Particulate Membrane Preparations Research Articles

Galactosyl- and fucosyltransferases in etiolated pea epicotyls: product identification and sub-cellular localisation

Summary Particulate membrane preparations from etiolated pea epicotyls were found to contain fucosyltransferases, which transferred fucose from GDP-fucose onto xyloglucan and N-linked glycoprotein, and galactosyltransferases, which transferred galactose from UDP-galactose onto galactan, xyloglucan, and N-linked glycoprotein. The products were characterised by specific enzyme degradation and by acid and alkaline hydrolysis. All the enzymes were found to be concentrated in the Golgi apparatus. The Golgi apparatus was further fractionated into membranes of low, medium and high-density. The glycoprotein fucosyltransferase activity was present in highest amounts in the medium-density Golgi membranes, while the majority of the xyloglucan fucosyltransferase was present in the low-density Golgi membranes. The majority of the galactan galactosyltransferase (galactan synthase) was found in the low-density membranes, while the glycoprotein galactosyltransferase was equally distributed in all three subfractions.

A Biotin-Streptavidin Enzyme Immunoassay for Detection of Antibodies to Porcine Granulosa Cell Antigens

A colorimetric solid-phase enzyme immunoassay has been developed which quantifies antibodies to porcine granulosa cell membrane antigens in rabbits immunized with porcine granulosa cells. A cell-free, particulate membrane preparation of porcine granulosa cells was used as coating antigen. A biotinylated second antibody in conjunction with a streptavidin-beta-galactosidase conjugate was utilized to amplify reactivity. The enzyme beta-galactosidase was used due to high background obtained using peroxidase, presumably due to endogenous peroxidase activity of the tissue. Sigmoidal serum dilution curves were obtained with immune rabbit sera indicating that absorbance was related to the concentration of antibodies. Assay activity was reduced by preincubation of immune serum with granulosa cell membranes. Sera from ovariectomized or pre-immune rabbits did not yield any specific binding in the assay. This assay has potential applicability for quantifying antiovarian and antigranulosa cell antibodies in women suspected of having autoimmune premature ovarian failure.

Age-related alterations in prolactin binding sites in the female rat

Aging is associated with various neuroendocrine alterations, including in the rat a hypersecretion of PRL with maintained ovulations (repetitive pseudo-pregnancy) and a reduced activity of the hypothalamic dopaminergic neurons with loss of the neuron responsiveness to PRL, suggestive of age-related alterations in PRL receptors. In this study we have investigated PRL binding sites in the hypothalamus as well as in the mammary glands, the ovaries and the liver of young and old nulliparous female rats. The old rats (26-28 months) displayed spontaneous repetitive pseudopregnancies and they were compared with young (4-6 months) pseudopregnant rats; the binding studies were performed by saturation analysis using 125I-oPRL as ligand and particulate membrane preparations. In the hypothalamus, a negligible binding of PRL was observed in all fragments studied, mediobasal hypothalamus, median eminence, in both young and old rats and no characterization of the binding sites could be achieved. In the mammary glands, the number of PRL binding sites was appreciable in spite of the nulliparity of the rats, but it was smaller in the old than in the young rats (9.0 +/- 1.4 vs 14.9 +/- 1.2 fmol/mg protein; mean +/- SEM; p less than 0.02). In the ovaries, the density of PRL binding sites was similar in the old and young rats (112.6 +/- 9.7 vs 115.0 +/- 8.9 fmol/mg protein), illustrative of a maintained luteotropic effect of PRL with age in the rat. In contrast, in the liver a greater number of binding sites was found in the old than in the young rats (261.9 +/- 36.6 vs 63.6 +/- 5.8 fmol/mg protein; p less than 0.001), supportive of the ability of PRL to induce its own receptors in that tissue. The affinity constant of PRL binding was not altered with age in the tissues studied. These results are illustrative of tissue-specific modifications in the number of PRL binding sites with age and they are suggestive of a sustained biological activity of PRL in the old rats.

