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F S Gorelick

Publications and source records attributed to F S Gorelick.

At least 55 records · Page 3Linked to original sources
PubMed1990

Regulated phosphorylation of secretory granule membrane proteins of the rat parotid gland.

An antiserum raised against purified rat parotid secretory granule membrane proteins has been used to identify organelle-specific protein phosphorylation events following stimulation of intact cells from the rat parotid gland. After lobules were prelabeled with [32P]orthophosphate and exposed to secretagogues, phosphoproteins were immunoprecipitated with the granule membrane protein antiserum, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. Parallel studies of stimulated amylase release were performed. Isoproterenol treatment of parotid lobules resulted in an increase in the phosphate content of immunoprecipitable 60- and 72-kDa proteins that correlated with amylase release in a time-dependent manner. Forskolin addition mimicked these effects, but only the isoproterenol effects were reversed by propranolol treatment. To confirm the specificity of the antiserum to the secretory granule membrane fraction, subcellular isolation techniques were employed following in situ phosphorylation. The 60- and 72-kDa phosphoproteins were immunoprecipitated from both a particulate fraction and a purified secretory granule fraction. Furthermore, the extraction properties of both species suggest that they are integral membrane proteins. These findings support the possibility that stimulus-regulated secretion may involve phosphorylation of integral membrane proteins of the exocrine secretory granule.

Amylases↗
PubMed1989

Presence of calcium/calmodulin-dependent protein kinase II in nerve terminals of rat brain.

Calcium/calmodulin-dependent protein kinase type II, a multimeric 550-650 kilodalton enzyme composed of major alpha (50 kilodalton) and beta/beta' (60/58 kilodalton) subunits, is present in high concentrations in mammalian brain. Previous immunocytochemical studies indicated that the enzyme is enriched in cell bodies and dendrites, but did not show a clear-cut localization in nerve terminals. The present study presents evidence, using lesion-induced degenerations of pre- and postsynaptic neuronal populations in the neostriatum and substantia nigra, that calcium/calmodulin-dependent protein kinase II, as measured both by autophosphorylation of enzyme subunits and by synapsin I kinase activity, is present in high concentrations in several populations of presynaptic terminals. Lesions of the corticostriatal tract decreased the amount of enzyme by 30-40% in the neostriatum, a decrease similar to that seen in the same region of synapsin I, a general nerve terminal marker. Lesions of the striatonigral tract induced an even more pronounced decrease of the enzyme in the substantia nigra; this decrease was larger than the lesion-induced change of synapsin I seen in the same region. Our data therefore indicate that certain nerve terminal populations in the rat brain contain high levels of calcium/calmodulin-dependent protein kinase II.

Animals↗
PubMed1989

Improving the quality of immunoblots by chromatography of polyclonal antisera on keratin affinity columns.

Unwanted reactivity of polyclonal antisera against keratins ("fingerprint proteins") is a problem commonly encountered when proteins transferred to nitrocellulose are studied by immunoblotting. Immunoreactivity against keratins is generally accompanied by a spotted background. This antikeratin immunoreactivity could be removed by adsorption of the antisera to human keratin bound to nitrocellulose. Larger amounts of antisera were purified from contaminant antikeratin antibodies by a single passage over a column of human keratin coupled to activated CH-Sepharose 4B. In contrast to nonpurified antisera and their IgG fractions, the column effluent no longer recognized the Mr 55,000-70,000 keratin proteins and exhibited a marked decrease in background labeling. We propose this simple method as a valuable alternative when affinity purification of polyclonal antisera on antigen columns is not practical.

Animals↗
PubMed1989

Translocation of synapsin I in response to depolarization of isolated nerve terminals.

