Distribution of protein I in mammalian brain as determined by a detergent-based radioimmunoassay.

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121

106

Protein tyrosine phosphorylation in purified synaptic vesicles from rat forebrain has been studied in the presence of Mn2" and orthovanadate. High levels of endogenous protein tyrosine phosphorylation were observed. Four major phosphoproteins, with apparent molecular masses of 105, 94, 38, and 30 kDa, were shown to contain phosphotyrosine. The 38-kDa phosphoprotein was identified as synaptophysin (p38), a well-characterized integral membrane protein of synaptic vesicles. The three other phosphotyrosinecontaining proteins distributed in the same manner as synaptophysin in all subcelular fractions. Like synaptophysin, the two high molecular weight phosphotrosine proteins (105 and 94 kDa) were found to be glycoproteins by lectin chromatography. Tyrosine phosphorylation of synaptophysin was an intravesicular reaction and reached 50% of maximal level within 3 min. Triton X-100, a nonionic detergent, inhibited tyrosine phosphorylation of endogenous protein substrates but not the phosphorylation ofan exogenous substrate, poly(Gluse,-Tyrn0). Tyrosine phosphorylation of synaptophysin was also demonstrated in synaptosomes, indicating that tyrosine phosphorylation of synaptic vesicle proteins occurs in intact nerve terminals.There is now a great deal of evidence that protein phosphorylation is involved in the regulation of neuronal function (for reviews, see refs. 1 and 2). Serine/threonine phosphorylation of proteins in synaptic vesicles appears to play an important regulatory role' in nerve terminals. It has been demonstrated that serine phosphorylation of synapsin I, a synaptic vesicle-specific protein, is regulated by depolarizing reagents and by neurotransmitters (1). Phosphorylation of synapsin I by calcium/calmodulin-dependent protein kinase II appears to facilitate'neurotransmitter release (3).Phosphorylation of proteins on tyrosine residues has generally been thought to be associated with the regulation of cell growth and transformation (4). The intrinsic tyrosine kinase activities of certain hormone receptors and oncogene protein products have been shown to be necessary for their biological activities (5-7). However, the presence of high levels of tyrosine kinase activity in adult brain (8-11) and other nonproliferative tissues (8, 12, 13) were incubated at 30'C for 15 min. The reaction was terminated by adding 100 1.L of a "stop-solution" containing 12% (vol/vol) NaDodSO4, 20o (vol/vol) glycerol, 0.25 M TrisHCl (pH 7), and 0.01% bromophenol blue, followed by boiling for'3 min. The phosphorylated proteins were analyzed by NaDodSO4/PAGE (10% acrylamide) (15). After staining and destaining, the gels were dried, and the phosphoproteins were visualized by autoradiography.Alkali treatment of gels was carried out essentially as described (16). The destained gel was shaken in 250 ml of 1 M KOH at room temperature for 15 min, then in 500 ml of 1 M KOH at 60'C for 2 hr. and finally in 800 ml of destaining solution at room temperature for 1 hr. The dried gel was autoradiographed.Phosphoamino Acid Analysis. To det...

Section: Methodsmentioning

confidence: 99%

Protein tyrosine phosphorylation in synaptic vesicles.

Pang¹,

Wang²,

Valtorta³

et al. 1988

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121

106

“…Proteins in samples were phosphorylated as described above except that the reaction was terminated by NaDodSO4 (final concentration, 1%). Immunoprecipitation was performed with minor modifications of the described procedure (13) except that 40 ,l4 of 87-kDa protein antiserum was used. Proteins in the final supernatant were subjected to NaDodSO4/polyacrylamide gel electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

Widespread occurrence of "87 kDa," a major specific substrate for protein kinase C.

