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H Zwiers

Publications and source records attributed to H Zwiers.

At least 55 records · Page 3Linked to original sources
PubMed1985

Characterization of alpha-MSH-induced changes in the phosphorylation of a 53 kDa protein in Xenopus melanophores.

alpha-Melanotropin has been shown to induce specific changes in the degree of phosphorylation of a 53 kDa melanophore protein, concomitant with pigment dispersion. To further characterize the alpha-MSH-induced changes in 53 kDa phosphorylation in melanophores from the ventral tail-fin of Xenopus tadpoles, we investigated the concentration and time dependency of the effect. A significant increase in 53 kDa phosphorylation was detectable at 5 X 10(-8) M alpha-MSH. The maximal increase in 53 kDa phosphorylation was found after an incubation time of 10-15 min, whereas pigment dispersion was optimal after 60 min. The phosphorylated 53 kDa band showed clear cross-reactivity with monoclonal anti-beta-tubulin, and migrates as a single protein after two-dimensional (2D) separation. On a 2D-separation system the 53 kDa protein (IEP 5.1) migrated in the acidic tail of purified beta-tubulin. Our data strongly indicate that the 53 kDa protein is a beta-tubulin-like protein. We suggest that the degree of 53 kDa phosphorylation may be an important factor in the regulation of microtubule function in melanophores.

Animals↗
PubMed1985

Resolution of rat brain synaptic phosphoprotein B-50 into multiple forms by two-dimensional electrophoresis: evidence for multisite phosphorylation.

Phosphoprotein B-50 was extracted from rat brain membranes by alkaline extraction and purified by ammonium sulphate precipitation and flat-bed isoelectric focusing. The purified protein shows microheterogeneity upon isoelectric focusing in a narrow pH gradient (pH 3.5-5.0). As visualized by two-dimensional gel electrophoresis, B-50 resolved into four clearly separated forms which differ slightly in isoelectric point. The forms are in part mutually convertible by exhaustive phosphorylation (using protein kinase C) and dephosphorylation (using Escherichia coli alkaline phosphatase). Proteolysis with Staphylococcus aureus protease yielded two radioactive peptides. Analysis of their molecular weights and the time course of their formation suggests that B-50 was cleaved at only one specific site. Our data indicate the presence of more than one phosphorylatable site. The possibility that the heterogeneity of B-50 was in part due to a glycoprotein nature of B-50 was studied extensively. However, none of the six different methods used revealed the presence of glyco-moieties in B-50.

Alkaline Phosphatase↗
PubMed1984

Purification and partial characterization of the phosphatidylinositol 4-phosphate kinase from rat brain.

Phosphatidylinositol 4-phosphate (PtdIns4P) kinase was purified from cytosolic and particulate material of rat brain. The purification procedure of the enzyme from cytosol consisted of (NH4)2SO4 precipitation. DEAE-cellulose column chromatography and preparative isoelectric focusing. Other methods after DEAE-cellulose column chromatography failed to achieve further purification of the PtdIns4P kinase, probably caused by the tendency of the enzyme to aggregate with contaminating proteins. The final purification was 67-fold, and the recovery was 0.6%. After isoelectric focusing the fraction containing the highest PtdIns4P kinase activity showed only one protein as visualized by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and silver staining. The apparent Mr of this protein was 45 kDa and the isoelectric point about 5.8. The activity of PtdIns4P kinase was dependent on the concentration of divalent cations in the incubation medium. PtdIns4P kinase activity was found to be optimal at 10-30 mM-Mg2+. In an attempt to compare the cytosolic with the membrane-derived kinase activity, a Triton/KCl extract from synaptic membranes was subjected to the same purification procedure as the cytosolic enzyme. A difference in isoelectric focusing was observed, possibly due to a higher tendency to form aggregates. However, we tend to conclude that also in the membranes the PtdIns4P kinase activity is present as a 45 kDa protein, identical with that found in the cytosol.

Animals↗
PubMed1984

Adrenocorticotropic hormone (ACTH)-lipid interactions. Implications for involvement of amphipathic helix formation.

