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J A Babitch

Publications and source records attributed to J A Babitch.

At least 37 records · Page 2Linked to original sources
PubMed1983

Endogenous protein phosphorylation in chick and rat brain synaptic membranes.

The protein kinase activities endogenous to synaptic membranes prepared by an identical procedure from avian (chick) and mammalian (rat) brains were compared. Both species showed similar responses towards both protein kinase effector molecules cyclic adenosine monophosphate and Ca2+. Kapp for cyclic adenosine monophosphate-dependent protein kinase activity occurred at 0.4-0.8 microM cAMP and Kapp for Ca2+-dependent, calmodulin-requiring protein kinase activity occurred at 1-2 microM Ca2+ (free ion concentration) both in the absence or presence of calmodulin added to the reaction mixture. This suggests that endogenous calmodulin in these membranes was able to modulate the Ca2+-dependent, calmodulin requiring protein kinase activity. After EGTA-treatment of the membranes to remove endogenous Ca2+ and calmodulin, no significant response towards Ca2+ on the phosphorylation of the membrane polypeptides was measured unless exogenous calmodulin was added after which the Kapp for Ca2+ was increased to 15 microM Ca2+ (free ion concentration). There was a difference in the maximal levels of kinase activity in these membranes with chick membranes containing 57% less cyclic adenosine monophosphate-dependent protein kinase activity, but 65% more Ca2+-dependent, calmodulin-requiring protein kinase activity than the rat membranes. Similar results were determined when either low (5 microM) or high (5.8 microM) concentrations of adenosine 5'-triphosphate were added to the reaction mixtures. Besides certain species differences in the molecular weights of the resulting phosphoproteins, we observed several major differences with respect to the absence or presence of some of the phosphoproteins. Chick synaptic membranes may lack the cyclic adenosine monophosphate-requiring, microtubule-associated phosphoprotein, MAP2, one of the 2 neurospecific, cyclic adenosine monophosphate-requiring and Ca2+, calmodulin-requiring phosphoproteins (Protein Ib, although Protein Ia apparently is present), and the Ca2+-requiring, calmodulin-independent, ACTH-sensitive phosphoprotein, B50. The phenothiazines, trifluoperazine, fluphenazine and chlorpromazine were found to inhibit the Ca2+-dependent, calmodulin-requiring protein kinase activities of both the chick and rat synaptic membranes. This inhibition appeared to be specific for calmodulin because at the same concentrations the phenothiazine analogue, chlorpromazine-sulfoxide, had no effect on this activity. Also found to inhibit Ca2+-dependent calmodulin-requiring protein kinase activity were dibucaine and adrenocorticotropin. These data suggest that rat forebrain synaptic plasma membranes are activated by cyclic adenosine monophosphate

Adenosine Monophosphate↗
PubMed1981

Identification of myosin in isolated synaptic junctions.

A monospecific antibody prepared against chicken gizzard myosin reacted with only one peptide corresponding to myosin heavy chain (Mr = 200,000) in gels of synaptic plasma membranes (SPM) and synaptic junctions (SJ) prepared from several species. Preadsorption of antisera with purified brain myosin eliminated antibody reactivity to SPMs and SJs. SJs were found to contain approximately 3 times the concentration of myosin found in SPMs when assayed by an indirect immunoradiometric assay. Postsynaptic density and myelin fractions contained no myosin detectable by immunoradiometric assay, antibody binding to gels, or Coomassie blue staining. The band identified as myosin in SJ fraction yielded peptide fingerprints indistinguishable from fingerprints of purified brain myosin but distinct from fingerprints of purified smooth and skeletal muscle myosins. The distribution of exogenous [125I]myosin during subcellular fractionation indicated that myosin in isolated synaptic junction could not have resulted from artifactual re-distribution of soluble myosin. Together these results show that a non-muscle myosin is an endogenous component of CNS asymmetric synapses.

