Adenosine triphosphate in the bovine chromaffin granule.

1. pH and potential gradients are generated across the membranes of chromaffin granule 'ghost' by incubating them with MgATP: the inside of the 'ghosts' is positive and acid with respect to the incubation medium. 2. The pH gradient is partially dissipated by inclusion of a substrate for the catecholamine pump, or a mitochondrial uncoupling agent, but is enhanced by reserpine. 3. An imposed pH gradient leads to amine uptake by the 'ghosts': a potential gradient leads to ATP uptake. Studies with inhibitors confirm that amine accumulation by chromaffin granules is dependent on the former, and that ATP uptake results from ATPase-induced potential difference generation. 4. ATP has two known roles in chromaffin granule structure: the first is as a substrate for a membrane-bound proton-translocating ATPase; the second is as a component of the intragranular catecholamine storage complex.     

Identification of chromaffin granule-binding proteins. Relationship of the chromobindins to calelectrin, synhibin, and the tyrosine kinase substrates p35 and p36

At least 23 soluble proteins (chromobindins) bind to chromaffin granule membranes in the presence of Ca2+. In order to further the identification of the chromobindins and to determine the roles they may play in exocytosis or other aspects of chromaffin cell biology, several of these proteins were compared to other known membrane-binding proteins. Chromobindin 4 was identified as a 32-kDa protein called calelectrin or endonexin. Immunologically related proteins were detected in bovine brain and human platelets. Chromobindin 20 was identified as a 67-kDa variant of calelectrin and was found to have the activities of the synexin inhibitory protein, synhibin. Chromobindin 8 was identified as p36, a substrate for the tyrosine-specific kinase, pp60v-src. Chromobindin 8 was also demonstrated to undergo phosphorylation predominantly on alkali-sensitive sites during stimulation of the chromaffin cell with 20 microM nicotine. Chromobindin 6 was identified as p35, a substrate for the tyrosine kinase activity associated with the epidermal growth factor receptor. Chromobindin 9, which is known to be a substrate for protein kinase C (Ca2+/phospholipid-dependent enzyme), was found to be immunologically related to p35 and may be a precursor of chromobindin 6. The identification of these proteins from the chromaffin system may be useful in the characterization of similar, complex groups of membrane-binding proteins that have been observed in other systems.     

Chromogranin B: isolation from pheochromocytoma, N-terminal sequence, tissue distribution and secretory vesicle processing

The chromogranins/secretogranins are a family of neuroendocrine vesicle secretory proteins. Immunohistology and immunoblotting have suggested that a major soluble protein in human chromaffin granules may be chromogranin B (CgB). We purified from pheochromocytoma chromaffin granules an SDS-PAGE 110-120 kDa protein whose N-terminal sequence matched that previously deduced from a human CgB cDNA. An antibody directed against a synthetic human CgB N-terminal region specifically recognized the CgB N-terminus, though not the chromogranin A (CgA) N-terminus or the CgB C-terminus on immunoblots. An antiserum directed against CgB's C-terminus also visualized CgB but not CgA. By immunoblotting, CgB was a quantitatively major protein in human pheochromocytoma chromaffin granules, but a relatively minor in normal bovine adrenal medullary chromaffin granules. In a variety of normal bovine neuroendocrine tissues, the relative abundance of CgB immunoreactivity on immunoblots was: adrenal medulla greater than anterior pituitary greater than pancreas greater than small intestine, hypothalamus. Immunoblotting of neuroendocrine tissues (or their hormone storage vesicle cores) with both anti N-terminal and anti C-terminal CgB antisera suggested bidirectional cleavage or processing of CgB; in the anterior pituitary, a unique 40 kDa C-terminal fragment was observed. Bidirectional CgB cleavage was also suggested on immunoblots of chromaffin tissue from three species (human, bovine, rat). C-terminal processing of CgB was also confirmed by amino acid sequencing of SDS-PAGE-separated, polyvinylidene difluoride membrane-immobilized CgB fragments from pheochromocytoma chromaffin granules. Whether such fragments possess biological activity remains to be investigated.     

