Multiple Forms of Choline-O-Acetyltransferase in Mouse and Rat Brain: Solubilization and Characterization

Three forms of acetyl coenzyme A: choline-O-acetyltransferase (EC 2.3.1.6, ChAT) have been isolated from mouse and rat forebrain synaptosomes with a 100 mM sodium phosphate (NaP) buffer of pH 7.4, a high-salt solution (500 mM NaCl), and a 2% Triton DN-65 solution, respectively. The Triton-solubilized form of ChAT differed from the other two forms in its capacity to acetylate homocholine, its pH profile, and its sensitivity to denaturation. NaCl-solubilized ChAT could be distinguished from the other two forms with respect to pH profile, sensitivity to inhibition by 4-(1-naphthylvinyl) pyridine (in the presence of Triton), and apparent Km value for choline acetylation. The caudate and putamen of rat brain contained the highest amount of ChAT activity, based on tissue wet weight, and the cerebellum contained the least of the brain regions examined; only the cerebellum had more membrane-bound than soluble ChAT. Septal lesion reduced ChAT activity in the NaP- and Triton-solubilized fractions prepared from hippocampus by 68% and 64%, respectively, whereas it reduced the activity of the NaCl-solubilized fraction by only 21%. These results suggest that three different forms of ChAT may exist in both mouse and rat brain.     

Properties and Ontogenic Development of Membrane-Bound Histidine Decarboxylase from Rat Brain

Abstract: Histidine decarboxylase (HD) activity was determined in high-speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates. Twenty-eight percent of the HD activity was associated with membranes, and the remaining was soluble. Several properties of the soluble and membrane-bound HD were compared. No significant differences in the values of K _m for histidine and pyridoxal 5'-phosphate were observed. The solubilization of membrane-bound HD with Triton X-100 resulted in an increase of 60% over the nonsolubilized activity with no changes in the K _m for substrate and cofactor. The proportion of free pyridoxal 5'-phosphate-independent activity was identical in both fractions. The soluble and membrane-bound forms of the enzyme differ slightly in their pH-activity profiles, although both enzymes showed an optimum pH near 6.5. The HD activities present in soluble and membrane fractions were determined at different postnatal ages. The soluble activity increased until day 90, whereas the membrane-bound activity became stabilized from day 20.     

Comparison of the Soluble and Membrane-Bound Forms of the Puromycin-Sensitive Enkephalin-Degrading Aminopeptidases from Rat

Enkephalin degradation in brain has been shown to be catalyzed, in part, by a membrane-bound puromycin-sensitive aminopeptidase. A cytosolic puromycin-sensitive aminopeptidase with similar properties also has been described. The relationship between the soluble and membrane forms of the rat brain enzyme is investigated here. Both of these aminopeptidase forms were purified from rat brain and an antiserum was generated to the soluble enzyme. Each of the aminopeptidases is composed of a single polypeptide of molecular mass 100 kilodaltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography. The antisoluble aminopeptidase antiserum reacts with both enzyme forms on immunoblots and inhibits both with nearly identical inhibition curves. The isoelectric points (pI = 5.0) of both forms were shown to be identical. N-terminal sequencing yielded a common sequence (P-E-K-R-P-F-E-R-L-P-T-E-V-S-P-I-N-Y) for both enzyme forms, and peptide mapping yielded 26 peptides that also appeared identical between the two enzyme forms. Studies on the nature of the association of the membrane enzyme form with the cell membrane suggest that this enzyme form does not represent the soluble form trapped during the enzyme preparation. It is suggested that the membrane form of the puromycin-sensitive aminopeptidase is identical to the soluble enzyme and that it associates with the membrane by interactions with other integral membrane proteins.     