A 1,4-?-D-glucan-synthase system fromDictyostelium discoideum

Particulate membrane preparations have been isolated from culminatingDictyostelium discoideum cells. The preparations incorporated glucose from uridine 5′-diphosphate-glucose into a glucose polymer or polymers. These have been shown to be homopolymers ofβ-linked glucose. A high percentage (78% by methylation analysis) of the linkages formed are 1,4-linkages and a lower percentage (12%) are 1,3-linkages. The glucan-synthase complex present in the particulate membrane preparation has an apparent Km of 0.28 mM and a Vmax of 1.59 nmol·min−1·(mg protein)−1. The enzyme system is dependent upon Mg2+ and cellobiose for maximal activity, but is inhibited by millimolar levels of Ca2+. Particulate membrane preparations were made from cells at various times during a synchronous developmental time course and demonstrated that the glucan-synthase activity appeared at the tight-aggregate stage of development.

A 1,4-?-D-glucan-synthase system from Dictyostelium discoideum

Particulate membrane preparations have been isolated from culminating Dictyostelium discoideum cells. The preparations incorporated glucose from uridine 5'-diphosphate-glucose into a glucose polymer or polymers. These have been shown to be homopolymers of β-linked glucose. A high percentage (78% by methylation analysis) of the linkages formed are 1,4-linkages and a lower percentage (12%) are 1,3-linkages. The glucan-synthase complex present in the particulate membrane preparation has an apparent Km of 0.28 mM and a Vmax of 1.59 nmol·min(-1)·(mg protein)(-1). The enzyme system is dependent upon Mg(2+) and cellobiose for maximal activity, but is inhibited by millimolar levels of Ca(2+). Particulate membrane preparations were made from cells at various times during a synchronous developmental time course and demonstrated that the glucan-synthase activity appeared at the tight-aggregate stage of development.

Adrenergic Receptors in Aging and Alzheimer's Disease: Increased β2‐Receptors in Prefrontal Cortex and Hippocampus

Loss of pigmented noradrenergic locus ceruleus neurons occurs in Alzheimer's disease (AD) and, to a lesser extent, in aging. We studied beta-adrenergic receptors and their subtypes, beta 1 and beta 2, by the specific binding of 125I-pindolol to particulate membrane preparations from prefrontal cortex, hippocampus, putamen, and cerebellum and to sections from frontal cortex by in vitro autoradiography. In prefrontal cortex from controls, numbers of total beta- and beta 2-adrenoceptors did not significantly correlate with age, but number of beta 1-adrenoceptors showed a weak but significant negative correlation. Binding in tissue particulate preparations to total beta-receptors did not reveal significant differences in samples from prefrontal cortex between AD subjects and age-matched controls. However, beta 1-adrenoceptors were decreased and beta 2-adrenoceptors were increased in number by approximately 30-50% in AD subjects. Thus, the relative ratio of beta 1-/beta 2-receptors was decreased in AD. Binding by in vitro receptor autoradiography performed in a subset of samples of frontal cortex also showed beta 2-adrenoceptors, and less consistently total beta- and beta 1-receptors, to be increased significantly in number in cortical laminae II, III, IV, and V of tissue sections from AD subjects. In these subjects, number of locus ceruleus cells and norepinephrine concentrations in putamen and frontal cortex were markedly reduced compared with values in controls. In the hippocampus, total beta- and both beta 2- and beta 1-adrenoceptors were increased in number in AD. In contrast, in the putamen, where beta 1-receptors predominate, total beta- and beta 1-receptors were significantly decreased in number with no consistent change in content of beta 2-receptors in AD. There were no significant changes in the cerebellum. Specific pindolol binding was not affected by interval between death and sampling of tissue at autopsy. Our results indicate selective changes in number of beta-receptors in AD. These changes in the cortex and hippocampus suggest receptor upregulation in response to noradrenergic deafferentation from the locus ceruleus or may simply reflect glial proliferation in AD.

