceptor and glycogen and at pH 5.5 for transfer on collagen. None of these enzymes require divalent cations for activity. While transfers on endogenous acceptor and glycogen are inhibited by the presence of a non-ionic detergent, Triton X-100, the transfer on collagen is activated by the same detergent. Glycogen-synthase activity requires glucose 6-phosphate at an optimal concentration of 1 mM. The Km values for UDP-glucose are respectively: 0.5 mM, 0.33 mM, and 1 mM for transfer on endogenous acceptor, glycogen and collagen. Characterisation of the product indicates that a protein-bound alpha1-4 glucan is formed when no primer is added. Enzymatic and acidic hydrolyses of radioactive glycogen and collagen show only glucose as a radioactive sugar identified by thin layer chromatography on cellulose. Pre-treatment of microsomal membranes by alpha-amylase demonstrates that glucosyltransferases are not adsorbed on endogenous glycogen and seem to be really membranous enzymes.     

Acidic pH generated by H+-ATPase pumps triggers the activity of a fusogenic protein associated with rat liver endoplasmic reticulum.

Fusogenic protein (FP) is a glycoprotein ( approximately 50 kDa), previously purified by us from rat liver endoplasmic reticulum, which explicates fusogenic activity at acidic pH in vitro. To suggest a possible role of FP in membrane fusion, the topology of the protein in the membrane and the conditions in which FP is operating in microsomes have been investigated. Anti-FP polyclonal antibodies inhibited pure FP activity, but not the protein activity in microsomes, suggesting interaction of antibodies with a part of FP concealed in intact membranes. FP activity in microsomes was lost after treatment with Pronase. Western blot analysis of Pronase-treated microsomes showed that the proteolysis removed a fragment ( approximately 5 kDa). This fragment is exposed on the outer surface of microsomes and involved in fusogenic activity, whereas the largest part of FP is embedded in microsomal vesicles. Therefore, FP can be affected by modifications on the cytosolic and luminal sides of microsomal membranes. Indeed, when microsomal lumen was acidified by H+-ATPase activity, binding and fusion of fluorescent labelled liposomes to microsomes occurred. Direct involvement of FP in the fusogenic event was observed by reconstituting pure FP in liposomes with a preformed H+ gradient. FP triggered a fusion process in response to the acidic interior of liposomes, despite an exterior 7.4 pH unable to promote fusogenic protein activity. As intracellular membrane fusion occurs at neutral pH involving the cytosolic sides of membranes, FP may participate in this event by exploiting the acidic pH formed in the lumen of endoplasmic reticulum through H+-translocating ATPase activity.     

Chemical modification of rat hepatic microsomes with N-ethylmaleimide results in inactivation of both UDP-N-acetylglucosamine-dependent stimulation of glucuronidation and UDP-glucuronic acid uptake.

Chemical modification of rat hepatic microsomes with N-ethylmaleimide (NEM) resulted in inactivation of UDP-N-acetylglucosamine (UDP-GlcNAc)-dependent stimulation of glucuronidation of p-nitrophenol. Inactivation kinetics and pH dependence were in agreement with the modification of a single sulfhydryl group. NEM also inactivated the uptake of UDP-glucuronic acid (UDP-GlcUA) but not UDP-glucose. With various sulfhydryl-modifying reagents, the inactivation of UDP-GlcUA uptake was linked to that of glucuronidation. UDP-GlcUA protected against NEM-sensitive inactivation of both UDP-GlcNAc-dependent stimulation of glucuronidation and UDP-GlcUA uptake, suggesting that the sulfhydryl group is located within or near the UDP-GlcUA binding site of the microsomal protein involved in the stimulation. Using microsomes labeled with biotin-conjugated maleimide and immunopurification with anti-peptide antibody against UDP-glucuronosyltransferase family 1 (UGT1) isozymes, immunopurified UGT1s were found to be labeled with the maleimide and UDP-GlcUA protected against the labeling as it did with the NEM-sensitive inactivation. These data suggest the involvement of a sulfhydryl residue of microsomal protein in the UDP-GlcNAc-dependent stimulation mechanism via the stimulation of UDP-GlcUA uptake into microsomal vesicles.     