Stimulatory Effect of Ascorbic Acid on Norepinephrine Biosynthesis in Digitonin-Permeabilized Adrenal Medullary Chromaffin Cells

The regulatory role of ascorbic acid in norepinephrine biosynthesis was studied using digitonin-permeabilized chromaffin cells. When permeabilized chromaffin cells were incubated with [3H]3,4-dihydroxyphenylethylamine ([3H]dopamine) in calcium-free medium, the amounts of radioactive dopamine and norepinephrine measured in the cell fraction were increased as a function of incubation time and dopamine concentration. Both the accumulation of dopamine and the formation of norepinephrine were shown to require the presence of Mg-ATP in the medium. These results indicate that the permeabilization of chromaffin cells by digitonin treatment does not disrupt the functions of chromaffin granules, including dopamine uptake, norepinephrine formation, and storage of these amines. Using this permeabilized cell system, the effect of ascorbic acid on the rates of dopamine uptake and hydroxylation was investigated. The formation of norepinephrine was stimulated by ascorbic acid at concentrations of 0.5-2 mM in the presence of Mg-ATP. By contrast, dopamine uptake was not affected by the presence or absence of ascorbic acid in the medium. These findings provide evidence that ascorbic acid may stimulate the conversion of dopamine to norepinephrine by increasing dopamine beta monooxygenase activity rather than by increasing the substrate supply of dopamine. These observations also suggest that the rate of norepinephrine biosynthesis in adrenal medullary cells may be regulated by the concentration of ascorbic acid within the cell cytoplasm.     

The assignment of the Ca2+-ATPase activity of chromaffin granules to the proton translocating ATPase

CaATP is shown to function as a substrate for the proton translocating ATPase of chromaffin granule ghosts at concentrations which are comparable to that of MgATP. Using the initial rate of the proton pump activity as the measure (delta pH/delta t), an apparent Km-value of 139 +/- 8 microM was estimated for CaATP and 59 +/- 3 microM for MgATP. The maximal rate was markedly higher with MgATP than with CaATP, partly due to an inhibition of the hydrolytic activity at the higher concentrations of CaATP. The proton pump activity with CaATP was inhibited by N-ethylmaleimide and N,N'-dicyclohexylcarbodiimide at concentrations similar to that found for MgATP. No inhibition was observed with sodium vanadate in the concentration range 0-15 microM. Calmodulin and trifluoperazine had no effect on the overall ATPase activity with CaATP. These findings establish this activity as an intrinsic property of the chromaffin granules, i.e., linked to the H+-ATPase. No evidence was obtained for the presence of a Ca2+-translocating ATPase [Ca2+ + Mg2+)-ATPase) in the chromaffin granules.     

Detection of proteolytic activity by fluorescent zymogram in-gel assays

Proteases are involved in the regulation of many biological functions. This study describes a novel method for detecting protease activity by fluorescent zymogram in-gel protease assays, using SDS polyacrylamide gels copolymerized with a peptide-MCA (4-methyl-coumaryl-7-amide) substrate. This method allows simultaneous determination of protease cleavage specificity and molecular weight. Trypsin was electrophoresed in SDS polyacrylamide gels copolymerized with Boc-Gln-Ala-Arg-MCA, the gel was then incubated in assay buffer, and trypsin cleavage of the peptide-MCA substrate generated fluorescent AMC (7-amino-4-methyl-coumarin), which was subsequently detected under UV transillumination. Chymotrypsin activity was detected in gels copolymerized with Suc-Ala-Ala-Pro-Phe-MCA substrate. Selective detection of these proteases was demonstrated by the absence of trypsin activity in gels containing the chymotrypsin substrate, and the lack of chymotrypsin activity in gels containing the trypsin substrate. Detection of proteolytic activity from secretory vesicles of adrenal medulla (chromaffin granules) was observed with the trypsin substrate, Z-Phe-Arg-MCA, but not with the chymotrypsin substrate. Overall, this sensitive fluorescent zymogram in-gel protease assay method can be used for rapid determination of protease cleavage specificity and enzyme molecular weight in biological samples. This assay should be useful for many research disciplines investigating the role of the many proteases that control cellular functions.     