A membrane-bound enzyme complex synthesising glucan and glucomannan in pine tissues

Particulate membrane preparations isolated from cambial cells and differentiating and differentiated xylem cells of pine (Pinus sylvestris L.) trees synthesised [(14)C]glucans using either guanosine 5'-diphosphate (GDP)-D-[U-(14)C]glucose or uridine 5'-diphosphate (UDP)-D-[U-(14)C]glucose as glycosyl donors. Although these glucans had β-(1→3) and β-(1→4) linkages in an approximate ratio 1:1, the distribution of the linkages in the glucan synthesised from GDP-D-glucose was different from that synthesised from UDP-D-glucose. The synthesis of the mixed β-(1→3) and β-(1→4) glucan from GDP-D-[U-(14)C]glucose was changed to that of β-(1→4) glucomannan in the presence of increasing concentrations of GDP-D-mannose. The glucan formed from UDP-D-[U-(14)C]glucose was not affected by any concentration of GDP-D-mannose. The membrane preparations epimerized GDP-D-glucose to GDP-D-mannose; however, the low amount of GDP-D-mannose formed was not incorporated into the polymer becaus the affinity of the synthase for GDP-D-glucose was much greater than that for GDP-D-mannose. The glucan formed from GDP-D-glucose and the glucomannan formed from GDP-D-glucose together with GDP-D-mannose were characterized. The apparent K m and V max of the glucan synthase for GDP-D-glucose were 6.38 μM and 5.08 μM·min(-1), respectively. No lipid intermediates were detected during the synthesis of either glucan or glucomannan. The results indicated that an enzyme complex for the formation of the glucomannan was bound to the membrane.

Glucomannan-synthase activity in differentiating cells of Pinus sylvestris L.

Particulate membrane preparations have been isolated from cambial cells, and from differentiating and differentiated xylem cells of the main stem of pine trees. These preparations synthesise a β1→4 glucomannan from guanosine 5'-diphosphate-mannose. The polysaccharide and the synthase have been characterized and the Km and Vmax for the synthase determined as 85 μM and 52.9 μM·min(-1), respectively. The enzymic activity was inhibited by the addition of guanosine 5'-diphosphate-D-glucose so that the presence of an epimerase on the particulate fraction in conjunction with the synthase probably allowed the heteropolymer to be formed with the optimal ratio of the concentrations of the nucleoside-diphosphate sugar donors. No evidence for a polyprenyl-phosphate derivative as an intermediate during the polymer synthesis was obtained. Part of the control mechanism for the deposition of the large amounts of the glucomannan during the secondary thickening of the tracheids of the vascular system is by an increase in the amount of synthase activity at the endomembrane system of the cells. This probably occurs by an increase in the amount of enzyme which is modulated by gene regulation during differentiation.

Phosphatidylglycerol as biosynthetic precursor for the poly(glycerol phosphate) backbone of bifidobacterial lipoteichoic acid.

Phosphatidylglycerol functions as donor of the sn-glycerol 1-phosphate units in the synthesis in vitro of the 1,2-phosphodiester-linked glycerol phosphate backbone of the lipoteichoic acids of Bifidobacterium bifidum subsp. pennsylvanicum. The incorporation was catalysed by a membrane-bound enzyme system. After addition of chloroform/methanol the product formed coprecipitated with protein. The material was phenol-extractable and was co-eluted with purified lipoteichoic acid on Sepharose 6B. The reaction was stimulated by Triton X-100, UDP-glucose and UDP-galactose, but Mg2+ ions had no effect. The apparent values for Km and Vmax. of the phosphatidylglycerol incorporation were 1.4 mM and 3.1 nmol/h per mg of membrane protein, respectively. Labelled UDP-glucose and UDP-galactose were not incorporated into the lipoteichoic acid fraction by the particulate membrane preparation.