Acetylcholinesterase and nonspecific cholinesterase activities in rat liver: subcellular localization, molecular forms, and some extraction properties

Subcellular distribution and some extraction properties of acetylcholinesterase (AchE) (EC 3.1.1.7) and nonspecific cholinesterase (ChE) (EC 3.1.1.8) were studied in rat liver employing subcellular fractionation techniques. All purified subcellular fractions were enriched in total cholinesterase activity over the homogenate. Plasma membrane and Golgi fractions showed a significant enrichment in AchE activity, while ChE activity was enriched in both rough and smooth endoplasmic reticulum. Subcellular fractions were subjected to conditions that selectively release proteins having varying degrees of association to membranes. High-pH treatment (known to release peripheral and soluble proteins) extracted ChE activity, but more than 90% of AchE activity remained associated to the pellet. Solubility properties and molecular forms of AchE and ChE in this tissue were studied by extraction in high-salt medium with and without Triton X-100, followed by velocity sedimentation centrifugation. Most of AchE activity (88%) (41% G4 and 59% G2 + G1) was detergent soluble; 42% of ChE activity (detected only as G2 + G1) was high-salt soluble, whereas remaining ChE activity was detergent soluble. These results indicate not only a different subcellular location for both enzymes, but also point to a differential association to membranes. AchE behaves as an integral membrane protein and ChE behaves as a peripheral or a luminal soluble protein.     

5'-Nucleotidase in rat liver lysosomes

5'-Nucleotidase was found in purified rat liver tritosomes. When tritosomes were subfractionated into the membrane and soluble contents fractions, 73% of the total 5'-nucleotidase activity was found in the membrane fraction and 24% in the soluble contents fraction. Immunoblotting using specific polyclonal antibodies against the rat liver plasma membrane 5'-nucleotidase showed that the mobilities on SDS-polyacrylamide gel electrophoresis of both 5'-nucleotidases in the membrane and contents fractions were identical to that of the enzyme in the plasma membranes (Mr = 72,000). 5'-Nucleotidases in the membrane and contents fractions were sensitive to neuraminidase and converted into a form that was 4 kDa smaller after digestion, as observed in the case of plasma membrane enzyme. 5'-Nucleotidases, both from the membrane and contents fractions, were purified using immunoaffinity chromatography, and the isoelectric points, heat stability, and oligomeric structure of the purified enzymes were compared. Isoelectric focusing and the heat stability test indicated the resemblance of the soluble enzyme to the membrane-bound enzyme. However, the membrane-bound enzyme aggregated in the absence of Triton X-100, whereas the soluble enzyme behaved as a dimer. The topography of 5'-nucleotidase in the tritosomal membranes was studied using antibodies against 5'-nucleotidase and neuraminidase treatment. The inhibition of 5'-nucleotidase were not observed in the intact tritosomal fraction until the tritosomes had been disrupted by osmotic shock. These results show that the active sites and the oligosaccharide chains of 5'-nucleotidase are located on the inside surface of the tritosomal membranes.     

Membrane-bound choline acetyltransferase from human brain: Purification and properties

Choline acetyltransferase (ChAT; EC 2.3.1.6) was separated from human caudate/putamen into three fractions by successive extractions into apotassium phosphate buffer, a high salt (NaCl) buffer and a buffer containing 0.6% Triton X-100. The Triton-X-solubilized fraction is the membrane-bound ChAT (mChAT) and represents about 40% of the total ChAT. After centrifugation, mChAT was precipitated by ammonium sulfate at 35–65% saturation. The crude enzyme preparation was fractionated in turn on a DEAE-Sepharose, a hydroxylapatite and a phosphocellulose columns. Finally, mChAT was applied to a CoA-Sepharose column equilibrated with buffer containing 100 mM choline chloride and was specifically eluted with buffer containing acetyl-CoA. The presence of both substrates greatly stabilized the enzyme and ChAT was recovered almost quantitatively. The final preparation of mChAT has a specific activity of 37.2 mol of acetylcholine synthesized per min-mg protein. The purified mChAT has a pH optimum of 8.3. It migrated as two bands on SDS-PAGE with molecular weights of 67,000 and 62,000 daltons, respectively. Immunoblot autoradiography showed that an antiserum prepared previously against soluble ChAT also cross-reacted with both bands of mChAT, indicating that both forms of this enzyme are related. Furthermore, as previously reported for soluble ChAT, Fab-Sepharose chromatography could be used for the purification of mChAT and this preparation also resolved into two bands of 10% SDS gel.Special Issue dedicated to Prof. Eduardo De Robertis.     