Beta-amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for beta-secretase activity in chromaffin vesicles

A key factor in Alzheimer's disease (AD) is the beta-secretase activity that is required for the production of beta-amyloid (Abeta) peptide from its amyloid precursor protein (APP) precursor. In this study, the majority of Abeta secretion from neuronal chromaffin cells was found to occur via the regulated secretory pathway, compared with the constitutive secretory pathway; therefore, beta-secretase activity in the regulated secretory pathway was examined for the production and secretion of Abeta in chromaffin cells obtained from in vivo adrenal medullary tissue. The presence of Abeta(1-40) in APP-containing chromaffin vesicles, which represent regulated secretory vesicles, was demonstrated by radioimmunoassay (RIA) and reverse-phase high-performance liquid chromatography. These vesicles also contain Abeta(1-42), measured by RIA. Significantly, regulated secretion of Abeta(1-40) from chromaffin cells represented the majority of secreted Abeta (> 95% of total secreted Abeta), compared with low levels of constitutively secreted Abeta(1-40). These results indicate the importance of Abeta production and secretion in the regulated secretory pathway as a major source of extracellular Abeta. Beta-secretase activity in isolated chromaffin vesicles was detected with the substrate Z-Val-Lys-Met-/MCA (methylcoumarinamide) that contains the beta-secretase cleavage site. Optimum beta-secretase activity in these vesicles required reducing conditions and acidic pH (pH 5-6), consistent with the in vivo intravesicular environment. Evidence for cysteine protease activity was shown by E64c inhibition of Z-Val-Lys-Met-MCA-cleaving activity, and E64c inhibition of Abeta(1-40) production in isolated chromaffin vesicles. Chromatography resolved the beta-secretase activity into two distinct proteolytic pathways consisting of: (i) direct cleavage of the beta-secretase site at Met-/Asp by two cysteine proteolytic activities represented by peaks Il-A and Il-B, and (ii) an aminopeptidase-dependent pathway represented by peak I cysteine protease activity that cleaves between Lys-/Met, followed by Met-aminopeptidase that would generate the beta-secretase cleavage site. Treatment of chromaffin cells in primary culture with the cysteine protease inhibitor E64d reduced the production of the beta-secretase product, a 12-14 kDa C-terminal APP fragment. In addition, BACE 1 and BACE 2 were detected in chromaffin vesicles; BACE 1 represented a small fraction of total beta-secretase activity in these vesicles. These results illustrate that multiple cysteine proteases, in combination with BACE 1, contribute to beta-secretase activity in the regulated secretory pathway. These results complement earlier findings for BACE 1 as beta3-secretase for Abeta production in the constitutive secretory pathway that provides basal secretion of Abeta into conditioned media. These findings suggest that drug inhibition of several proteases may be required for reducing Abeta levels as a potential therapeutic approach for AD.     

Dopamine β-hydroxylase of bovine adrenal medullae. A rapid purification procedure

1. A rapid purification procedure for dopamine β-hydroxylase from bovine adrenal-medulla chromaffin granules is presented. The homogeneity of the purified enzyme was demonstrated by means of three independent criteria. The specific activity of the enzyme compares favourably with that obtained by more involved procedures. 2. The stability of the enzyme was investigated and storage in polypropylene tubes was found preferable to storage in glass. 3. The soluble and particulate forms of dopamine β-hydroxylase appear to be identical, since membrane-bound and membrane-enclosed forms of the enzyme exhibit similar properties as regards size, charge and amino acid composition. 4. Ca²⁺ was found to stimulate the release of dopamine β-hydroxylase from bovine chromaffin granules in vitro. 5. An endogenous inhibitor of the enzyme was found in the chromaffin granules. This inhibitor was not inactivated either by heating at 100°C or by pretreatment with p-chloromercuribenzoate or Cu²⁺ ions.     

Effect of secretagogues on chromogranin A synthesis in bovine cultured chromaffin cells. Possible regulation by protein kinase C.

