Dopamine-β-Hydroxylase: Structural Comparisons of Membrane-Bound Versus Soluble Forms from Adrenal Medulla and Pheochromocytoma

Dopamine-beta-hydroxylase (DBH) in membrane-bound (mDBH) and water-soluble (sDBH) forms was isolated from chromaffin granules of bovine adrenal medullae and a human pheochromocytoma tumor. sDBH was purified by concanavalin A-agarose column chromatography followed by DEAE-Sepharose column chromatography. The final bovine preparation had a specific activity of 16.27 IU/mg; the human preparation had a specific activity of 9.16 IU/mg. mDBH was isolated in enzymatically inactive form by preparative polyacrylamide gel electrophoresis. The proteins were subjected to amino acid analysis, as well as digestion with trypsin, followed by separation of the resulting peptides by two-dimensional TLC/electrophoresis. No intraspecies differences between sDBH and mDBH were found from comparisons of amino acid composition or peptide maps. Thus the basis of the difference between sDBH and mDBH cannot easily be explained by differences in primary structure, within the resolution of these techniques.     

Purification of the insulin receptor from human placental membranes

Insulin receptors were purified from human placental microsomal membranes by solubilisation with Triton X-100 followed by Sepharose 6B chromatography, phosphate gradient elution from hydroxyapatite and affinity chromatography on concanavalin A-Sepharose. 2000-fold purification was achieved with 63% overall recovery. The purified receptor gave a single band on 3.75% polyacrylamide (0.1% Triton X-100) gel electrophoresis. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis there was a major band at 75,000 and a minor band at 80,000 daltons. The purified receptor rechromatographed on Sepharose 6B with an apparent molecular weight of 300,000.     

Complete purification of choline kinase from rat kidney and preparation of rabbit antibody against rat kidney choline kinase

Choline kinase was purified from rat kidney to apparent homogeneity with respect to both native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme showed a minimum molecular weight of 42,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On the other hand, the molecular size of 75,000-80,000 was estimated through Sephadex G-150 gel filtration, indicating that the enzyme in rat kidney exists most likely in a dimeric form. Specific antibody was raised in rabbit against the highly purified rat kidney choline kinase protein, then immunochemical cross-reactivity was investigated between rabbit antiserum and choline kinase preparations from various rat tissues. The antiserum inhibited choline kinase activity almost completely in the crude preparation not only from kidney but also from lung, intestine, and normal untreated liver cytosol, but it could inhibit only partially the activity from either 3-methylcholanthrene- or carbon tetrachloride-induced rat liver cytosol. The overall results demonstrated that, although choline kinase protein appears to exist in multiple forms in rat tissues, most of them are immunochemically identical, and that either 3-methylcholanthrene- or carbon tetrachloride-inducible form(s) of choline kinase in rat liver could be quite different from a form or forms existing in normal untreated rat liver cytosol.     

Purification of soluble acetylcholinesterase from Japanese quail brain by affinity chromatography.

The purification of a soluble acetylcholinesterase from Japanese quail brain using affinity chromatography on concanavalin A-Sepharose and edrophonium-Sepharose is described. The affinity matrix was synthesized by coupling an inhibitor edrophonium to epoxy-activated Sepharose. Acetylcholinesterase was purified 10,416-fold with a specific activity of 2500 U/mg protein. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol gave only one band with a molecular weight of 62.5 kDa. The molecular weight of the purified acetylcholinesterase was estimated to be 245.5 kDa by gel chromatography on Sephacryl S-200 under nondenaturing conditions. Based on the molecular weight obtained by both SDS-PAGE and gel filtration the purified acetylcholinesterase was assumed to be a tetrameric form.     