Animals↗
PubMed1981

Synaptic plasma membrane tubulin may be an integral constituent.

Mild detergent extraction of chick brain synaptic plasma membranes followed by gel electrophoresis suggests that synaptic plasma membrane tubulin is an integral component. Although some of the synaptic plasma membrane tubulin might be aggregates, that possibility is not supported by the observation that tubulin aggregates that are added to synaptosomes before synaptic subfractionation do not partition with synaptic plasma membranes during membrane isolation.

Animals↗
PubMed1980

Two-dimensional analysis of autophosphorylated chick brain synaptic subfractions.

Chick brain synaptosomes were autophosphorylated upon incubation with [32PO4] and glucose, exposed to depolarizing ionic conditions, subsequently lysed, and respective synaptic subfractions were isolated. Analysis of these subfractions by two-dimensional electrophoresis revealed complex and unique phosphopeptide patterns. The phosphorylation levels of several soluble and membrane polypeptides appeared to change upon exposure to the depolarizing conditions.

Animals↗
PubMed1980

Externally disposed polypeptides of chick synaptosomal plasma membrane. Identification with pyridoxal phosphate and sodium borotritide.

The topographical distribution of polypeptides in chick brain synaptic plasma membrane was studied using pyridoxal phosphate-sodium borotritide labeling. Labeling of intact synaptosomes was restricted to the external surface only by very careful adjustment of the reaction conditions. Fourteen major external polypeptides were labeled. These had apparent molecular weights of 210 000, 160 000, 130 000, 100 000, 92 000, 82 000, 60 000, 52 000, 42 000, 34 000, 29 000, 26 000, 24 000, and 19 000. Most of the label was incorporated into the 42 000, 29 000 and 26 000 dalton polypeptides.

Animals↗
PubMed1979

Autophosphorylation of chick brain synaptic polypeptides. Phosphorylation of proteins during incubation of intact synaptosomes with 32PO4.

Chick brain synaptosomes incorporated phosphate into proteins when incubated in physiological buffer containing energy sources. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that three synaptosomal polypeptides were significantly phosphorylated after 15 sec incubation while at least fifteen polypeptides were active kinase substrates after 15 min incubation. Labeled synaptosomes were hypotonically lysed and separated by centrifugation into soluble, membrane, and mitochondrial fractions. Every fraction exhibited significant phosphate incorporation. Electrophoresis revealed that each fraction had several unique phosphorylated polypeptides and a distinctive phosphorylation pattern. The same polypeptides appear to be labeled whether MgATP was added to synaptic plasma membranes or synaptic plasma membranes were isolated after synaptosomal autophosphorylation.

Animals↗
PubMed1978

Topography of glycoproteins in the chick synaptosomal plasma membrane.

Chick brain synaptosomes or synaptic subfractions were treated with neuraminidase (EC 3.2.1.18) and/or galactose oxidase (EC 1.1.3.9) preparations in which proteolytic activity was inhibited with phenylmethanesulfonyl fluoride followed, after washing, by reductive incorporation of sodium boro[3H]hydride to identify galactose residues exposed on the synaptosomal external surface. Control experiments to demonstrate restriction of labeling to the external surface involved comparing the radioactivity in synaptoplasmic, soluble polypeptides isolated after labeled, isolated synaptoplasm and examining incorporation into fractions incubated without enzymes. Intactness of the synaptic plasma membrane after labeling was shown by trypsin digestion studies. Polypeptides were separated on sodium dodecyl sulfate polyacrylamide gels and were detected by a liquid scintillation counting procedure. Eleven major radioactive peaks were found after galactose oxidase treatment and reduction of isolated synaptic membranes. When intact synaptosomes were labeled, the same components were detected. When isolated synaptic membranes or intact synaptosomes were treated with neuraminidase before galactose oxidase treatment, three additional components were labeled. These results suggest that (a) chick synaptic membranes have a complex mixture of glycoproteins, (b) all major chick synaptic membrane glycoproteins labeled by galactose oxidase have most or all carbohydrate groups exposed at the exterior surface of the synaptosome, (c) all major, externally-disposed polypeptides of these synaptic membranes are glycoproteins.