Depolarization of isolated nerve terminals (synaptosomes) has been shown to stimulate neurotransmitter release and to increase the phosphorylation state of a number of proteins, including synapsin I, in a Ca2+-dependent manner. Synapsin I, a prominent nerve terminal phosphoprotein, interacts with the cytoplasmic surface of small synaptic vesicles and with cytoskeletal elements in a phosphorylation-dependent manner. In the present study we have found that depolarization of synaptosomes resulted in a rapid (2-5 sec) translocation of synapsin I from the particulate to the cytosolic (soluble) fraction. This translocation of synapsin I correlated with its phosphorylation state and was dependent on the presence of Ca2+ in the incubation medium. The stoichiometry of phosphorylation of soluble synapsin I was considerably higher than that of synapsin I in the particulate fraction, under both basal and depolarizing conditions. These data are consistent with the hypothesis that, in situ, the phosphorylation of synapsin I promotes its translocation from synaptic vesicles/cytoskeleton to the cytosol. This phosphorylation/translocation may be instrumental in regulating the release of neurotransmitter.

Animals↗
PubMed1988

Autophosphorylation and activation of Ca2+/calmodulin-dependent protein kinase II in intact nerve terminals.

The autophosphorylation of purified Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) on a threonine-containing phosphopeptide common to both the alpha and beta subunits was previously shown to convert this enzyme into a catalytically active Ca2+-independent species. We now have examined the phosphorylation and activation of Ca2+/CaM kinase II in synaptosomes, a Ca2+-dependent neurosecretory system consisting of isolated nerve terminals. Synaptosomes were prelabeled with 32Pi and the alpha subunit of Ca2+/CaM kinase II was immunoprecipitated. Under basal incubation conditions the alpha subunit was phosphorylated. Depolarization of synaptosomes produced a rapid (2-5 s) Ca2+-dependent increase of about 50% in the state of phosphorylation of the alpha subunit. This was followed by a slower increase in the 32P content of the alpha subunit over the next 5 min of depolarization. The enhanced phosphorylation was characterized by an initial rise (2 s) and subsequent decrease (30 s) in the phosphothreonine content of the alpha subunit. In contrast, the phosphoserine content of the alpha subunit slowly increased during the course of depolarization. Thermolytic two-dimensional phosphopeptide maps of the alpha subunit demonstrated that depolarization stimulated the labeling of a phosphopeptide associated with autoactivation. In parallel experiments, unlabeled synaptosomes were depolarized, and lysates of these synaptosomes were assayed for Ca2+/CaM kinase II activity. Depolarization produced a rapid (less than or equal to 2 s) increase in Ca2+-independent Ca2+/CaM kinase II activity. This activity returned to basal levels by 60 s. Thus, depolarization of intact synaptosomes is associated with the transient phosphorylation of Ca2+/CaM kinase II on threonine residues, presumably involving an autophosphorylation mechanism and concomitantly the transient generation of the Ca2+-independent form of Ca2+/CaM kinase II.

Animals↗
PubMed1988

Cell-specific localization of the alpha-subunit of calcium/calmodulin-dependent protein kinase II in Purkinje cells in rodent cerebellum.

Brain calcium/calmodulin-dependent protein kinase type II, a multimeric 600-650 kDa enzyme composed of alpha- (50 kDa) and beta/beta' (60 and 58 kDa) subunits, may be formed by alpha- and beta-subunits combining in variable proportions in different types of neurons. This study presents evidence, using cerebella from mutant mice, that the alpha-subunit displays a restricted localization in the rodent cerebellum, being detectable only in Purkinje cells. Immunocytochemical analysis of normal rat cerebellum with an antibody selective for the alpha-subunit confirmed that this subunit was detectable only in Purkinje cells. In contrast, the beta/beta'-subunits appeared to be present in all types of cerebellar mutants examined. These results indicate that different cells of the cerebellum express distinct isozymic forms of the multifunctional calcium/calmodulin-dependent protein kinase type II. It appears that Purkinje cells primarily contain an isoenzyme formed by both alpha- and beta/beta'-subunits, and that non-Purkinje cells contain an isoenzyme formed primarily by beta/beta'-subunits.