Effect of Phospholipase C from Bacillus cereus on the Release of Membrane-Bound Choline-O-Acetyltransferase from Rat Hippocampal Tissue

Some of the enzyme choline-O-acetyltransferase (ChAT) associated with central cholinergic nerve terminals appears to be non-ionically associated with membranes. In the present study, we tested the possibility that some membrane-bound ChAT might be anchored to membranes by a phosphatidylinositol linkage by incubating rat hippocampal tissue with phospholipase C (PLC) from Bacillus cereus. The PLC selectively augmented the release of ChAT; also, the glycosylphosphatidylinositol-PLC inhibitor, zinc, blocked this increase in release. When control and PLC-treated hippocampal tissues were subjected to Triton X-114 phase separation, a procedure that separates amphiphilic from hydrophilic proteins, the detergent-soluble, membrane-bound fraction of tissue ChAT appeared to be the source of the ChAT released by PLC into the incubation medium. Zinc also blocked the temperature-dependent release of ChAT, but not lactic dehydrogenase, from hippocampal tissue. Extracellular membrane-bound ChAT appeared to be the source of the ChAT released by a low exogenous concentration of PLC, as well as that released by a temperature-dependent process during tissue incubation. Phosphatidylinositol-specific PLC from Bacillus thuringiensis released ChAT, but not lactic dehydrogenase, from a crude synaptosomal fraction prepared from rat hippocampal tissue. These results suggest that some of the membrane-bound ChAT in rat hippocampal tissue may be extracellular and anchored to the membrane by phosphatidylinositol, and also that an endogenous factor in hippocampal tissue may function to remove this extracellular ChAT from the membrane.     

Immunological,isoelectric, hydrophobic and molecular weight differences between soluble and ionically membrane-bound fractions of choline-o-acetyltransferase prepared from mouse and rat brain

Choline-O-acetyltransferase (EC 2.3.1.6; ChAT) was prepared from synaptosomal fractions (P₂) of mouse and rat brain in the presence of proteolytic inhibitors by the method of Gray and Whittaker (1962) as modified by (Salehmoghaddam and Collier, 1976). The P₂ fraction was hypo-osmotically shocked with glass distilled water and centrifuged to separate the cytoplasmic (S₃) and vesicle-bound (P₃) fractions. Fraction S₃ was saved for ChAT assay and compared with the ChAT fraction eluted from the P₃ by salt at a pH 7.4 or by detergent (Benishin and Carroll, 1983). These three fractions of ChAT were then compared by molecular weights, isoelectric points, immunoblotting with monoclonal or polyclonal antibodies and hydrophobicity. The results show that the S₃ fraction of ChAT has a molecular weight of 66 K_d, whereas the ionically-bound fraction of ChAT has a molecular weight of 73–78 K_d. SDS-PAGE of these two ChAT fractions followed by immunoblotting revealed the presence of two immunoreactive bands at 28–29 K_d and 50–51 K_d for the ionically bound ChAT fraction. Conversely, none of these antibodies immunostained any protein bands for the S₃ ChAT fraction even though one monoclonal antibody had been prepared against this ChAT fraction and the S₃ ChAT fraction had a similar specific activity prior to SDS-PAGE as did the salt solubilized ChAT fraction. However, anti-ChAT monoclonal antibody MB16 binds the native S₃ ChAT fraction in the co-precipitation assay.The S₃ fraction of ChAT had only one isoelectric point at pH 7.8, whereas the ionically bound and detergent soluble ChAT fractions had two isoelectric points at pH 8.1–8.15 and 7.45–7.5. The S₃ ChAT fraction also differed in hydrophobicity from the other two ChAT fractions. These differences between the S₃ and salt soluble ChAT fractions were not obviated by addition of Triton X-100 and thus could not be attributed to the association of lipids with either of the fractions. We conclude that the water soluble fraction of ChAT in central nerve terminals differs in its physical properties and its subcellular location from that which ionically binds to membranes.     