Animals↗
PubMed1978

Use of cis-Pt(II)-uracil for electron microscopic cytochemistry of rat brain nucleic acids.

Cis-Pt(II)-uracil staining reveals nucleic acids in a relatively specific manner when applied alone for 1 h to rat brain tissue specimens. Poor contrast and resolution are observed when glutaraldehyde-fixed and epoxyembedded thin sections are post-stained with cis-Pt(II)-uracil alone. Counterstaining thin sections with uranyl acetate decreases staining specificity by revealing many tissue proteins. Synapses did not stain, which suggests that they do not contain significant amounts of large RNA molecules. Technical procedures must be carefully regulated to avoid artifactual staining of cellular components other than nucleic acids.

Animals↗
PubMed1976

On using cis-Pt (II)-uracil as a stain for nucleic acids in brain slices and subcellular fractions.

Chick brain cortical slices and crude mitochondrial fractions were fixed with glutaraldehyde, stained only with cis-Pt (II)-uracil and processed for electron microscopy. The optimal time of staining was determined to be 10 min. Results show that this platinum-pyrimidine complex is a relatively specific stain for the nucleic acids of brain slices. However, staining of crude mitochondrial fractions apparently resulted in some protein staining and other artifacts. The method should be helpful identifying ribosomal contamination of subcellular preparations and if its specificity can be increased it may prove a useful addition to staining methods of the electron microscopist.

Animals↗
PubMed1976

Preparation of chick brain synaptosomes and synaptosomal membranes.

A method is described for the preparation of synaptosomes and synaptosomal membranes from chicken brain. Procedures for isolating rat synaptosomal membranes could not be used directly; several modifications of existing procedures are reported. Purity of the subcellular and subsynaptosomal fractions was monitored by electron microscopy and measurements of ferrocytochrome c: oxygen oxidoreductase (EC 1.9.3.)), monoamine: oxygen oxidoreductase (deaminating) EC 1.4.3.4), rotenone-insensitive NADH: cytochrome c oxidoreductase (EC 1.6.99.3), NADPH: cytochrome c oxidoreductase (EC 1.6.99.1), orthophosphoric monoester phosphohydrolase (EC 3.1.3.2), ATP phosphohydrolase (EC 3.6.1.4), and levels of RNA. Microsomes are the main contaminant of the synaptosomal membrane fraction. Mitochondrial and lysosomal enzymes occur in lesser amounts. No myelin contamination was observed. Marker enzymes for contaminants suggest that these synaptosomal membranes are as pure as membranes described by others, and the specific activity of a neuronal membrane marker, (Na+ -K+)-activated ATPase, is as high as other preparations. Levels of this enzyme in the membrane fraction are enriched 13-fold over homogenate ATPase levels.

Animals↗
PubMed1975

Amino acid incorporation into neurons and glia of guinea pigs with experimental allergic encephalomyelitis.

After in vitro incubation of brain slices from guinea pigs in the intermediate (10 days postinduction) stage of experimental allergic encephalomyelitis (EAE), incorporation of [14C]leucine into protein was increased in a glial-enriched fraction. By the late (17-18 days postinduction) stage of the disease, when EAE symptoms were manifest, both the neuronal- and glial-enriched fractions showed increased specific activity of their total protein. SDS polyacrylamide gel electrophoresis showed that the increased leucine incorporation occurred in those proteins which labeled in a control material. After intraperitoneal injection of [3H]leucine the incorporated radioactivity was slightly increased in unfractionated brains from EAE animals. The neuronal-glial ratios for protein-bound radioactivity indicated that the increased incorporation resided mainly in the glial population.

Animals↗