Animals↗
PubMed1988

Development of secretagogue responsiveness in the pancreas.

Epithelial cells are bound by a plasma membrane that is divided into apical and basolateral domains. In turn, the species of membrane proteins within these polarized plasmalemmal domains define the structural and functional specializations of the epithelium. Currently, research into the mechanisms whereby epithelial cell polarity is generated is a major thrust in cell biology. The topic discussed here will focus on recent studies carried out by us on the genesis of regulated secretion in the developing pancreatic acinar cell--that is, the secretory pathway responsive to second-messenger elevation after hormonal stimulation. In the developing rat pancreas, acinar cells complete cytodifferentiation 1 day before birth yet do not discharge secretory proteins from zymogen granules in response to the secretagogue cholecystokinin (CCK). The cause of this refractoriness to CCK does not lie at the receptor level, since the number, affinity, and plasmalemmal location of the receptor and its ability to generate second messengers after interaction with the ligand has already appeared. However, 1 day after birth, the acinar cell is able to discharge secretory proteins by exocytosis through the regulated secretory pathway in response to CCK. The acquisition of distal stimulus-secretion coupling events in the perinatal period has been found to correlate with developmental changes in Ca2+/calmodulin-dependent protein kinase II and its phosphorylated substrates. The relevance of the studies reported here to the complex and coexistent regulated and constitutive secretory pathways in the pancreatic acinar cell will be discussed.

Animals↗
PubMed1987

Purification and properties of a multifunctional calcium/calmodulin-dependent protein kinase from rat pancreas.

A calcium/calmodulin-dependent protein kinase (Ca/calmodulin protein kinase) was purified from rat pancreas using hydrophobic chromatography followed by gel filtration and affinity chromatography. Ca/calmodulin protein kinase from pancreas resembled previously described multifunctional Ca/calmodulin protein kinases from other tissues with respect to substrate specificity, autophosphorylation on serine and threonine residues, and catalytic and hydrodynamic properties. While Ca/calmodulin protein kinase from other tissues contains subunits of 53-60 kDa with variable proportions of a smaller 50-52 kDa subunit, pancreatic Ca/calmodulin protein kinase was found to contain a single component of 51 kDa. Experiments mixing brain Ca/calmodulin protein kinase with pancreatic homogenate suggest that the absence of a larger subunit in the pancreatic Ca/calmodulin protein kinase is not due to proteolytic degradation during enzyme preparation. Ca/calmodulin protein kinase binding to 125I-labeled calmodulin in solution was demonstrated using the photoaffinity cross-linker, N-hydroxysuccinimidyl-4-azidobenzoate. 125I-labeled calmodulin binding to Ca/calmodulin protein kinase was also demonstrated using filters containing Ca/calmodulin protein kinase transferred from polyacrylamide gels after two-dimensional gel electrophoresis. Finally, the ribosomal substrate for Ca/calmodulin protein kinase was identified as the ribosomal protein, S6. The purification procedure presented in this study promises to be useful in characterizing Ca/calmodulin protein kinase in other tissues and in clarifying the role of these enzymes in cellular function.

Animals↗
PubMed1987

Calcium-calmodulin-stimulated protein kinase in developing pancreas.