A particulate membrane preparation from ovaries of preovulatory rainbow trout ( Salmo gairdnerii): Binding studies with 125I-human chorionic gonadotropin

1. 1. In order to obtain a particulate membrane preparation from complete ovaries of previtellogenic and vitellogenic trout, gonads were homogenized in a phosphate-buffered 0.5 M NaCl solution. 2. 2. Radio-receptor assay studies were carried out with these preparations using 125I-hCG as the ligand and binding-sites showed high-affinity ( K A = 1.6−2.9 × 10 9), low-capacity (15.3–149.7 pg hCG/mg freshweight) and target-organ specificity. 3. 3. We conclude that the method of membrane preparation is suitable for processing yolk-laden ovaries and that the binding sites can be assayed—with some restrictions—using 125I-hCG as the ligand.

A mecillinam-sensitive peptidoglycan crosslinking reaction in [formula omitted

The amidinopenicillin, mecillinam, induces the formation of spherical cells of Escherichia coli by inactivation of penicillin-binding protein 2 (PBP2). A mecillinam-sensitive peptidoglycan crosslinking reaction has been demonstrated in particulate membrane preparations from this organism. The activity was detected in membranes that contained elevated levels of PBP2 and in which crosslinking reactions due to all other PBPs had been inactivated with the cephamycin antibiotic, cefmetazole. The particulate membrane preparation catalyzed synthesis of peptidoglycan that was up to 20% crosslinked from nucleotide precursors. Crosslinkage of the peptidoglycan was inhibited 50% by 0.2 μg mecillinam per ml but was not inhibited by much higher concentrations of cephamycins, which have very low affinity for PBP2. The crosslinking reaction appears to be due to the transpeptidase activity of PBP2, which is implicated in the mechanism of cell shape determination, and is the killing target for mecillinam.

Adenylate cyclase of human articular chondrocytes. Responsiveness to prostaglandins and other hormones.

Adenylate cyclase [ATP pyrophosphate lyase (cyclizing), EC 4.6.1.1] was shown to be present in cultured human articular chondrocytes. Optimal conditions of incubation time, protein and substrate concentrations and pH were determined in whole cell lysates. Maximal activity occurred at pH 8.5 with no decrease in activity up to pH 10.0. Adenylate cyclase activity of particulate membrane preparations was enhanced by the addition of crude cytosol preparations. The prostaglandins E1, E2, F1 alpha, F2 alpha, D2, B1, B2, A1 and A2, as well as adrenaline and isoprenaline, stimulated adenylate cyclase derived from either adult or foetal chondrocytes. No significant stimulation was observed in the presence of human calcitonin or glucagon. Bovine parathyroid hormone always significantly stimulated the adenylate cyclase derived from foetal chondrocytes, but not from adult chondrocytes. Preincubation of the chondrocytes in culture with indomethacin and with or without supernatant medium from cultured mononuclear cells increased the responsiveness of the adenylate cyclase to prostaglandin E1.

Relaxin receptors in the myometrium and cervix of the pig.

The binding of I2uI relaxin to particulate membrane preparations of myometrium and cervix from the pig has been characterized and investigated in different physiological and experimental states. Binding properties were very similar for the two tissues: binding was rapid, exhibited a limited steady.state, and had comparable rates of dissociation at 27#{176}C; divalent cations in the order 2* > Ca2 enhanced I2SI relaxin binding. Scatchard analysis yielded curvilinear plots compatible with the presence of two classes of binding sites. The high affinity binding sites in fractions from both tissues exhibited similar affinity constants Ka (in the order of l0’#{176} M ) although with differing binding capacities. Unlabeled relaxin had the greatest inhibitory action on the binding of labeled relaxin to both tissues whereas NGF > proinsulin > insulin were less effective in this respect. Myometrial and cervical fractions from PMSG-hCG-treated cyclic and prepubertal pigs exhibited high affinity binding sites. PMSG-hCG-treated prepubertal pigs exhibited slightly more binding in the myometrium than cervix which was unchanged 24 h after ovariedtomy. Similar animals examined 10 days after ovariectomy had a significantly decreased number of myometrial binding sites (P<0.01) which decreased to undetectable levels at 16 days postovariectomy. Cervical fractions, on the other hand, showed complete lack of binding by 10 days postovariectomy. Similar ovariectomized animals, treated with estrogen daily for 7 days, showed recovery of relaxin receptors in the myometrial fractions comparable in number to those of control animals; cervical receptors remained undetectable however. The data suggest a different hormonal control of the myometrium and cervix with regard to their sensitivity to relaxin as expressed by receptor concentration. It is suggested that this may have biological significance in view of the differential time scale for the need of relaxin action on uterine quiescence and cervical dilatation during pregnancy.