Mouse bone collagenase inhibitor: purification and partial characterization of the inhibitor from mouse calvaria cultures

The organ culture of neonatal mouse calvaria produced both collagenase and collagenase inhibitor. The inhibitor was purified by a series of column chromatographies: DEAE-cellulose and CM-cellulose ion-exchange chromatography, concanavalin A-Sepharose and heparin-Sepharose affinity chromatography, and finally by Sephacryl S-200 gel filtration. The purified inhibitor migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular mass of 28,000. The inhibitor was purified 140-fold to a specific activity of 163 units/mg with a yield of 18% over the first step of the purification by DEAE-cellulose chromatography. The inhibitor stained positively for carbohydrate with periodic acid-Schiff's reagent indicating, in conjunction with its affinity to concanavalin A, that the inhibitor is a glycoprotein. In addition to mouse bone collagenase, this inhibitor also inhibited chick bone, rat bone, rabbit corneal, and human gingival collagenase, but did not inhibit bacterial collagenase.     

Affinity purification and some properties of nucleoside diphosphatase from rat liver cytosol.

Nucleosidediphosphatase (nucleosidediphosphate phosphohydrolase, EC 3.6.1.6) of rat liver cytosol was purified up to 336--fold by the procedure including affinity chromatographies of concanavalin A- and alanine-Sepharose. The final purified enzyme showed a single protein band upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Its native form was found to be a tetramer with molecular weight of 120 000 which consists of subunit with molecular weight of 30 000. The enzyme was found to be a glycoprotein on the basis of its chromatographic behaviour with concanavalin A-Sepharose and positive staining with periodate-Schiff reaction in polyacrylamide gels. Furthermore, the two molecular forms with isoelectric points of 4.7 and 5.0 were demonstrated by electrofocusing, though they did not show any significant difference with respect to their catalytic properties.     

Affinity purification and some molecular properties of human liver alkaline phosphatase.

Alkaline phosphatase from human liver was purified to homogeneity. The purification procedure included solubilization with butanol, fractionation with acetone, and chromatography on concanavalin A-Sepharose, DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex. Purity was established by standard and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The isoelectric point of the protein was determined to be 4.0. Sephadex-gel filtration gave a mol.wt. of 146000, although a higher value was obtained in the presence of 100mM-NaC1. The subunit mol.wt. 76700, was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Neuraminidase treatment resulted in two enzyme-activity bands on isoelectric-focused gels with isoelectric points of 6.6 and 6.8. The desialylated enzyme gave only one protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with a subunit molecular weight indistinguishable from that of the non-neuraminidase-treated protein. The desialylated enzyme was more readily denatured by sodium dodecyl sulphate in the presence of mercaptoethanol than was the native enzyme.     

Neurons and neuroendocrine cells contain chromogranin: detection of the molecule in normal bovine tissues by immunochemical and immunohistochemical methods

Gel-eluted bovine chromogranin (CG), the 75,000 dalton acidic protein abundantly present in adrenal chromaffin granules, was used as immunogen to prepare anti-CG serum. The specificity of the antiserum was demonstrated in immunoblots of electrophoresed bovine CG and in immunohistochemical studies of bovine adrenal medulla. In the immunoblots, the predominant immunoreactive band had a molecular weight of 75,000 daltons. Bands with a higher or lower molecular weight were also immunoreactive and may represent CG precursors or breakdown products. In the adrenal gland, only adrenal chromaffin cells contained CG immunoreactivity. Immunoblots and immunohistochemistry were also used to characterize the distribution of CG in bovine tissues. CG was expressed by cells of the diffuse neuroendocrine system (DNS) including: adrenal chromaffin cells, enterochromaffin cells, pancreatic islet cells, cells of the adenohypophysis, thyroid C cells, parathyroid cells, and submandibular gland. CG was also seen in four locations not previously recognized to express this antigen: thymic epithelial cells, neurons, the inner segment of rods and cones, and the submandibular gland. We demonstrate a wider distribution of CG than previously recognized and that the molecule detected in tissue by immunohistochemistry is indeed CG. We conclude that CG is expressed by neurons, cells of the DNS, and by a few other cells that may or may not be related to the DNS. The antiserum described here should prove valuable in developing an understanding of the function(s) of CG.     