Chromogranin A is a major component of storage granules in many different secretory cell types. After [35S]methionine labelling of proteins from cultured bovine chromaffin cells, chromogranin A was immunoprecipitated with specific antibodies, and the radioactivity incorporated into chromogranin A was determined and used as an index of its synthesis rate. Depolarization of cells with nicotine or high K+ evoked a Ca2+-dependent increase in chromogranin A synthesis, whereas muscarine, which does not evoke significant Ca2+ influx from bovine chromaffin cells, had no effect on chromogranin A synthesis. Forskolin, an activator of adenylate cyclase, affected neither the basal nor the nicotine-stimulated rate of chromogranin A synthesis. In contrast, 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, significantly enhanced the incorporation of radioactivity into chromogranin A. Sphingosine, an inhibitor of protein kinase C, abolished both nicotine-stimulated and TPA-induced chromogranin A synthesis. In addition, long-term treatment of chromaffin cells with TPA decreased protein kinase C activity and inhibited the nicotine-stimulated chromogranin A synthesis. These results suggest that protein kinase C may play an important role in the control of chromogranin A synthesis.     

Effects of mono-ADP-ribosylation on cytoskeletal actin in chromaffin cells and their release of catecholamine.

To better understand the physiological role of mono-ADP-ribosylation in animals, we examined its role in chromaffin cells. Monoclonal antibodies against rat brain ADP-ribosylhydrolase were prepared, one of which (9E7) completely inhibited the enzyme's activity with ADP-ribosylated actin as the substrate. After actin monomers were polymerized by the addition of Mg2+, mono-ADP-ribosylation induced actin depolymerization. After mono-ADP-ribosylation, the actin monomers did not polymerize by the addition of Mg2+. Polymerized actin cosedimented with chromaffin granules but mono-ADP-ribosylated actin did not. After ADP-ribosylhydrolase on the membrane of chromaffin granules was incubated with 9E7, mono-ADP-ribosylated actin did not cosediment with chromaffin granules. When chromaffin cells permeabilized with saponin were incubated with NAD and 9E7, actin and rho protein was mono-ADP-ribosylated and stimulated catecholamine release from the cells. In histochemical experiments, catecholamine and actin filaments disappeared when the permeabilized chromaffin cells were treated with NAD and 9E7. These findings indicate that mono-ADP-ribosylation breaks the actin barrier in order to move granules during exocytosis, and ADP-ribosylactin hydrolase may keep the granules within the actin barrier.     

Purification and characterization of a putative proenkephalin cleaving enzyme.

A putative proenkephalin-cleaving enzyme (PCE) extracted from bovine adrenal chromaffin granules was purified with soybean trypsin inhibitor high-performance affinity chromatography. The 12,600-fold purified enzyme was maximally active at pH 8.0. The enzyme was completely inhibited with lima bean trypsin inhibitor (0.1 mg/ml), soybean trypsin inhibitor (0.1 mg/ml), and p-(chloromercuri)benzenesulfonic acid (1.0 mM), indicating PCE is a serine protease with cysteine residues likely to be involved in its structure or activity. It exhibited significant autoproteolysis without specific substrates present. The substrate specificity and kinetic constants with the enkephalin-containing (EC) peptides Leu-9 and proenkephalin Peptides B, E, and F as substrates were studied. The cleavage patterns were substantially different than with trypsin digestion. PCE specifically recognized the paired basic amino acid residues and predominantly cleaved the peptide bonds between Lys and Arg sites and peptide bonds after Lys-Lys and Arg-Arg sites. Different Km and Vmax values for the different Lys-Arg sites indicate sequences in addition to the paired basic residues can affect enzyme activity. Also, the lower Km and Vmax of Peptide E suggest a higher affinity for this peptide but much slower cleavage. The C-terminally located Lys-Arg site appears responsible for this high affinity. Based on these observations, we propose the following: (a) the primary structure of these peptides contains enough information to be processed correctly by PCE and (b) PCE may be regulated by pH and Peptide E to prevent extensive processing of the intermediate EC peptides which are the major opioid peptides found in the adrenal chromaffin granules.     