To implicate the Ca2+-calmodulin-dependent protein kinase (CDPK) in the secretory response of pancreatic acinar cells to postnatal neurohumoral stimulation, we examined enzymatic protein kinase activity during pancreatic development. CDPK was investigated by measuring the phosphorylation of endogenous proteins and an exogenous substrate, synapsin I. Ca2+-calmodulin (CaM)-stimulated phosphorylation of endogenous proteins as well as synapsin I progressively increased in pancreatic supernatants from rats of increasing age. When compared with embryonic pancreas (1 day before birth), Ca2+-CaM-stimulated protein kinase activity increased two- and sixfold in neonatal (1 day after birth) and adult pancreas (60 days after birth), respectively, when normalized to protein content. To further characterize CDPK activity, soluble fractions prepared from embryonic, neonatal, and adult pancreas were separated by gel filtration chromatography. CDPK activities from embryonic and neonatal pancreas were contained in fractions of relative molecular weights (Mr) less than 200,000. In contrast, kinase activity from adult pancreas was predominantly comprised of a Mr approximately 550,000 species that coeluted with a type II CDPK. Increasing amounts of a Mr = 51,000 CaM-binding protein as a function of increasing age were consistent with the developmental appearance of the CaM-binding subunit of type II Ca2+-CaM-stimulated protein kinase. CaM levels increased in parallel with total Ca2+-CaM-stimulated protein kinase activity. Coordinate enhancement of type II CDPK activity and secretagogue responsiveness during pancreatic maturation may suggest a role for this enzyme in the secretory process.

Animals↗
PubMed1987

Calcium-calmodulin-dependent protein kinase in hyperplastic human parathyroid glands.

The function of the parathyroid gland is closely linked to intracellular and extracellular Ca2+ concentrations. As a step toward understanding the mechanism of action of Ca2+ on the parathyroid, we examined hyperplastic human parathyroid tissue for Ca2+ and calmodulin-dependent protein kinase activity. In parathyroid homogenates, Ca2+ stimulates the phosphorylation of substrate protein in the presence of calmodulin or phospholipid. The calmodulin (CaM)-stimulated activity is present in a soluble fraction of parathyroid and can be separated from other protein kinase activities by gel filtration chromatography. The concentration dependence of CaM kinase on Ca2+ and CaM was determined using the gel filtration. The Ka values for CaM and calcium were 100 nM and 5 microM, respectively. The fraction containing the CaM kinase activity had a calculated mol wt of 5.5 X 10(5). It contained a protein with a mol wt of 4.9 X 10(4) whose phosphorylation was Ca2+ CaM dependent and a CaM-binding protein of mol wt 4.9 X 10(4) which we suggest may be the catalytic subunit of a type II Ca2+-CaM dependent protein kinase. Hyperplastic human parathyroid tissue contains a type II Ca2+-CaM dependent protein kinase which may serve an important function in Ca2+-directed metabolism.

Calcium↗
PubMed1986

Phytohemagglutinin from red kidney bean (Phaseolus vulgaris) inhibits sodium and chloride absorption in the rabbit ileum.

Phytohemagglutinin (PHA), derived from red kidney bean (Phaseolus vulgaris), can induce malabsorption and diarrhea when fed to rats. In this study, we determined the effect of PHA on ion transport in the rabbit ileum in vitro. Compared with control tissues, PHA (1 mg/ml) added to the mucosal solution increased short-circuit current (1.1 +/- 0.2 microEq/cm2 X h, p less than 0.001), decreased net Na (-1.0 +/- 0.5 microEq/cm2 X h, p less than 0.02) and Cl (-1.2 +/- 0.6 microEq/cm2 X h, p less than 0.025) absorption, and decreased tissue conductance (-1.8 +/- 0.5 mS/cm2, p less than 0.001). Serosal addition of PHA had no effect on the short-circuit current or tissue conductance. Mucosal PHA did not increase mucosal levels of cyclic adenosine monophosphate or cyclic guanosine monophosphate. Removal of serosal calcium did not affect the increase in short-circuit current induced by mucosal PHA. Utilizing fluorescent microscopy, rhodamine-labeled PHA was found to bind to the luminal border of villus cells, but not to crypt cells, in the ileum. In the descending rabbit colon, PHA did not affect either the short-circuit current or conductance, and rhodaminated PHA did not bind to the epithelial surface. Using the increase in short-circuit current as an indicator of absorption, PHA did not affect Na-coupled glucose or amino acid absorption in the ileum. This study suggests that dietary lectins may play a role in regulating intestinal fluid and electrolyte transport.