SOLUBLE AND MEMBRANE BOUND CARBONIC ANHYDRASES FROM RAT CNS: REGIONAL DEVELOPMENT

Abstract— The distribution of the soluble, membrane bound and myelin carbonic anhydrase in different regions of the rat CNS was examined as a function of age. A neuraxial progression from spinal cord to upper brain stem was observed for all three enzyme fractions in the 90 day old rat: upper brain stem > lower brain stem and cerebellum > spinal cord. The membrane bound fraction accounted for close to 60% of the total carbonic anhydrase in all regions except the cerebellum where it accounted for only 40%. The developmental pattern of the total membrane bound and soluble fractions were virtually parallel in all regions studied suggesting that they are derived from a common enzyme pool. The myelin enzyme accounts for a small but significant part of the membrane bound fraction and is present at adult levels by 16 days of age indicating it is an early and specific myelin component.     

Solubilization and some properties of a semicarbazide-sensitive amine oxidase in brown adipose tissue of the rat.

A semicarbazide-sensitive clorgyline-resistant amine oxidase (SSAO) was solubilized from membrane fractions of rat brown adipose tissue by the non-ionic detergent, Triton X-100. Alteration of ionic strength or addition of chelating agents alone failed to release the enzyme from its membrane. Lipid-depletion led to loss of enzyme activity and alteration of substrate affinity. Over 80% of the activity of the solubilized enzyme was found in gel filtration fractions corresponding to an Mr of between 160 000 and 180 000. The glycoprotein nature of SSAO was established from affinity chromatography with either immobilized concanavalin A or Lens culinaris lectin. Elution of over 50% SSAO activity from the lentil lectin was achieved with 0.25M-alpha-methyl D-mannoside to give 80-90-fold purification of the enzyme. Irradiation inactivation gave a value for Mr of around 183 000 for both soluble and membrane-bound SSAO. Substrate affinity and inhibitor sensitivity of the enzyme were unaltered by solubilization. The copper-chelating agent, diethyldithiocarbamate, did not affect the enzyme, shedding doubt on the suggestion that SSAO is a copper-requiring enzyme. The significance of these findings in relation to the nature of SSAO and to its disposition within the cell membrane is discussed.     

Developmental Differences Between Soluble and Membrane-Bound Fractions of Choline-O-Acetyltransferase in Neonatal Mouse Brain

Three fractions (one soluble and two membrane-bound) of choline acetyltransferase (ChAT) isolated from a nerve ending fraction of mouse forebrain, which have previously been reported to differ in several biochemical and physical aspects, were also found to differ in their rates of postnatal development. At 2 days of age, the activity in all three fractions was very low. Sodium phosphate buffer-soluble (cytoplasmic) ChAT activity increased significantly by 8 days of age, whereas the ChAT activity of the two membrane-bound fractions (NaCl- and Triton-soluble) did not increase until 13 days of age. These results suggested that the differences observed between the three fractions of ChAT prepared from mouse brain are not solely artifacts of the isolation procedure.     

Changes in Hexokinase Isoenzymes in Regions of Rat Brain During Insulin-Induced Hypoglycemia

Activities of hexokinase isoenzymes were determined during insulin-induced hypoglycemia in soluble and total particulate fractions from three regions of rat brain. Type I hexokinase isoenzyme activity showed a small decrease in both soluble and particulate fractions from the cerebral hemispheres. In cerebellum and brain stem, however, Type I isoenzyme showed a decrease only in the soluble fraction. A significant increase was observed in hexokinase Type II isoenzyme from both the fractions, in all the three brain regions 1 h after insulin administration.     

Characterization of a lipid-associated gonadotropin receptor from the luteinized rat ovary