Purification of (Ca2+-Mg2+)-ATPase from rat liver plasma membranes

The Ca2+-stimulated, Mg2+-dependent ATPase from rat liver plasma membranes was solubilized using the detergent polyoxyethylene 9 lauryl ether and purified by column chromatography using Polybuffer Exchanger 94, concanavalin A-Sepharose 4B, and Sephadex G-200. The molecular weight of the enzyme, estimated by gel filtration in the presence of the detergent on a Sephadex G-200 column, was 200,000 +/- 15,000. The enzyme was purified at least 300-fold from rat liver plasma membranes and had a specific activity of 19.7 mumol/mg/min. Polyacrylamide gel electrophoresis under nondenaturing conditions of the purified enzyme indicated that the enzymatic activity correlated with the major protein band. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one major band in the molecular weight range of 70,000 +/- 5,000 was seen. The isoelectric point of the purified enzyme was 6.9 +/- 0.2 as determined by analytical isoelectric focusing. The enzyme was activated by Ca2+ with an apparent half-saturation constant of 87 +/- 2 nM for Ca2+. Calmodulin and trifluoperazine at the concentration of 1 microgram/ml and 100 microM, respectively, had no effect on the enzymatic activity.     

Phosphodiesterase from cultured tobacco cells. Physical and chemical properties.

Phosphodiesterase isolated from suspension cultures of tobacco cells showed high affinity for concanavalin A-Sepharose and gave single superimposed bands of protein and carbohydrates on disc gel electrophoresis, suggesting that it is a glycoprotein. It contains 14% carbohydrate by weight, and has relatively high contents of basic and aromatic amino acids. Its isoelectric point is at pH 8.8, and the molecular weight of its subunits was estimated as 72 000 from a plot of the retardation coefficient on sodium dodecyl sulfate gel electrophoresis versus the molecular weight. The enzyme was catalytically active in an immobilized state on a concanavalin A-Sepharose column.     

Purification and partial characterization of a thiol proteinase activating prokallikrein from the rat kidney cortex

The rat kidney cortex contains at least three kinds of prokallikrein-activating proteinase, and among these the one with the highest molecular weight was purified by a procedure including chromatography on CM-cellulose, concanavalin A-Sepharose, organomercurial-Sepharose 4B and Sephadex G-100. The resulting preparation was apparently homogeneous, as assessed by SDS-polyacrylamide gel electrophoresis. The molecular weight was estimated to be 57,000. The optimal pH for the activation of prokallikrein by the preparation, termed activator I, was around 4.5. Activator I was inhibited by E-64, iodoacetate and leupeptin, but not by PMSF and phosphoramidon. In immunodiffusion analysis, the antiserum to activator I formed an immunoprecipitin arc with the extract from the kidney cortex or submandibular gland, but not with that from the pancreas. These results indicate that activator I is a thiol proteinase with a molecular weight of 57,000. A proteinase immunologically identical with activator I appears to be present in the submandibular gland.     

Purification of human milk bile salt-activated lipase by cholic acid-coupled sepharose 4B affinity chromatography

Sequential chromatography of human milk whey on concanavalin A—Sepharose 4B followed by cholate—Sepharose 4B yielded a bile salt-activated lipase with 150-fold purification. The lipase was not retained by concanavalin A—Sepharose 4B but was retained by the cholate—Sepharose 4B, from which it was eluted with 2% sodium cholate. The affinity chromatography procedure on cholate—Sepharose 4B was based on the specific structural requirement of the enzyme for a 7-hydroxyl group of bile salt. Sodium deoxycholate, which lacks the 7-hydroxyl group, was effective in removing the nonspecifically bound proteins without affecting the binding of the enzyme. Bile salt-activated lipase showed a single band on urea-sodium dodecyl sulfate—polyacrylamide gel electrophoresis with an apparent molecular weight of 125,000, and based on densitometric measurement accounted for 0.5–1.0% of the protein mass of human whole milk. A rabbit antiserum to the purified bile salt-activated lipase caused no inhibition of human milk lipoprotein lipase activity but completely inhibited bile salt-activated lipase activity.     