Binding of S-adenosylhomocysteine to various domains of the plasma membrane and to the endoplasmic reticulum from rat liver: Relation between binding and phospholipid methyltransferase activity

S-Adenosylhomocysteine (AdoHcy) binding to various membrane fractions of rat liver was determined at pH 7.4, using an oil centrifugation technique. The highest binding activity was found in the heavy microsomal (M-H) fraction enriched in endoplasmic reticulum, but high binding activity was also observed in the light microsomal fractions enriched in blood sinusoidal membranes (M-L fraction), and the heavy nuclear fraction (N-H fraction) containing the contiguous area. A substantial portion of AdoHcy binding activity in the M-L fraction may be ascribed to contamination of this fraction with endoplasmic reticulum, as indicated by the distribution of NADPH cytochrome c reductase activity. Binding activity was low in the light nuclear (N-L) fraction corresponding to the bile canaliculi. Phospholipid methyltransferase activity was determined in the same membrane fractions under similar conditions (pH 7.4), and in the absence and presence of added phospholipids. The distribution of the enzyme activity was dependent on the presence of exogenous phospholipids, and grossly similar to AdoHcy binding, the highest activities being observed in the M-H and the M-L fractions. The N-H fraction, rich in AdoHcy-binding activity, demonstrated, however, a very low phospholipid methyltransferase activity. It is concluded that AdoHcy-binding activity is not confined to the plasma membranes, and a major fraction of the binding activity resides on membranes derived from the endoplasmic reticulum. Also, the present results add to previous data suggesting that phospholipid methyltransferase does not totally account for the AdoHcy-binding sites on rat liver membranes.     

Demonstration and characterization of the specific binding of growth hormone-releasing peptide to rat anterior pituitary and hypothalamic membranes.

Since the growth hormone-releasing peptide (GHRP), His-D-Trp-Ala-Trp-D-Phe-Lys-NH2, was found to specifically release growth hormone by a complementary but yet not clearly defined action on the pituitary as well as the hypothalamus, in vitro studies have been performed to demonstrate and characterized GHRP binding sites on peripheral membranes of both the rat anterior pituitary and hypothalamus. Optimum binding assay conditions were established using [125I]Tyr-Ala-GHRP as the radioligand. The membrane binding sites were specific, reversible, saturable and time, temperature, pH and concentration dependent. Computerized analyses of competition experiments suggested two classes of binding sites in both pituitary and hypothalamic membranes. The maximum specific binding was observed at pH 5.0 than the physiological pH in both tissues. Pretreatment of the membranes with trypsin prevented specific binding. The increase in Bmax was statistically significant and showed a 2.0- to 8.9-fold and 5.8- to 11.2-fold in pituitary and hypothalamus, respectively, whereas the affinity constants (Kds) were not significant. Of the synthetic and natural neuropeptides that influence the release of GH from somatotrophs, only (D-Lys3)GHRP, substance P antagonists and growth hormone-releasing factor analog were potent inhibitors of GHRP binding in both tissues.     

Subcellular Distribution of Basal and Cation-sensitive ATPases in Roots of Phaseolus vulgaris

A smooth microsomal fraction isolated from homogenates of Pbaseolus vulgaris root tissue has been found to possesss a highly active basal ATPase (measured in the absence of added cations). The microsomal membranes also feature a cation-sensitive ATPase which responds to Mg²⁺, Na⁺ and K⁺, but in a manner that is highly variable with pH. In contrast, membrane fragments prepared by a technique designed to yield purified plasma membrane were capable of little or no hydrolysis of ATP either in the presence or absence of added cations. This suggests that the microsomal activity is a reflection of membrane-bound ATPase which has been derived from cytoplasmic membranes, possibly the tonoplast, rather than plasma membrane.     

Photoactivated cross-linking of proteins within the erythrocyte membrane core.