ACTH-lipid interactions were investigated by: (1) lipid-monolayer studies using several zwitterionic and anionic phospholipids and gangliosides, (2) permeability experiments by following the swelling rate of liposomes in isotonic glycerol solutions by light scattering, using liposomes of synthetic lipids and liposomes made of lipids extracted from light synaptic plasma membranes, and (3) by steady-state fluorescence anisotropy measurements on liposomes derived from light synaptic plasma membranes employing 1,6-diphenyl-1,3,5-hexatriene as fluorescent probe. (1) The monolayer experiments demonstrated an interaction with gangliosides GT1, GM1, dioleoylphosphatidic acid and phosphatidylserine, but little or no interaction with phosphatidylcholine or sphingomyelin. The interaction with monolayers of GT1 or phosphatidic acid decreased for ACTH1-13-NH2 and ACTH1-10. (2) The liposome experiments showed that 2 X 10(-5) M ACTH1-24 increased the glycerol permeability by 20% and decreased the activation energy only when liposomes derived from light synaptic plasma membranes were used. Treatment of the liposomes with neuraminidase abolished the ACTH-induced permeability increase. (3) Steady-state fluorescence depolarization measurements revealed that ACTH1-24, ACTH1-16-NH2 and ACTH1-10 did not change the fluidity of liposomes derived from light synaptic plasma membranes as sensed by diphenylhexatriene. It is concluded that ACTH1-24 can bind to negatively charged lipids and can form an amphipathic helix aligned parallel to the membrane surface involving the N-terminal residues 1 to 12, possibly to 16. Polysialogangliosides will favorably meet the condition of a high local surface charge density under physiological circumstances. It is suggested that ACTH-ganglioside interactions will participate in ACTH-receptor interactions.

Adrenocorticotropic Hormone↗
PubMed1984

Dopamine-induced changes in protein phosphorylation and polyphosphoinositide metabolism in rat hippocampus.

Effects of dopamine (DA) on endogenous phosphorylation of hippocampal proteins and polyphosphoinositides were studied in subcellular fractions from a crude mitocondrial/synaptosomal preparation. DA induced a concentration-dependent decrease in the in vitro phosphorylation of the protein B-50 (-22.1% at 10(-5) M DA), whereas no changes were found in phosphoproteins in other subcellular fractions. Treatment of hippocampal slices with 5 X 10(-4) M DA resulted in a 45.8% increase in post hoc phosphorylation of B-50 in SPM and it affected post hoc phosphorylation of several proteins in a cytosolic fraction. In vitro phosphorylation of SPM with DA (5 X 10(-4) M) increased endogenous TPI phosphorylation (+51.6%), whereas treatment of slices with DA (5 X 10(-4) M) resulted in a 39.4% decrease in post hoc TPI phosphorylation. This decrease could be blocked by haloperidol. Significant changes induced by DA (5 X 10(-4) M) were also found in 32P-incorporation into PA (in vitro: -32.4% and post hoc: +39.3%), but were not found in DPI labeling. The data provide evidence for DA-induced changes in phosphorylation of proteins and polyphosphoinositides in rat hippocampal SPM.

Animals↗
PubMed1984

Alteration of hippocampal B50 phosphorylation after adrenalectomy.

Adrenalectomy alters endogenous phosphorylation of the presynaptic protein, B50, in the hippocampus. Three and four days following adrenalectomy decreases are seen, relative to control values, in the in vitro phosphorylation of B50 when a synaptic plasma membrane fraction from the hippocampus is incubated with [gamma-32P]ATP. At four days post-adrenalectomy, the percent decrease in B50 phosphorylation is -49.8 +/- 6.8%. No alteration is seen in the level of B50 phosphorylation when comparing hippocampal membrane preparations from sham-operated and intact animals. Fourteen days following adrenalectomy, hippocampal B50 phosphorylation was restored to normal levels. Hypophysectomy did not alter the degree of in vitro B50 phosphorylation, but the effect of adrenalectomy occurred in hypophysectomized rats. Following adrenalectomy, no differences are seen in the phosphorylation of any hippocampal cytosolic proteins. Changes in B50 phosphorylation seen in hypothalamic synaptic plasma membranes are likely due to the effects of sham-operation. The results indicate that a transient neurochemical or neuroendocrine event following adrenalectomy modulates in vivo the degree of B50 phosphorylation in hippocampal synaptic membranes.

Adrenal Glands↗
PubMed1984

Cross-reaction of anti-rat B-50: characterization and isolation of a "B-50 phosphoprotein" from bovine brain.

Antibodies to the phosphoprotein B-50 of rat brain were used to trace cross-reacting brain proteins of vertebrates. With the SDS-gel-immunoperoxidase method, a cross-reacting protein (CP) of apparent Mr 53,000 was demonstrated in the homogenate and the synaptic plasma membrane fraction of bovine brain. Sequence 1-24 of adrenocorticotropin (ACTH1-24) (10(-5) M and 10(-4) M) inhibited endogenous phosphorylation of CP in synaptic plasma membranes. The protein was partially characterized and purified to homogeneity from bovine brain by procedures previously described for rat B-50. CP was enriched in ammonium sulfate precipitated protein (ASP) fractions and phosphorylated by an endogenous protein kinase. Two-dimensional gel analysis of bovine and rat ASP showed that the cross-reacting protein had an isoelectric point less acidic than B-50. Limited proteolysis by Staphylococcus aureus protease yielded a "peptide map" analogous to B-50. Two major fragments of Mr 30,000 and 17,000 were produced. In addition, CP exhibited other similarities to rat B-50: phosphorylation by rat brain protein kinase C, microheterogeneity observed after isoelectric focusing, and possibly degradation by endogenous proteolysis. Cross-reaction of proteins in brain homogenates of other mammalian species and of chicken was demonstrated: the Mr of the proteins ranged from 47,000 to 53,000. We conclude that (1) the cross-reacting bovine protein is a "B-50 protein," and (2) the Mr of the "B-50 protein" varies from species to species.