Purification and initial characterization of guinea pig testicular acrosin

Guinea pig (GP) acrosin was purified following acid extraction of testicular acetone powder, pH precipitation of the soluble extract, gel filtration on Sephadex G-100, ion-exchange chromatography on SP-Sephadex, and affinity chromatography on Concanavalin A-Sepharose. Final purification was achieved by re-chromatography on Sephadex G-100. Enzymatic activity was detected by following the hydrolysis of N-benzyloxycarbonylarginyl amide of 7-amino-4-trifluoromethylcoumarin at 37 degrees C, pH 8.0, before and after activation. GP testicular acrosin exhibited a molecular weight of 48,000 by gel filtration and 34,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Following SDS-PAGE in gels containing 0.1% gelatin, protease activity was observed to comigrate with the major protein detected by silver staining. The purified GP acrosin showed cross-reactivity with a monospecific polyclonal rabbit antiserum directed against boar sperm acrosin and exhibited reversible pH-dependent activation. The physiochemical characteristics of the purified protein, including the amino acid composition, resemble those reported for acrosins from other species.     

Purification and characterization of microsomal and lysosomal beta-glucuronidase from rat liver by use of immunoaffinity chromatography.

Rat liver beta-glucuronidase (EC 3.2.1.31), both from microsomal and lysosomal fractions, were purified about 9500-fold over the homogenate with high yield using affinity chromatography prepared by coupling purified specific immunoglobulin G against rat preputial gland beta-glucuronidase to Sepharose 2B and isoelectric focusing. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of approximately 310000. In dodecylsulfate polyacrylamide gel electrophoresis, the microsomal beta-glucuronidase showed a single band corresponding to a molecular weight of 79000, while the lysosomal beta-glucuronidase had three distinct bands which consisted of one major and two minor bands corresponding to molecular weight of 79000, 74000, and 70000, respectively. A broad pH activity curve with a single optimum at pH 4.4 was observed in both the microsomal and the lysosomal beta-glucuronidases. Immunological gel diffusion technique with rabbit antiserum against rat liver lysosomal beta-glucuronidase revealed that both enzymes had the same or quite similar antigenic determinants.     

Partial purification of a boar sperm membrane protein inducing sperm agglutinating antibody

For the development of immunological contraception, attention is being concentrated on the possibility of using a sperm membrane antigen. Boar sperm membrane was extracted with triton-X 100 and fractionated by Sephadex G-150 column chromatography. The glycosylated and nonglycosylated portions of protein peaks from the gel filtration were obtained by fractionating on concanavalin A-Sepharose and eluting the bound protein with 0.3 M methyl mannoside. A glycosylated fraction was found to induce sperm agglutinating antibodies in rabbit. The partially purified protein has a molecular weight of 30 kilodaltons, as determined by sodium dodecyl polyaccyrlamide gel electrophoresis. Further work is planned on the histochemical determination of the origin of this protein and species cross-activity of the antibody.     

Soluble guanylate cyclase from rat lung exists as a heterodimer

The soluble form of guanylate cyclase (EC 4.6.1.2) from rat lung has been purified to homogeneity by a one-step immunoaffinity chromatographic procedure. The purified soluble guanylate cyclase has specific activities of 432 and 49.1 nmol of cyclic GMP formed per min/mg protein with manganese and magnesium ions as a cofactor, respectively. This represents a purification of approximately 2,000-fold with a 50% recovery. The native enzyme has a molecular weight of 150,000 and a Stokes radius of 4.8 nm as determined on Spherogel TSK-G3000SW gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results in two protein-staining bands with molecular weights of 82,000 and 70,000. The purified soluble guanylate cyclase was also subjected to native polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, ion exchange chromatography, and GTP-agarose affinity chromatography. These additional purification procedures confirmed the presence of a single protein peak coincident with enzyme activity. The two subunits separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were shown to have different primary structures by immunoblotting with monoclonal and polyclonal antibodies prepared against purified soluble guanylate cyclase and by peptide mapping with papain or Staphylococcus aureus V8 protease treatment. These data demonstrate that soluble guanylate cyclase purified from rat lung is a heterodimer composed of 82,000- and 70,000-dalton subunits with different primary structures.     