Gonadotropin receptors which bind luteinizing hormone (lutropin) and human chorionic gonadotropin (hCG) in the ovaries of immature female rats showed a 30-fold increase after treatment of animals with pregnant mare serum gonadotropin (PMSG) and hCG. This marked induction of lutrophin/hCG receptors in the rat ovary was not accompanied by a change in binding affinity for labeled hCG. Such luteinized ovaries have been found consistently to contain a small proportion of soluble receptor sites, which comprised about 5% of the total receptor population. The soluble receptor sites were present in the floating lipid fraction of the 360 000 × g supernatant of homogenate prepared from luteinized ovaries, and could not be detected in similar fractions prepared from interstitial cells or homogenates of the normal rat testis.The physico-chemical properties of the spontaneously soluble ovarian receptors were similar to those derived for detergent-solubilized receptors prepared by extraction of particulate ovarian binding fractions with Triton X-100. The affinity constant to the soluble ovarian receptor sites for [¹²⁵I]hCG was 0.70 · 10¹⁰ M^?1, and that of the receptors solubilized by Triton X-100 was 0.72 · 10¹⁰ M^?1. The sedimentation pattern of the soluble receptors during sucrose density gradient centrifugation showed extensive aggregation into rapidly sedimenting forms. However, centrifugation of the cytosol receptor in the presence of Triton X-100 gave a single 6.5 S component, corresponding to the solubilized receptors previously characterized in detergent extracts of the rat ovary and testis.The pesence of a spontaneously soluble lutropin/hCG receptor in ovarian cytosol fractions suggests that rapid synthesis and assembly of receptors in ovaries of PMSG-hCG-treated rats is accompanied by increased production of cytoplasmic receptor precursors; alternatively, this receptor population may represent a fraction that has been internalized or processed as during receptor turnover in the cell membrane.     

Pancreatic beta cells express two autoantigenic forms of glutamic acid decarboxylase, a 65-kDa hydrophilic form and a 64-kDa amphiphilic form which can be both membrane-bound and soluble

The 64-kDa pancreatic beta-cell autoantigen, which is a target of autoantibodies associated with early as well as progressive stages of beta-cell destruction, resulting in insulin-dependent diabetes (IDDM) in humans, has been identified as the gamma-aminobutyric acid-synthesizing enzyme glutamic acid decarboxylase. We have identified two autoantigenic forms of this protein in rat pancreatic beta-cells, a Mr 65,000 (GAD65) hydrophilic and soluble form of pI 6.9-7.1 and a Mr 64,000 (GAD64) component of pI 6.7. GAD64 is more abundant than GAD65 and has three distinct forms with regard to cellular compartment and hydrophobicity. A major portion of GAD64 is hydrophobic and firmly membrane-anchored and can only be released from membrane fractions by detergent. A second portion is hydrophobic but soluble or of a low membrane avidity, and a third minor portion is soluble and hydrophilic. All the GAD64 forms have identical pI and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results of pulse-chase labeling with [35S]methionine are consistent with GAD64 being synthesized as a soluble protein that is processed into a firmly membrane-anchored form in a process which involves increases in hydrophobicity but no detectable changes in size or charge. All the GAD64 forms can be resolved into two isoforms, alpha and beta, which differ by approximately 1 kDa in mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis but are identical with regard to all other parameters analyzed in this study. GAD65 has a shorter half-life than the GAD64 forms, remains hydrophilic and soluble, and does not resolve into isomers. Comparative analysis of the brain and beta-cell forms of GAD show that GAD65 and GAD64 in pancreatic beta-cells correspond to the larger and smaller forms of GAD in brain, respectively. The expression of different forms and the flexibility in subcellular localization of the GAD autoantigen in beta-cells may have implications for both its function and autoantigenicity.     

Characterization of Postmortem Human Brain Proteins by Two-Dimensional Gel Electrophoresis

Abstract: The proteins of membrane and cytosol fractions from frozen human postmortem brain were analyzed by two-dimensional gel electrophoresis (isoelectric range: 5.1–6.0) and both Coomassie-blue and ammoniacal silver staining. Cytosol preparations were analyzed from six different postmortem brains from patients with various neurologic diagnoses and immediate causes of death. Intervals between death and brain freezing (−70^oC) ranged from 2 to 20 h. The vast majority of proteins detected in these cytosol fractions had identical molecular weights and isoelectric points in each of six human brains examined. However, in some tissue samples tubulin was either quantitatively decreased or undetectable. The possibility that this partial or complete depletion of tubulin was related to postmortem interval and/or brain freezing was studied using rat forebrain tissue. Rat brain incubated at room temperature for up to 24 h did not reproduce the changes seen in the region of human cytosol tubulin. However, other changes seen in the two-dimensional electrophoretic pattern of rat cytosol proteins did relate to postmortem interval, brain freezing, or both. Rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum were prepared from three human brains, with highly reproducible two-dimensional patterns. Protein analysis of these membrane fractions revealed that human RER contained significant amounts of tubulin, in contrast to rat RER which contained no detectable tubulin. This discrepancy was elucidated by allowing rat brains to remain at room temperature for 24 h before freezing; gels of rat RER prepared from this tissue showed that tubulin subunits were present.     