Animals↗
PubMed1983

Somatostatin and analogs inhibit endogenous synaptic plasma membrane protein phosphorylation in vitro.

The addition of somatostatin to hippocampal synaptic plasma membrane (SPM) preparations in vitro decreased subsequent phosphorylation of specific protein bands. 10(-4)M somatostatin inhibited the phosphorylation of protein bands with apparent molecular weights between 10 000 and 20 000 daltons and, to a lesser extent, 48 000 daltons (B-50) and 52 000. Increasingly greater degrees of inhibition were seen in response to somatostatin-28 and [D-Trp8]somatostatin. Inhibition of B-50 protein phosphorylation in the presence of [D-Trp8]somatostatin was most prominent in SPM preparations from the hippocampus and amygdala, with lesser degrees of inhibition seen in the cortex and hypothalamus. Addition of [D-Trp8]somatostatin to an ammonium sulfate-precipitated fraction (ASP 55-80) from cortex only slightly inhibited endogenous B-50 phosphorylation. The injection of [D-Trp8] somatostatin intracerebroventricularly into rats did not induce excessive grooming behavior but resulted in barrel rotation. These results suggest that somatostatin and congeners affect SPM protein phosphorylation in a manner different from that of ACTH, presumably involving membrane sites that bind somatostatin.

Adrenocorticotropic Hormone↗
PubMed1983

Affinity-purified anti-B-50 protein antibody: interference with the function of the phosphoprotein B-50 in synaptic plasma membranes.

Affinity-purified anti-B-50 protein antibodies were used to study the previously proposed relationship of the phosphorylation state of B-50 protein and polyphosphoinositide metabolism in synaptic plasma membranes. Antibodies were raised against a membrane extract enriched in the B-50 protein and its adrenocorticotropin-sensitive protein kinase, obtained from rat brain. Anti-B-50 protein immunoglobulins were purified by affinity chromatography on a solid immunosorbent prepared from B-50 protein isolated by an improved procedure. The purified antibodies reacted only with the B-50 and B-60 protein, a proteolysis derivative (of B-50), as assessed by the sodium dodecyl sulfate-gel immunoperoxidase method. These antibodies inhibited specifically the endogenous phosphorylation of B-50 protein in synaptic plasma membranes, without affecting notably the phosphorylation of other membrane proteins. This inhibition was accompanied by changes of the formation of phosphatidylinositol 4,5-diphosphate and phosphatidic acid in synaptic plasma membranes, whereas formation of phosphatidylinositol 4-phosphate was not altered. Inhibition by ACTH 1-24 of the endogenous phosphorylation of B-50 protein in membranes was associated only with an enhancement of the phosphorylation of phosphatidyl-inositol 4-phosphate to phosphatidylinositol 4,5-diphosphate. These data support our hypothesis on the functional interaction of B-50 protein and phosphatidylinositol 4-phosphate kinase in rat brain membranes. The evidence shows that purified anti-B-50 protein antibodies can be used to probe specifically the function of B-50 protein in membranes.

Adrenocorticotropic Hormone↗
PubMed1983

Phosphorylation of B-50 protein by calcium-activated, phospholipid-dependent protein kinase and B-50 protein kinase.

B-50 is a brain-specific phosphoprotein, the phosphorylation state of which may play a role in the regulation of (poly)phosphoinositide metabolism. Several kinases were tested for their ability to phosphorylate purified B-50 protein. Only calcium-activated, phospholipid-dependent protein kinase (kinase C) and B-50 protein kinase were able to use B-50 protein as a substrate. Furthermore, kinase C specifically phosphorylates B-50 when added to synaptic plasma membranes. We further characterized the sensitivity of kinase C and B-50 kinase to ACTH (and various fragments), phospholipids, chlorpromazine, and proteolytic activation. Since the sensitivities of both kinases were similar, we conclude that B-50 protein kinase is a calcium-dependent, phospholipid-stimulated protein kinase of the same type as kinase C.

Adrenocorticotropic Hormone↗
PubMed1982

The effect of ACTH on rat brain synaptic plasma membrane lipid fluidity.