Animals↗
PubMed1983

Duodenal epithelial thymidine uptake in patients with duodenal ulcer or endoscopic duodenitis.

To evaluate the relationship between duodenal ulcer disease and duodenitis, duodenal epithelial cell renewal was measured in mucosal biopsies by the incorporation of [3H]thymidine. When 14 patients with duodenal ulcer were compared to 13 control subjects or 7 with endoscopic duodenitis alone, the crypt size was the same in all groups. Similar to other inflammatory processes of the gastrointestinal tract, patients with endoscopic duodenitis showed increased proliferative indices including a greater number of cells incorporating [3H]thymidine. In contrast, the proliferative indices from the duodenal mucosa of patients with duodenal ulcers did not differ from a control group. In a group of 6 patients with both endoscopic duodenitis and duodenal ulcer, the [3H]thymidine incorporation was intermediate between control subjects or patients with duodenal ulcer alone and those with endoscopic duodenitis alone. When subjects were divided according to the histologic appearance of the duodenal mucosa, those having chronic duodenitis demonstrated enhanced [3H]thymidine incorporation in comparison to a control group or patients with chronic active duodenitis (polymorphonuclear leukocytes present). Although there are many possible explanations of these findings, one may speculate that duodenal ulceration does not stimulate duodenal epithelial proliferation.

Adolescent↗
PubMed1983

Calmodulin-stimulated protein kinase activity from rat pancreas.

Previous work from our laboratory has demonstrated that neurohumoral stimulation of the exocrine pancreas is associated with the phosphorylation of the Mr 29,000 ribosomal protein S6. In a cell-free system using pancreatic postmicrosomal supernatant as the kinase donor, we found that the following co-factors stimulate the phosphorylation of the Mr 29,000 ribosomal protein: calcium with calmodulin, calcium with phosphatidyl serine, and cAMP. These findings suggest that the pancreas contains a calcium-calmodulin-dependent protein kinase (CaM-PK) that can phosphorylate the Mr 29,000 ribosomal protein. A CaM-PK activity was partially purified sequentially by ion exchange, gel filtration, and calmodulin-affinity chromatography. Phosphorylation of the Mr 29,000 ribosomal protein by the partially purified CaM-PK was dependent on the presence of both calcium and calmodulin and not on the other co-factors. The CaM-PK fraction contained a phosphoprotein of Mr 51,000 whose phosphorylation was also dependent on calcium and calmodulin. When 125I-calmodulin-binding proteins from the CaM-PK fraction were identified using electrophoretic transfers of SDS-polyacrylamide gels, a single Mr 51,000 protein was labeled. The preparation enriched in CaM-PK activity contained an Mr 51,000 protein that underwent phosphorylation in a calcium-calmodulin-dependent manner and an Mr 51,000 calmodulin-binding protein. It is therefore possible that the CaM-PK may comprise a calmodulin-binding phosphoprotein component of Mr 51,000.

Animals↗
PubMed1983

ERCP and bacteremia.

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Anti-Bacterial Agents↗
PubMed1982

Biliary tract abnormalities in patients with arteriohepatic dysplasia.

Arteriohepatic dysplasia is a congenital syndrome associated with life-long cholestasis. Because of symptoms suggesting extrahepatic biliary tract obstruction, we studied three patients with this syndrome by endoscopic retrograde cholangiopancreatogram (ERCP). All patients showed a decrease in the number of intrahepatic ducts. In addition, the intrahepatic ducts show attenuation with focal areas of dilatation. In one subject, this latter finding appeared to be a localized process. The extrahepatic ducts were also narrowed. One patient in this series was found to have gallstones and another cirrhosis. Although the relationship of these anomalies to the cholestasis seen in these patients is unclear, arteriohepatic dysplasis can be added to the list of processes associated with biliary tract abnormalities.

Adolescent↗