Characterization of protein kinases from adrenal medulla. A study of cytosol and nuclear enzymes.

Since phosphorylation of chromosomal proteins by cyclic AMP-dependent protein kinases (EC 2.7.1.37) enhances template activity of adrenal medulla chromatin (9), we have studied the properties and regulation of protein kinases isolated from chromaffin cell cytosol and nuclei. DEAE-cellulose chromatography revealed three peaks of kinase activity in the nucleus (nPKI, nPKII, nPKIII) and two in the cytosol (cPKI, cPKII). The three nuclear enzymes, as well as cPKII, did not require cyclic AMP to express their catalytic activity. nPKI and nPKIII preferred acidic substrates as PO3-4 acceptors, while nPKII and the cytosol enzymes preferred basic PO3-4 acceptors. Enzyme recombination experiments using protein kinase regulatory subunits from cytosol suggested that cPKII was the catalytic subunit of cPKI. In contrast, the nuclear enzymes were not catalytic subunits of the cyclic AMP-dependent protein kinase in the cytosol (cPKI). Only the cytosol protein kinases could be inhibited by endogenous heat-stable protein kinase inhibitors. The nuclear and cytosol cyclic AMP-independent protein kinases were distinguishable on the basis of their sedimentation constants as well as Mc2+ and Mn2+ requirements.     

Characterization of tyrosine hydroxylase from bovine adrenal medulla

Tyrosine hydroxylase purified to apparent homogeneity from the soluble fraction of bovine adrenal medulla had an apparent Mr of about 280,000 by Bio-Gel A-1.5m chromatography, and gave a single band with a Mr of 60,000 by sodium dodesyl sulfate polyacrylamide gel electrophoresis. The enzyme is considered to be composed of four identical subunits. Isoelectric point of purified enzyme was pH 6.0. The amino acid composition of the enzyme was characterized by fairly high contents of glutamic acid and alanine residues. The N-terminal amino acid was determined to be glutamic acid.     

Characterization of myosin light-chain kinase from bovine adrenal medulla

Partially purified bovine adrenal medullary myosin light-chain kinase (MLCK) possesses a Stoke's radius of 79 A and a sedimentation coefficient of 3.95 +/- 0.45 S, yielding a native molecular weight of 150,000 +/- 17,000 g/mol and a frictional ratio of 2.24. It catalyzes the phosphorylation of the isolated light chain of skeletal muscle myosin and the light chain of intact adrenal medullary myosin, but not phosphorylase b or histone. The activation of MLCK by calmodulin is specific and dose dependent, yielding a K0.5 value of 9.0 nM; the dose response curve with respect to free Ca2+ is biphasic, exhibiting a stimulatory phase at low free Ca2+ concentrations (K0.5 = 0.17 microM) and an inhibitory phase at higher free Ca2+ concentrations (400-3000 microM). Michaelis-Menten kinetics are observed for ATP, yielding a Km for ATP of 25 microM and a Vmax of 23.2 nmol/min/mg. However, positive cooperative kinetics are observed for the skeletal muscle myosin light chain, yielding a Hill coefficient of 3.57, a K0.5 for light chain of 27 microM and a Vmax of 16.6 nmol/min/mg. A stoichiometry of phosphorylation of approximately 1 mol of phosphate/mol of skeletal muscle myosin light chain was observed. Therefore, adrenal medullary MLCK is similar in most, but not all, of its physical and kinetics properties to MLCKs isolated from other sources and may serve to regulate actin-myosin contractile activity in the adrenal medulla.     

Characterization of protein kinases from Blepharisma intermedium.

Three protein kinases (EC 2.7.1.37) were detected in Blepharisma and partially purified. The enzymes were most active with histone as substrate protein. The stability of the bond between phosphate and protein acceptor showed the characteristics of seryl- or threonylphosphate. Protein kinase I was solubilized by ultrasonication or freezing and thawing, while the enzymes II and III were readily solubilized by mild homogenization. Protein II and III were noticeably activated by cAMP and cGMP, while protein kinase I was inhibited by cAMP. Associated with protein kinase II and III activity was the ability to bind labeled cAMP. The following molecular weights were determined: 90000 for enzyme I, 280000 for enzyme II, and 95000 for enzyme III. Various apparent Michaelis constants were estimated.     