亚热带砂岩发育森林土壤酚氧化酶活性测定方法优化

酚氧化酶在土壤有机质降解过程中起重要作用,然而,目前用于测定土壤酚氧化酶活性的方法尚未统一。本研究以亚热带地区砂岩发育的3种不同林分的森林土壤为对象,探讨底物类型、pH值、土壤储存条件、储存时间、底物浓度、水土比、培养时间和温度对土壤酚氧化酶活性的影响,以期建立统一、可比较的测定亚热带森林土壤酚氧化酶活性的方法。结果表明: 浸提液pH值显著影响土壤酚氧化酶活性,且与目前普遍使用的左旋多巴胺(L-DOPA)相比,2,2′-联氨-双(3-乙基苯并噻唑啉-6-磺酸)-二胺盐(ABTS)所测得的氧化酶活性更高、适用pH值范围更广,说明ABTS可能更适合作为测定亚热带森林酸性土壤酚氧化酶活性的底物。储存方式显著影响酚氧化酶活性,3种供试土壤样品酚氧化酶活性均随时间呈降低的趋势,降幅表现为风干> 4 ℃冷藏> -20 ℃冷冻> -80 ℃冷冻,表明在无法保证快速测定土壤酚氧化酶活性的情况下,冷冻保存方式更有利于维持土壤酚氧化酶活性。底物浓度、水土比以及培养时间和温度均影响土壤酚氧化酶活性。当土壤样品与浸提液比例为1∶100时,选择2 mmol·L^-1浓度的ABTS为底物,在25~30 ℃下培养4 h,测定酚氧化酶活性结果重复性好、灵敏度高,是测定亚热带森林酸性土壤酚氧化酶活性的最优条件。     

Vesicular neurotransmitter transporters

Neurotransmission depends on the regulated release of chemical transmitter molecules. This requires the packaging of these substances into the specialized secretory vesicles of neurons and neuroendocrine cells, a process mediated by specific vesicular transporters. The family of genes encoding the vesicular transporters for biogenic amines and acetylcholine have recently been cloned. Direct comparison of their transport characteristics and pharmacology provides information about vesicular transport bioenergetics, substrate feature recognition by each transporter, and the role of vesicular amine storage in the mechanism of action of psychopharmacologic and neurotoxic agents. Regulation of vesicular transport activity may affect levels of neurotransmitter available for neurosecretion and be an important site for the regulation of synaptic function. Gene knockout studies have determined vesicular transport function is critical for survival and have enabled further evaluation of the role of vesicular neurotransmitter transporters in behavior and neurotoxicity. Molecular analysis is beginning to reveal the sites involved in vesicular transporter function and the sites that determine substrate specificity. In addition, the molecular basis for the selective targeting of these transporters to specific vesicle populations and the biogenesis of monoaminergic and cholinergic synaptic vesicles are areas of research that are currently being explored. This information provides new insights into the pharmacology and physiology of biogenic amine and acetylcholine vesicular storage in cardiovascular, endocrine, and central nervous system function and has important implications for neurodegenerative disease.     

Phosphatidylinositol-specific phospholipase C activity of chromaffin granule-binding proteins

Using [U-14C]phosphatidylinositol as substrate, Ca2+-dependent phospholipase C activity was detected in a group of bovine adrenal medullary proteins that bind to chromaffin granule membranes in the presence of Ca2+ ("chromobindins," Creutz, C. E., Dowling, L. G., Sando, J. J., Villar-Palasi, C., Whipple, J. H., and Zaks, W. J. (1983) J. Biol. Chem. 258, 14664-14674). The activity was maximal at neutral pH and represented an 80- to 240-fold enrichment of adrenal medullary cytosol phospholipase C activity measured at pH 7.3. The stimulation of activity by Ca2+ was complex; no activity was present in the absence of Ca2+, 25% activation occurred at 1 microM Ca2+, and full activation at 5 mM Ca2+. The enzyme bound to chromaffin granule membranes in the presence of 2 mM Ca2+ but was released at 40 microM Ca2+, suggesting that intrinsic enzyme activity may be regulated by [Ca2+] at 1 microM, but additional activation at higher concentrations of Ca2+ is seen in vitro as a result of Ca2+-dependent binding of the active enzyme to substrate-containing membranes. This enzyme may generate diacylglycerol and phosphorylated inositol to act as intracellular messengers in the vicinity of the chromaffin granule membrane during the process of exocytosis.     