Purification and characterization of alkaline phosphatase from cultured rat ascites hepatoma cells.

Alkaline phosphatase of cultured rat ascites hepatoma cells has been purified by butanol extraction, DEAE-cellulose column chromatography, gel filtration through Sephadex G-200, concanavalin A-Sepharose affinity chromatography, and polyacrylamide gel electrophoresis. Affinity chromatography confirmed the glycoprotein nature of alkaline phosphatase from cultured rat ascites hepatoma cells. Electrophoresis on polyacrylamide gels of various concentrations indicated a molecular weight of 290,000. The molecular weight of the subunit was estimated to be 72,000 by SDS-polyacrylamide gel electrophoresis. These findings suggest that alkaline phosphatase of cultured rat ascites hepatoma cells is a tetramer with a subunit molecular weight of 72,000.     

Cell-free synthesis of putative neurophysin precursors from rat and mouse hypothalamic poly (A)-RNA.

Poly(A)-containing RNA has been isolated from rat and mouse hypothalamic tissue and used to direct the synthesis of polypeptides in cell-free systems derived from wheat germ extract and rabbit reticulocyte lysate in the presence of [³⁵S]-L-cysteine and [³H]-L-proline. Translation products were subjected to immunoprecipitation using an antiserum to rat neurophysin proteins. Following purification of the immunoprecipitates by protein A-Sepharose chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a single polypeptide species of molecular weight 17,500 derived from both cell-free systems.     

Production and application of a polyclonal peptide antiserum for universal detection of StAR protein

We report production of a polyclonal antibody against the StAR (steroidogenic acute regulatory) protein of steroidogenic cells and immunohistochemistry (IHC) staining of bovine adrenal gland tissue available in paraffin block. The epitope-specific polyclonal antibody was produced in a rabbit immunized against a synthetic 26 amino acid peptide (82AMQRALGILKDQEGWKKESRANGDE107) derived from the coding sequences reported for the bovine StAR gene (Gene Bank Accession No. Q28918). Western blots were developed using the StAR-specific peptide antiserum with an alkaline-phosphatase-conjugated anti-rabbit IgG second antibody chromogenic system. The antiserum was found to be highly specific for StAR, which exhibited an estimated molecular weight of about 30 KDa for all species analyzed. Finally, the peptide antiserum was successfully employed to localize StAR protein by immunohistochemical staining of thin sections prepared from bovine adrenal gland tissue. This study is the first to report a polyclonal peptide antiserum that apparently recognizes native StAR protein, regardless of the species of origin. The successful production of the antibody has provided a useful tool for studying regulation of StAR protein.     

Affinity purification and properties of cathepsin-E-like acid proteinase from rat spleen.

A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin-Sepharose 4B and concanavalin A-Sepharose 4B, chromatography on Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing. A purification of 4200-fold over the homogenate was achieved and the yield was 11%. The purified enzyme appeared to be homogeneous on electrophoresis in polyacrylamide gels. The isoelectric point of the enzyme was determined to be 4.1-4.4. The enzyme hydrolyzed hemoglobin with a pH optimum of about 3.1. The molecular weight of the enzyme was estimated to be about 90000 by gel filtration on Sephadex G-100. In sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified enzyme showed a single protein band corresponding to a molecular weight of about 45000. The hydrolysis of bovine hemoglobin by the enzyme was much higher than that of serum albumin. Various synthetic and natural inhibitors of the enzyme were tested. The enzyme was inhbited by Zn2+, Fe3+, Pb2+, cyanide, p-chloromercuribenzoate, iodoacetic acid and pepstatin, whereas 2-mercaptoethanol, phenylmethyl-sulfonyl fluoride and leupeptin showed no effect.