Characterization of the rat adrenal medulla cultured in vitro

Summary A wide variety of experimental animal models have been used to investigate the mechanisms of synthesis, storage, and release of catecholamines. Whereas in vivo experimental models are situated at one end of the spectrum, cell culture models are situated at the other end. In the present study, we have characterized various aspects of the rat adrenal medulla cultured in vitro as a whole tissue, aiming to establish a new experimental model in between in vivo animal models and cell culture models. We adapted a bottle rotator system commonly used for culturing rodent whole embryos. Changes in histology, activities and mRNA levels of catecholamine-synthesizing enzymes, and concentrations of catecholamines in the adrenal medulla were studied. In addition, the effects of cholinergic stimulation on catecholamine release from the adrenal medulla were examined. Overall the results indicate that various aspects of the adrenal medulla become stable after 4 d of culture and the adrenal medulla at this stage releases catecholamines in response to cholinergic stimulation. The whole adrenal medulla culture system may be a useful tool for investigating catecholamine-related functions dependent on intercellular reactions or communications.     

Characterization of specific insulin binding sites on chromaffin cells from bovine adrenal medulla

1. Insulin receptors were investigated in isolated chromaffin cells from bovine adrenal medulla. 2. The cells were incubated with [125I]insulin in HEPES buffer, pH 7.8 at 15 degrees C for 180 min to obtain steady state binding. Specific binding was linearly related to the number of cells in the range 0.5-10 x 10(6) cells/ml. Insulin and proinsulin caused half maximal displacement of specifically bound tracer in concentrations of 0.18 and 2.46 nM, respectively. 3. Computer analysis of the binding data gave a linear Scatchard plot, consistent with a single class of non-interacting receptors with an affinity constant of 5.6 nM-1, the total number of receptors per cell being 1700. 4. The apparent MW of the insulin binding subunit of the receptor was 135,000, determined by affinity crosslinking and SDS gel electrophoresis under reducing conditions.     

Rapid purification and properties of protein kinase C from bovine adrenal medulla

Protein kinase C was purified from bovine adrenal medulla using a rapid procedure which resulted in an approximate 1500-fold increase in specific activity. The characteristics of the enzyme are reported and for the first time it is possible to compare the effects of TPA on secretion from intact and permeabilized cells with the effect of TPA on protein kinase C purified from the same secretory tissue.     

Characterization of microtubule-associated protein 1-associated protein kinases from rat brain

The microtubule-associated protein (MAP) 1 preparation, MAP1A and 1B, obtained from rat brain microtubules was associated with protein kinases that were insensitive to cAMP, cGMP, calcium, calcium/calmodulin and calcium/phosphatidylserine. The fractionation of highly purified MAP1 by phosphocellulose chromatography revealed that protein kinase activity to phosphorylate phosvitin was separated into three major peaks (MAP1 kinases A, B and C). MAP1 was recovered in the MAP1 kinase A fraction and phosphorylated by the contained kinase. MAP1 kinase A is a novel protein kinase that is remarkably activated by poly- -lysine and poly- -arginine, but very insensitive to heparin among the kinases. Photoaffinity labeling using [-³²P]8-azido ATP indicated that the e65 kDa polypeptide is identified as an ATP-binding protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the highly purified MAP1 and MAP1 kinase A fractions. MAP1 kinases B and C may be identified as casein kinase I- and II-like kinases. The present results show that MAP1 is associated with at least three kinases and provide an insight for understanding thoroughly the MAP1-mediated microtubule functions.     