Characterization of Proenkephalin-Cleaving Proteinases in Bovine Adrenal Chromaffin Granules Using [35S]Proenkephalin Copolymerized into Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Proteinases capable of cleaving proenkephalin into smaller peptides have been identified in bovine adrenal chromaffin granules using [35S]methionine-labeled recombinant rat proenkephalin as a selective substrate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis proteinase radiozymography. This technique was used for the screening of subcellular fractions, general characterization of pH optima, and the mechanistic characterization of proteinases with both reversible and irreversible inhibitors. Two enzymes with approximate molecular masses of 76 and 30 kDa were shown to be localized to the highest-density fractions of chromaffin granules by sucrose density gradient fractionation. Both were enriched in a 1 M NaCl wash of purified chromaffin granule membranes, were active at high pH, and were characterized as serine proteinases based on inhibition by soybean trypsin inhibitor. The 30-kDa enzyme was also inhibited by diisopropyl fluorophosphate, D-Phe-Pro-Arg-CH2Cl, and D-Val-Phe-Lys-CH2Cl and appeared to be the previously described adrenal trypsin-like enzyme. A third enzyme, of 66 kDa, was also associated with the 1 M NaCl wash of purified chromaffin granule membranes but was not localized exclusively to chromaffin granules in sucrose gradients. This proteinase was found to be Ca2+ activated and inhibited by EDTA but not diisopropyl fluorophosphate, soybean trypsin inhibitor, p-chloromercuriphenylsulfonic acid, 1,10-phenanthroline, or pepstatin.     

Ascorbic acid and Mg-ATP co-regulate dopamine beta-monooxygenase activity in intact chromaffin granules

Ascorbic acid and Mg-ATP were found to regulate norepinephrine biosynthesis in intact secretory vesicles synergistically and specifically, using the model system of isolated bovine chromaffin granules. Dopamine uptake into chromaffin granules was shown to be unrelated to the presence of Mg-ATP and ascorbic acid at external dopamine concentrations of 7.5 and 10 mM. Under these conditions of dopamine uptake, norepinephrine biosynthesis was enhanced 5-6-fold by Mg-ATP and ascorbic acid compared to control experiments with dopamine only. Furthermore, norepinephrine formation was enhanced approximately 3-fold by ascorbic acid and Mg-ATP together compared to norepinephrine formation in granules incubated with either substance alone. The action of Mg-ATP and ascorbic acid together was synergistic and independent of dopamine content of chromaffin granules as well as of dopamine uptake. The apparent Km of norepinephrine formation for external ascorbic acid was 376 microM and for external Mg-ATP was 132 microM, consistent with the larger amounts of cytosolic ascorbic acid and ATP that are available to chromaffin granules. Other physiologic reducing agents were not able to increase norepinephrine biosynthesis in the presence or absence of Mg-ATP. In addition, maximum enhancement of norepinephrine biosynthesis occurred only with the nucleotide ATP and the cation magnesium. The mechanism of the effect of ascorbic acid and Mg-ATP on norepinephrine biosynthesis was investigated and appeared to be independent of a positive membrane potential. The effect was also not mediated by direct action of ADP, ATP, or magnesium on the activity of soluble or particulate dopamine beta-monooxygenase. These data indicate that Mg-ATP and ascorbic acid specifically and synergistically co-regulate dopamine beta-monooxygenase activity in intact chromaffin granules, independent of substrate uptake. Although the mechanism is not known, the data are consistent with the possibility that the chromaffin granule ATPase mediates these effects.     