We describe the reactions of three lipophilic, photoactivated cross-linking reagents, 1,5-diazidonapthalene, 4,4'-diazidobiphenyl, and the reversible 4,4'-dithiobisphenylazide, with erythrocyte membranes. Cross-linking occurs only upon photoactivation. At pH 7 to 8, only spectrin components are cross-linked by these reagents. At pH 5.0 to 5.5 several additional membrane proteins including the major "integral" membrane proteins are also cross-linked, despite equivalent binding of the cross-linkers at neutral and acid pH. The cross-linking rates of various membrane proteins at pH 5.0 to 5.5 depend distinctly upon duration of photoactivation. Bidimensional electrophoresis of membrane proteins after cross-linking with the reversible cross-linker, 4,4'-dithiobisphenylazide, has allowed for the identification of homopolymeric products of cross-linking (e.g. dimers and tetramers of Band 3) and heterocomplexes (spectrin plus other membrane proteins). The data suggest that at reduced pH, cross-linking can proceed not only at the membrane surface but also in the membrane core.     

Cotranslational glycosylation of proteins in systems depleted of protein disulphide isomerase.

The role of protein disulphide isomerase (PDI) and other resident proteins of the endoplasmic reticulum (ER) lumen in co- and post-translational modification of secretory proteins has been studied in experiments on translation in vitro. We have devised procedures for extracting the lumenal content proteins of dog pancreas microsomal vesicles by alkaline buffer, or detergent washing, and for reconstitution of the depleted membrane fraction. When microsomal membranes are depleted of content by washing at pH 9.1, they are able to co-translationally glycosylate human interferon-gamma (IFN-gamma) and yeast pro-alpha-factor and the products appear to be identical to those produced by control microsomes. However, when microsomal membranes are depleted of content by washing with saponin they are still able to co-translationally translocate and glycosylate human IFN-gamma, but the products were of higher apparent Mr than those generated by control microsomes. When saponin-washed microsomal membranes were reconstituted with homogeneous protein disulphide isomerase (PDI), the generated vesicles gave the same pattern of co-translationally glycosylated IFN-gamma as saponin-washed microsomal membranes lacking PDI. These results are discussed in relation to the roles of resident ER proteins in co-translational modification; they suggest that PDI is not an essential component of the machinery of co-translational N-glycosylation, but that detergent washing may inactivate or remove some ER glycosidases.     

The beta-adrenergic receptor-regulated 1,4-dihydropyridine calcium channel receptor sites in coronary artery smooth muscle.

The cross-regulatory communication from beta-adrenergic receptors to 1,4-dihydropyridine (DHP) Ca2+ channel agonist and antagonist binding sites and cooperativity between DHP binding sites were studied in microsomal membranes of canine coronary artery (purified to a factor 2.9 for DHPs). The maximal number of binding sites (Bmax) identified in coronary artery microsomal membranes (CAM) with Ca2+ channel agonist (-)-S-(3H)BAY K 8644 was two times higher than Bmax of sites labelled with Ca2+ channel antagonist (+)-(3H)PN 200-110. The exposure of CAM to isoprenaline was accompanied with down-regulation of beta-adrenergic receptors and with increase in binding capacity for DHPs. The increase in Bmax was proportional in both groups of experiments and was related to increased affinity of DHPs. The 1,4-DHP binding sites identified in vascular smooth muscle showed characteristics typical for classification of specific 1,4-DHP receptor on Ca2+ channels. The binding was of high affinity, saturable and reversible, it showed stereoselectivity and it was positively modulated by beta-adrenergic stimulation and its showed cAMP and GTP sensitivity. The results support the hypothesis that beta-receptors also regulate the mode of Ca2+ channels in coronary artery smooth muscle.     

Amphomycin: A tool to study protein N-glycosylation

Radio-labelled amphomycin (³H-amphomycin) forms a complex with dolichylmonophosphate in presence of Ca²⁺. Complex formation has also been documented with retinylmonophosphate and perhydromonoeneretinylmonophosphate. Analysis of the space-filling model suggested both fatty acylated aspartic acid residue at the N-terminus of the lipopeptide and phosphate head group of dolichylmonophosphate are necessary for the complex formation. The binding ability of amphomycin is then utilized to localize dolichylmonophosphate in the microsomal membrane. Studies with microsomal membranes from hen oviduct suggested that dolichylmonophosphate is located in the cytoplasmic side of the membrane.