Cell-free biosynthesis of erythroglycan in a microsomal fraction from K-562 cells.

Particulate membrane preparations from K-562 [human CML (chronic-myelogenous-leukaemia)-derived] cells catalyse the transfer of [3H]galactose from UDP-[3H]-galactose and [3H]N-acetylglucosamine from UDP-[3H]N-acetylglucosamine into an endogenous product that on digestion with Pronase yields long-chain glycopeptides (mol.wt. 7000--10 000) called 'erythroglycan'. Incorporation of either labelled sugar increased up to 60 min of incubation time. The labelled erythroglycan was isolated by chromatography on Sephadex G-50 and characterized by digestion with endo-beta-galactosidase from Escherichia freundii, followed by analysis on Bio-Gel P-2 and paper chromatography. This digestion gave the following four products: (1) a disaccharide with the sequence beta GlcNAc-beta Gal; (2) a trisaccharide with the sequence betaGal-betaGlcNAc-beta Gal; (3) a larger oligosaccharide containing galactose and N-acetylglucosamine; and (4) a putative protein-linkage region.

Human parathyroid adenoma adenylate cyclase: Stimulation by histamine that is blocked by cimetidine

Recent evidence suggests that the histamine receptor blocking agent cimetidine can decrease parathyroid hormone release from human parathyroids. To determine the mechanism for inhibition we examined the ability of histamine 1 × 10 −5 moles/liter to stimulate adenylate cyclase in a particulate membrane preparation from 13 human parathyroid glands. Histamine significantly increased adenylate cyclase activity as compared to control; however, the degree of stimulation was variable among the individual tissue samples. Enzyme stimulation was dose dependent over the concentration range of 1 × 10 −7 to 1 × 10 −4 moles/liter. Cimetidine at 1 × 10 −4 moles/liter completely abolished the histamine mediated increase in activity, but did not block the epinephrine-induced stimulation. The identification of an adenylate cyclase system in certain human parathyroid adenomas that is stimulated by histamine and blocked by cimetidine may offer a basis for the pharmacologic alteration of parathyroid hormone secretion.

Generation of cytolytic T lymphocytes in vitro. XI. Accessory cell requirement in secondary responses to particulate alloantigen.

The non-T accessory cell requirement for cytolytic T lymphocyte (CTL) generation in vitro was studied in a system which makes use of particulate membrane preparations as a source of alloantigen, and spleen cells from alloimmune mice as a source of responding cells. It is shown that removal of nylon-adherent cells from the responding cell population strongly reduced CTL generation, whereas direct removal of Ig+, phagocytic or plastic-adherent cells had no effect. The CTL response of the nylon-nonadherent cell population could be reconstituted by the addition of normal spleen cells, which by themselves do not generate CTL in response to particulate alloantigen. The accessory cell function of normal spleen cells was not affected by depletion of T cells or of phagocytic cells, but was sensitive to gamma-irradiation (1000 rd). The system thus demonstrates the requirement for a nylon-adherent accessory cell population in the secondary CTL response to particulate alloantigens which does not exhibit the typical characteristics of T cells, B cells or macrophages.

Effects of prostaglandins on rat cardiac adenylate cyclase

A significant stimulatory action of PGE 1 on adenylate cyclase of the particulate membrane preparation of rat heart was observed at 1 × 10 −4 M. PGE 1 caused a positive inotropic effect on isolated spontaneously beating rat atria at 1 × 10 −5 M, and an increase in atrial cyclic AMP level at 1 × 10 −6 M. PGE 2α had no effect on these enzyme preparations. Our results suggest that: (i) The inotropic response of isolated spontaneously beating rat atria to PGE 1 may be connected with an increase in cyclic AMP level; (ii) the effect of prostaglandins on heart in vivo must be indirect, because the concentrations needed to stimulate isolated atria and particulate adenylate cyclase were high, compared with the doses effective in vivo.