The isolation and structure of membrane lipid rafts from rat brain

The action of detergents in the isolation of detergent-resistant membrane fractions from rat brain is reported. Triton X-100 treatment of whole rat brain homogenate at 4 degrees C produced detergent-resistant membranes with a density of 1.07g/ml compared with Brij96 where the density of the membrane was only 1.05g/ml. The DRM fractions isolated using Triton X-100 are considerably heavier than those isolated from homogenates treated with Brij96. The major polar lipid composition of DRMs derived from Brij96 treated homogenates have a higher proportion of aminophospholipids compared with choline phospholipids than Triton X-100 derived DRMs; this may indicate that DRMs from Brij96 treated homogenates are more closely related to the parent membrane in lipid composition. Solubilization by Triton X-100 at higher temperatures resulted in the appearance of a second detergent-resistant membrane fraction distinctly lighter in density than the membrane recovered at density 1.07g/ml. Analysis of phospholipid composition of the brain homogenate during detergent treatment for up to 30min at 37 degrees C showed a decreasing proportion of sphingomyelin. Treatment of homogenates at 37 degrees C appears to activate phospholipases/sphingomyelinases that may alter the lipid content of isolated DRMs. The presence of K+/Mg2+ with Brij96 treatment results in DRM fractions with significantly thicker bilayers and of larger vesicle diameter than DRMs isolated from either Triton X-100 or Brij96 treated homogenates in the absence of cations.     

Membrane-associated forms of peptidylglycine alpha-amidating monooxygenase activity in rat pituitary. Tissue specificity

Membrane-associated peptidylglycine alpha-amidating monooxygenase (PAM) activity was investigated in rat anterior and neurointermediate pituitary tissues and in pituitary AtT-20/D-16v and GH3 cell lines. A substantial fraction of total pituitary PAM activity was found to be membrane-associated. Triton X-100, N-octyl-beta-D-glucopyranoside, and Zwittergent were effective in solubilizing PAM activity from crude pituitary membranes. The distribution of enzyme activity between soluble and membrane-associated forms was tissue-specific. In the anterior pituitary lobe and pituitary cell lines, 40-60% of total PAM activity was membrane-associated while only 10% of the alpha-amidating activity in the neurointermediate lobe was membrane-associated. Soluble and membrane-associated forms of PAM shared nearly identical characteristics with respect to copper and ascorbate requirements, pH optima, and Km values. Upon subcellular fractionation of anterior and neurointermediate pituitary lobe homogenates on Percoll gradients, 12-18% of total PAM activity was found in the rough endoplasmic reticulum/Golgi fractions and 42-60% was localized to secretory granule fractions. For both tissues, membrane-associated PAM activity was enriched in the rough endoplasmic reticulum/Golgi pool, whereas most of the secretory granule-associated enzyme activity was soluble.     

Soluble and membrane-bound forms of dopamine beta-hydroxylase are encoded by the same mRNA.