Antibacterial Peptides Are Present in Chromaffin Cell Secretory Granules

1. Antibacterial activity has recently been associated with the soluble matrix of bovine chromaffin granules. Furthermore, this activity was detected in the contents secreted from cultured chromaffin cells following stimulation.2. The agents responsible for the inhibition of Gram+ and Gram– bacteria growth are granular peptides acting in the micromolar range or below. In secretory granules, these peptides are generated from cleavage of chromogranins and proenkephalin A and are released together with catecholamines into the circulation.3. Secretolytin and enkelytin are the best characterized; these two peptides share sequence homology and similar antibacterial activity with insect cecropins and intestinal diazepam-binding inhibitor. For some of the peptides derived from chromogranin A, posttranslational modifications were essential since antibacterial activity was expressed only when peptides were phosphorylated and/or glycosylated.4. The significance of this activity is not yet understood. It may be reminiscent of some primitive defense mechanism or may serve as a first barrier to bacteria infection during stress, as these peptides are secreted along with catecholamines.     

Immunoelectron microscopic localization of dopamine beta-hydroxylase and chromogranin A in adrenomedullary chromaffin cells

Dopamine beta-hydroxylase (DBH) was purified from bovine adrenal medullae. Rabbit IgG raised against DBH inhibited its activity by 80%. In an immunoblot analysis, the IgG specifically recognized two subunits of DBH the 72 and 75 KD components. Chromogranin A (CGA) also was purified from bovine adrenal medullae, and rabbit IgG against CGA recognized this chromogranin A in the immunoblot analysis. The intracellular distribution of DBH and CGA in bovine chromaffin cells was determined quantitatively by immunoelectron microscopy using post-embedding protein A-gold technique. DBH and CGA were localized exclusively on chromaffin granules. The binding of gold particles to these granules was saturable. The maximum number of gold particles bound to the granules roughly corresponded to the number of DBH or CGA molecules in the granules estimated biochemically. DBH was observed evenly in the periphery and in the dense matrix of the chromaffin granules.     

The Tyrosine Phosphorylation and Cytoskeletal Translocation of Phospholipase Cγ1 in Bovine Adrenal Medullary Chromaffin Cells

The tyrosine phosphatase inhibitor BpV(phen) stimulated a concentration-dependent increase of phospholipase C (PLC) activity in bovine adrenal medullary chromaffin cells. This response was accompanied by an increase in PLCgamma1 tyrosine phosphorylation and its cytosketetal translocation. Insulin, at high concentrations, stimulated PLC activity to a similar extent as BpV(phen), a response that was also accompanied by an increase in PLCgamma1 translocation but not its tyrosine phosphorylation. BpV(phen) strongly enhanced the insulin-stimulated increase in PLC activity and caused a small rise in PLCgamma1 translocation above that seen with insulin alone. Despite the synergistic rise in activity PLCgamma1 tyrosine phosphorylation did not increase beyond that seen with BpV(phen) alone. These results indicate that PLCgamma1 activation in chromaffin cells may be more closely associated with its cytoskeletal translocation than its tyrosine phosphorylation although other factors may also be important for activation of enzyme activity.     

Zwitterionic and anionic forms of a serotonin analog as transport substrates

4,6-Difluoroserotonin, a serotonin analog with an acidic 5-hydroxyl proton (pK alpha = 7.97) relative to serotonin (pK alpha = 10.73), was tested as a substrate for the biogenic amine transporter of bovine chromaffin granules and the plasma membrane serotonin transporter of human blood platelets. The platelet serotonin transporter transports this analog with identical rates as those for serotonin, both at pH 6.7, where the hydroxyl group is predominantly protonated and at pH 9, where it is largely dissociated. In contrast, the chromaffin granule biogenic amine transporter prefers the form of 4,6-difluoroserotonin with a protonated 5-hydroxyl group. Thus, the KM for 4,6-difluoroserotonin increases, and Vmax decreases (relative to the values for serotonin) as the pH increases from 7 to 9. This effect may reflect a specific requirement for the protonated hydroxyl group in substrate translocation, as opposed to binding, since the KI for 4,6-difluoroserotonin inhibition of serotonin transport is the same as the KM for serotonin from pH 7 to 9.