Biosynthesis ofSaccharomyces cerevisiae glycoproteins: Nature of some participating glycolipids

A particulate membrane preparation from Saccharomyces cerevisiae catalyzed the incorporation of mannose from GDP-mannose into lipids that were extractable in chloroform-methanol. One lipid has been previously characterized as dolichyl phosphomannose. Another one was purified by chromatography on silicic acid, DEAE-cellulose and Sephadex LH-20 was found to be alkali unstable. The lipid moiety was shown to be dolichol and the glycosydic part contained mannose, glucose and glucosamine. Radioactive mannose was also incorporated at a slower rate into more polar compounds. They were soluble in chloroform-methanol-water and were seen to liberate neutral oligosaccharides after alkaline hydrolysis. Radioactive mannose was also incorporated into substances which behave chemically as glycoproteins since they were insoluble in organic solvents, water and trichloroactic acid. Pronase treatment of the trichloroacetic acid-insoluble material released water-soluble oligosaccharides. When the particulate preparation which had been extracted with chloroform-methanol at-20 C, was incubated with GDP-(U-14C)mannose, radioactivity was incorporated into glycolipids that were soluble in chloroform-methanol-water and into glycoproteins. This result suggests that at least part of the mannose was transferred to endogenous acceptors independent of dolichyl phosphomannose.

SOLUBILIZATION OF A GROWTH HORMONE-SPECIFIC RECEPTOR FROM RABBIT LIVER

Membrane preparations from rabbit liver, known to possess GH-specific binding sites, have been solubilized with Triton X-100 and the binding characteristics of [125I]-human GH (hGH) and [125I]-bovine GH (bGH) subsequently studied. Specific binding of the hGH and bGH by the solubilized preparation was demonstrated of bound and free hormone by either polyethylene glycol precipitation or by Sephadex G-100 chromatography. Binding of hGH was both rapid and reversible and was displaced only by other growth hormones (bovine and ovine) and not by lactogenic hormones (ovine and human prolactins, human placental lactogen). As shown by Scatchard analysis specific binding of [125I]-bGH exhibited a lower binding affinity and capacity than did [125I]-hGH. Overall, the characteristics of the binding reaction for hGH were not significantly different from those reported for the particulate membrane preparation. The solubilization process did not appear to alter the binding protein(s) therefore, and permits a further study of the isolation, purification and properties of the binding protein(s) itself.

Studies of binding of parathyroid hormone to a detergent-dispersed preparation from bovine kidney cortex plasma membranes.

Bovine kidney plasma membranes containing parathyroid hormone-sensitive adenylate cyclase activity were dispersed with 1% Triton X-100 and centrifuged at 150,000 X g for 2 h. Approximately 40% of the total membrane protein was extracted by this procedure. The extraction greatly reduces the fluoride-stimulated and the parathyroid hormone-sensitive adenylate cyclase activity of the membranes and yields a supernatnat which binds biologically active, tritiated parathyroid hormone. Hormone binding is stable for up to 15 h and has a linear dependence on protein concentration in the extract. Binding of the labeled hormone at concentrations of 5 to 10 nM is inhibited by preincubation with unlabeled min, and displays a dependence on temperature, time, and pH. Binding specificity is maximal at physiological pH, being inhibited by only the native hormone or its synthetic 1-34 NH2-terminal, biologically active fragment. Binding increases dramatically at pH 6.0, but is nonspecific in character. Half-maximal inhibition of the binding was achieved at 3.2 X 10(-7) M concentrations of the native hormone and 5.0 X 10(-7) M concentrations of the synthetic 1-34 NH2-terminal fragment. Calcium does not inhibit either total or specific binding. Inhibition, kinetic, and pH dependence data suggest that the extracted component(s) represent the parathyroid hormone binding protein(s) formerly identified in particulate membrane preparations.