A soluble lipid.protein complex from bovine adrenal medulla chromaffin granules

A unique soluble lipoprotein has been isolated from aqueous lysates of bovine adrenal medulla chromaffin granules by DEAE-cellulose chromatography and gel filtration. Chloroform/methanol extracts of this complex contain sphingomyelin, lecithin, and cholesterol. Gel filtration in aqueous media indicate an approximate molecular weight of 900,000 for the complex. Incubation with sodium dodecyl sulfate causes dissociation to a low molecular weight polypeptide; prolonged treatment with guanidine HCl does not promote dissociation at all. Amino acid analysis revealed a high content of hydrophobic amino acids. Analysis of the tryptic fingerprint indicates that a single type of polypeptide chain is present. The complex appears to contain approximately five copies of polypeptide per aggregate.     

Characterization and localization of neutral sphingomyelinase in bovine adrenal medulla

Homogenates of bovine adrenal medullae hydrolyzed exogenous sphingomyelin at 4.3 +/- 1.6 nmol X mg-1 X min-1 and 97% of this sphingomyelinase activity was sedimentable at 110,000 g. The sphingomyelinase had a broad pH optimum centered at pH 7. Enzymatic activity was maximal with 80 microM added Mn2+; Mg2+ supported less than half maximal activity and both Ca2+ and EDTA inhibited activity. No activity was detected in the absence of Triton X-100. Response to detergent was biphasic with dose-dependent stimulation from 0.02% to 0.05% Triton X-100 followed by inhibition with increasing concentrations of detergent. Activity in response to detergent was also modulated by protein concentration. Sphingomyelinase activity was associated with a plasma membrane-microsomal fraction. Phosphatidylcholine was not hydrolyzed under optimal conditions for sphingomyelin hydrolysis and a variety of other conditions. Neutral-active sphingomyelinase activity in adrenal medulla was similar in magnitude to that observed in other non-neural bovine tissues. This study demonstrates the presence of a potent neutral-active sphingomyelinase in a plasma membrane-microsomal fraction of bovine adrenal medulla. This enzyme may be involved in membrane fusion and lysis during catecholamine secretion through its ability to alter membrane composition.     

Characterization of phospholipase activities in chromaffin granule ghosts isolated from the bovine adrenal medulla

Highly purified chromaffin granule membranes contain high levels (100 nmol/mg protein) of long-chain free fatty acids (Husebye, E.S. and Flatmark, T. (1984) J. Biol. Chem. 259, 15272-15276), as well as lysophosphatidylcholine (268 nmol/mg protein) and lysophosphatidylethanolamine (92 nmol/mg protein). The release of saturated and unsaturated long-chain fatty acids from endogenous phospholipids was 38 and 28 nmol/mg protein per h, respectively, at 37 degrees C and pH 7.5 (alkaline pH optimum). p-Bromophenacyl bromide inhibited the release of palmitate and oleate by 88 and 65%, respectively. The deacylation of membrane phospholipids was not significantly affected by micromolar free Ca2+. Based on experiments with pancreatic phospholipase A2, stearate and arachidonate were found to be suitable markers for deacylation at the sn-1 and sn-2 positions, respectively. Experiments with exogenously added labeled phosphatidylcholines confirmed that chromaffin granule ghosts contain a phospholipase A2 activity (alkaline pH optimum). The preparations also revealed a phospholipase A1 activity (acid pH optimum). Finally, the ghosts contain a lysophospholipase activity (alkaline pH optimum), that accounts for the major part of the deacylation of membrane phospholipids, notably the release of saturated fatty acids (stearate and palmitate). It is unlikely that the high content of lysophospholipids is an artifact of the procedure by which the granule ghosts are isolated.     