Albert¹,

Walaas²,

Wang³

et al. 1986

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162

An 87-kDa phosphoprotein, identified previously as a major, specific substrate for Ca2+/phospholipid/diacylglycerol-dependent protein kinase (protein kinase C) in broken cell preparations from rat brain, has been characterized with respect to its species, tissue, and subcellular distribution. A similar protein was present in monkey, human, mouse, and bovine brain and in Torpedo californica electric organ. The protein was also identified in a variety of nonneuronal rat and bovine tissues. The rat protein had an apparent molecular mass 4-7 kDa lower, and was slightly more acidic, than the protein in bovine tissues. The 87-kDa proteins from various bovine tissues were identical by the following criteria: each was phosphorylated by exogenous protein kinase C, was ofcomparable molecular mass, generated multiple spots within the pH range of 4.4-4.9 upon isoelectric focusing, yielded identical patterns upon digestion with Staphylococcus aureus V8 protease, and was recognized by a specific 87-kDa antiserum. The relative concentrations of the 87-kDa protein in bovine tissues were highest in brain, spleen, and lung, moderate in testis, pancreas, adrenal, kidney, and liver, and lowest in heart and skeletal muscle. In the brain, the 87-kDa protein was concentrated in the synaptosomal membrane and in the cytosol. The membrane-bound protein was extractable with nonionic detergents but not with NaCI. This species, tissue, and subcellular distribution of the 87-kDa protein is similar to that of protein kinase C.

“…The protein I content of peripheral nervous tissues or fractions of the tissues was determined by a detergent-based radioimmunoassay (12 (20) with bovine serum albumin as standard.…”

Section: Methodsmentioning

confidence: 99%

“…The state of phosphorylation of protein I has been shown to be regulated by impulse conduction (5), neurotransmitters (6,7), and depolarizing agents (6)(7)(8) in intact preparations of neuronal tissue. The distribution of protein I in the central nervous system has been studied by light and electron immunocytochemistry (9, 10), subcellular fractionation (11, t), and radioimmunoassay (12). Protein I is present throughout the central nervous system.…”

mentioning

confidence: 99%

Cellular and subcellular localization of protein I in the peripheral nervous system.

Fried¹,

Nestler²,

Camilli³

et al. 1982

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The cellular and subcellular distribution of protein I, a major brain phosphoprotein, has been studied in the peripheral nervous system. The levels of protein I in various peripheral nerves and innervated peripheral tissues were determined by radioimmunoassay and radioimmunolabeling of polyacrylamide gels. The results indicated that protein I is present throughout the peripheral nervous system. Denervation studies ofadrenal medulla and iris suggested that the protein I contained in peripheral tissues is localized to the neuronal elements innervating those tissues. Protein I was found to be enriched in neurotransmitter vesicle fractions of peripheral nervous tissue. Moreover, protein I appeared to be transported from cell bodies to axon terminals at least partly in association with neurotransmitter vesicles.Protein I is a neuron-specific protein (1) that is a prominent endogenous substrate in brain for both cyclic AMP-dependent (1) and calcium/calmodulin-dependent (2-4) protein kinases. The state of phosphorylation of protein I has been shown to be regulated by impulse conduction (5), neurotransmitters (6, 7), and depolarizing agents (6-8) in intact preparations of neuronal tissue. The distribution of protein I in the central nervous system has been studied by light and electron immunocytochemistry (9, 10), subcellular fractionation (11, t), and radioimmunoassay (12). Protein I is present throughout the central nervous system. It is concentrated in presynaptic nerve terminals, where it is associated at least partially with neurotransmitter vesicles. Protein I has also been detected immunocytochemically, apparently in axon terminals, throughout the peripheral nervous system (13).In the present study we have determined the cellular and subcellular distribution of protein I in various peripheral nervous tissues which have the advantage over brain of being better defined, less complex, and more easily manipulated experimentally. MATERIALS AND METHODSMaterials. Pure protein I was kindly supplied by Louis J.DeGennaro of our laboratory. "2I-Labeled protein A (specific activity, 30 mCi/mg; 1 Ci = 3.7 X 1010 becquerels) was purchased from Amersham and protein A-bearing Staphylococcus aureus cells (SAC) (Pansorbin) were from Calbiochem-Behring.Denervation. Rats with denervated adrenal glands (achieved by transsection of the splanchnic nerve), as well as sham-operated control rats, were obtained from Zivic-Miller Laboratories (Allison Park, PA). Individual adrenal glands were analyzed 2-4 weeks after surgery, at which time denervation is complete (14). Irides were sympathetically denervated by superior cervical ganglionectomy of Sprague-Dawley rats from Anticimex (Stockholm, Sweden). This procedure causes complete degeneration of iris sympathetic nerves within 48 hr (15). Sixteen normal irides and 30 denervated irides, taken 5 days after surgery, were pooled for protein I analysis. Subcellular Fractionation. All of the sucrose solutions used in the fractionation procedures were buffered with 5 mM potassium phosphat...