The effect of ACTH on the lipid fluidity was examined in synaptic plasma membranes from rat forebrain. ACTH1-24 increased the fluidity of the synaptic plasma membranes in a dose-dependent way, the lowest effective dose being 10(-5) M. The shorter N-terminal fragment ACTH1-10 was not effective. The significance of this finding is discussed in relation to the known effects of ACTH on synaptic membrane phosphorylation.

Adrenocorticotropic Hormone↗
PubMed1982

Membrane phosphoproteins of rat hippocampus: sensitivity to tetanic stimulation and enkephalin.

Hippocampal slices are electrically stimulated in the perforant path with a pulse-train, which can lead to long-term potentiation (LTP). Of the thus stimulated slices, subcellular fractions are prepared and used in an endogenous protein phosphorylation assay. A phosphoprotein band which was reported earlier to be sensitive to electric stimulation as well as to methionine-enkephalin is now further analyzed: it consists of two phosphoproteins only slightly differing in molecular weight: 50,000 Mr (50 K) and 52,000 Mr (52 K), but having distinct biochemical properties and subcellular localization. Their IEP is dissimilar (3.5-4.3 and 5.3, respectively), they display different sensitivity towards calcium when tested in the phosphorylation assay, but are both cAMP-independently phosphorylated. Only one of them responds to tetanic stimulation with an increased phosphorylation post hoc. This protein, the 52 K component, is localized in synaptic membranes. Moreover, this protein also responds to incubation of slices with methionine-enkephalin. The phosphorylation of the 50 K component is not influenced by electric stimulation, nor by incubations with neuropeptides; its phosphorylation takes place in material sedimenting with the mitochondrial cell fractions and is strongly calcium- and calmodulin-dependent.

Animals↗
PubMed1982

Specific proteolysis of a brain membrane phosphoprotein (B-50): effects of calcium and calmodulin.

The membrane bound phosphoprotein B-50 (MW 48K) was isolated from rat brain tissue. The fraction containing the highest endogenous B-50 phosphorylating activity (ASP 57-82%) contains protease activity. In the absence of calcium a time-dependent decrease of the protein B-50 is observed. Under these conditions another phosphoprotein B-60 (MW 46K) appears in the incubation medium. Addition of calcium and/or calmodulin enhances the protease activity whereas the substrate specificity is lost. Results of both isoelectric focussing and peptide mapping indicate the B-50 and B-60 are related proteins. These data support our hypothesis that the recently isolated behaviorally active peptide PIP (MW approx. 1600 D) is the smaller cleavage product of the proteolytic degradation of B-50 to B-60.

Animals↗
PubMed1982

Evidence that the synaptic phosphoprotein B-50 is localized exclusively in nerve tissue.

The localization of the phosphoprotein B-50 (molecular weight 48,000 isoelectric point 4.5) in the rat has been studied. Inspection of endogenous phosphorylation patterns of the particulate as well as the cytosolic subcellular fractions from a variety of peripheral organs failed to demonstrate phosphorylation of a molecular weight 48,000 protein. Only in the particulate fractions from brain tissue was there endogenous phosphorylation of the B-50 protein. Two-dimensional analysis (isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis) and in immunochemical detection method employing an anti B-50 antiserum revealed the presence of B-50 in particulate material from brain, but not in that of other tissues. Therefore the data were interpreted as pointing to the localization of B-50 in nervous tissue. In addition, the regional distribution of endogenous B-50 phosphorylation was studied using synaptosomal plasma membranes (SPM) obtained from individual rat brain regions. The highest value was found in SPM of septal origin, the lowest in SPM from the medulla spinalis. The relationship of the high value for B-50 phosphorylation in the septum to the sensitivity of that brain area to ACTH1-24 is discussed.

Animals↗
PubMed1982

ACTH1--24 releases a protein from synaptosomal plasma membranes.

Brain membranes contain several protein kinases, all of which appear to play a role in the regulation of neuronal functioning. These membranes also contain numerous (phospho) proteins. It has been proposed that the degree of phosphorylation of some of these proteins may affect neuronal membrane properties. In a series of previous reports we showed that ACTH1--24 inhibits the endogenous phosphorylation of several synaptosomal plasma membrane (SPM) proteins including the B-50 protein. Although we have speculated that the degree of phosphorylation of B-50 may be important in regulating the turnover of membrane (poly)-phosphoinositides, the exact nature of the interaction between ACTH1--24 and B-50/B-50 protein kinase is unknown. The purpose of the present study was to determine whether treatment of SPM with ACTH1--24 will lead to a specific release of proteins from SPM. We found that ACTH1--24 specifically releases a 41,000 Mr protein from rat brain SPM. Although we are not certain about the biological significance of the release of this polypeptide, it is of sufficient interest for further research in view of the lack of success of finding binding of labeled ACTh to brain membranes.

Adrenocorticotropic Hormone↗