Immunocytochemical localization of soluble protein in the adrenal medulla

Synopsis An indirect immunocytochemical technique (Nakane, 1970) was employed to localize the soluble proteins purified from lysates of catecholamine (CA)-containing vesicles of the bovine adrenal medulla. Antiserum to the proteins, produced in rabbits, was used for incubation of sections of bovine adrenal tissue prepared by fixation in glutaraldehyde and embedding in serum albumin (McLean & Singer, 1970). The site of the antigen-antibody complex was visualized by incubating the sections with anti-rabbit -globulin (goat) conjugated to peroxidase, followed by the deposition of electronopaque reaction product generated by the enzyme. The reaction product, also visible at the level of the light microscope, appeared to have a distribution similar to that of the CA-storage vesicles. Electron microscopic examination revealed that nearly all the reaction product was deposited over the electron-opaque core of the vesicles. The intravesicular localization is consistent with the proposal that these proteins exist primarily in the CA-containing granular vesicle and may function to stabilize the CA-storage complex.     

Purification and properties of an acidic protein from chromaffin granules of bovine adrenal medulla

1. A soluble protein has been purified from an aqueous extract of bovine adrenal chromaffin granules by chromatography on Sephadex G-200. This protein comprises 25% of the total protein of the granules and gave a single band on gel electrophoresis. 2. The protein is unusually rich in acidic amino acids, notably glutamic acid (26.0%, w/w); it is also relatively rich in proline (8.6%, w/w) but poor in cystine (0.35%, w/w). 3. A molecular weight of 77000 was obtained from sedimentation and diffusion measurements on the protein, and approach-to-equilibrium measurements gave apparent molecular weights of the same order. 4. A molecular weight 7 times that given above was estimated from the results of chromatography on a column of Sephadex G-200 that had been calibrated with globular proteins. However, good agreement between the ultracentrifuge and Sephadex experiments was obtained on the assumption that Sephadex chromatography depends on the effective hydrodynamic radii of proteins and not on their molecular weights. 5. The hydrodynamic properties of the protein differed from those of a typical globular protein. Thus the protein had a high intrinsic viscosity, a high frictional ratio and a large effective hydrodynamic volume. 6. The hydrodynamic properties of the protein, but not its molecular weight, were dependent on the ionic strength of the solvent. Increasing the ionic strength caused an increase in the sedimentation and diffusion coefficients, but a decrease in the intrinsic viscosity and in the frictional ratio of the protein. 7. Optical-rotatory-dispersion measurements indicated that only a small part of the polypeptide chain was in an alpha-helical conformation. 8. These results are compatible with the protein's having a conformation approaching that of a random-coil polypeptide, the volume occupied by the molecule being determined by electrostatic repulsion between the excess of negative charges.     

Purification and characterization of an inhibitor protein with cytochalasin-like acitvity from bovine adrenal medulla

A protein preparation with cytochalasin-like activity has been obtained from bovine adrenal medulla. Analysis by electrophoresis in SDS-polyacrylamide gels and chromatography in a Sephacryl S-200 column indicated that the inhibitor activity coincided with a 90 000 dalton polypeptide. The inhibitor decreased high-affinity binding of [³H]cytochalasin B to actin nuclei, apparently by competing with the drug for thesame binding site. At substoichometric levels, the inhibitor had a potent effect on actin filament elongation and on actin-dependent gelation of cell extracts in vitro. These results suggest that the inhibitor may be involved in the control of actin filament assembly and interaction in the adrenal medulla.     

Structures of gangliosides from bovine adrenal medulla.

Five gangliosides, accounting for over 95% of the total ganglioside fraction, were isolated from bovine adrenal medulla by preparative thin-layer chromatography and the carbohydrate structures determined by a combination of periodate oxidation and permethylation techniques. Partially methylated alditol acetates were generated from the neutral sugars of the fully methylated glycolipids and identified by gas-liquid chromatography. Substitution on N-acetylgalactosamine was determined by methanolysis of the permethylated ganglioside, acetylation of the products, and identification of the resulting substituted methyl glycosides by GLC. Periodate oxidation followed by borohydride reduction confirmed some of the linkages and demonstrated the absence of (2-8) linkages between sialic acid units. Mass spectrometry of the permethylated gangliosides gave information on sugar sequence at the nonreducing end.