A full length cDNA clone for bovine dopamine beta-hydroxylase was expressed in rat pheochromocytoma PC12 cells by stable transformation of this cell line with a plasmid expression vector. The recombinant protein exhibited dopamine beta-hydroxylase enzyme activity and was found in both the soluble and membrane fractions of the secretory vesicle. Immunoprecipitation of cell extracts from recombinant cell lines with dopamine beta-hydroxylase antisera followed by fractionation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two subunits, which migrated to relative molecular masses of 76 and 78 kDa. The recombinant protein co-fractionated with neurotransmitter when subcellular structures were separated by sucrose gradient density centrifugation, suggesting that the protein was routed to the secretory vesicles. Dopamine beta-hydroxylase immunoreactivity in those sucrose gradient fractions presumed to contain secretory vesicles was resistant to treatment with trypsin unless the nonionic detergent Triton X-100 was also present to disrupt membrane structure. The 76- and 78-kDa isoform were each found in both the membrane and soluble fractions of the secretory vesicle. Treatment of cultured cells with nerve growth factor or 8-(4-chlorophenylthio)-cyclic AMP alters the relative distribution of the subunits such that the 76-kDa form predominates. The subcellular distribution of a dopamine beta-hydroxylase cDNA clone lacking the first 16 nucleotide residues was also determined. The predicted amino acid sequence of the protein encoded by this cDNA would be deleted of the first 13 residues of the signal sequence, which were reported to be present in the membrane-bound form, but not the soluble form, of native dopamine beta-hydroxylase (Taljanidisz, J., Stewart, L., Smith, A. J., and Klinman, J. P. (1989) Biochemistry 28, 10054-10061). Immunoprecipitable dopamine beta-hydroxylase derived from expression of the deleted cDNA was found in both the membrane-bound and soluble fractions of the secretory vesicle. These experiments demonstrate that the membrane-bound and soluble forms of dopamine beta-hydroxylase are derived from one primary translation product, which is also sufficient to produce enzyme activity. In addition, the amino-terminal amino acids encoding residues 1-13, which compose the hydrophilic region of the signal sequence, are not necessary for the biogenesis of membrane-bound dopamine beta-hydroxylase.     

Evidence for Membrane-Associated Choline Kinase Activity in Rat Striatum

The distribution of choline kinase (EC 2.7.1.32) activity was investigated in subcellular fractions of rat striatum. Enzyme activity in the crude mitochondrial fraction, determined after dissolution in Triton X-100, was 5.90 mumol/g initial wet weight/h. When a crude mitochondrial preparation was hypoosmotically shocked and fractionated, followed by the addition of Triton X-100, choline kinase activity in the soluble and particulate fractions was 4.58 and 1.40 mumol/g initial wet weight/h, respectively. Enzyme activity in the particulate fraction was not detected in the absence of Triton X-100 or in the presence of NaCl (up to 1.5 M). Subcellular enzyme markers indicated that the membrane-associated activity was not attributable to mitochondrial or microsomal contamination. Kinetic analysis of the activity of soluble and membrane-solubilized choline kinase indicated Km values of 0.74 mM and 0.68 mM, respectively. Results indicate that choline kinase activity may be measured in both the soluble and the particulate fractions of rat striatum, the latter most likely involving enzyme associated with membrane through hydrophobic or covalent interactions. The specific function of the membrane-associated enzyme has not yet been determined.     

Identification of molecular aggregates containing glycoproteins III, J, K (carboxypeptidase H), and H (Kex2-related proteases) in the soluble and membrane fractions of adrenal medullary chromaffin granules.

An investigation of the molecular properties of glycoprotein III has shown this to be a major component of molecular aggregates present in the membrane and soluble fractions of secretory vesicles from bovine adrenal medulla. These aggregates also contain components identified as glycoproteins H, J, and K which are molecular forms of Kex2-related proteases (glycoprotein H) and carboxypeptidase H (glycoprotein components J and K) and which have functions concerned with the processing of prohormones. A number of experiments indicated that these glycoproteins were associated. These components were coimmunoprecipitated from the soluble and membrane fractions of chromaffin granules. Purification of soluble glycoprotein III using wheat germ agglutinin-Sepharose resulted in the recovery of similar proportions of glycoproteins H, J, and K and gel filtration of the eluted material in combination with immunoprecipitation revealed the presence of heteroaggregates containing all of the glycoproteins. Similar results were obtained following octylglucoside solubilization of chromaffin granule membranes. Glycoprotein components III, H, J, and K were also found to have identical distributions following fractionation of chromaffin granule membranes with Triton X-114. It was concluded that the aggregates seen in the soluble fraction reflect an association of these components in the chromaffin granule membrane. This raises the possibility that these interactions are important for the targetting of these glycoproteins to secretory granules.