alpha-Galactosidases II, III and IV from seeds of Trifolium repens. Purification, physicochemical properties and mode of galactomannan hydrolysis in vitro.

Five alpha-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) were identified by chromatography and by their different electrophoretic mobilities, in the germinated seeds of Trifolium repens (white clover). alpha-Galactosidases II, III and IV were purified to homogeneity, with increases in specific activity of approx. 4600-, 4900- and 2800-fold respectively. The enzymes were purified by a procedure that included (NH4)2SO4 precipitation, hydroxyapatite, Sephadex G-75 and DEAE-cellulose chromatography, and preparative polyacrylamide-gel disc electrophoresis. The purified enzymes showed a single protein band, corresponding to the alpha-galactosidase activity, when examined by polyacrylamide-gel electrophoresis. The pH optimum was determined with o-nitrophenyl alpha-D-galactoside and the galactomannan of T. repens To as substrate. All three enzymes are highly thermolabile. Hydrolysis of oligosaccharides and galactomannans was examined, including two galactomannans from the germinated seed of T. repens (T24 and T36). By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the mol.wts. of the multiple forms of enzyme were found to be identical (41 000).     

Pyrimidine biosynthesis in parasitic protozoa: purification of a monofunctional dihydroorotase from Plasmodium berghei and Crithidia fasciculata

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamoyl-L-aspartate (L-CA) to L-5,6-dihydroorotate (L-DHO), which is the third enzyme in de novo pyrimidine biosynthesis. The enzyme was purified from two parasitic protozoa, Crithidia fasciculata (about 16,000-fold) and Plasmodium berghei (about 790-fold). The C. fasciculata enzyme had a native molecular weight (Mr) of 42,000 +/- 5000, determined by gel filtration chromatography, and showed a single detectable protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Mr 44,000 +/- 3000. The DHOase from P. berghei had a native molecular weight of 40,000 +/- 4000 and a subunit molecular weight on SDS-PAGE of 38,000 +/- 3000. The DHOase from both parasites, in contrast to the mammalian enzyme which resides on a trifunctional protein of the first two enzymes of the pathway, carbamoyl-phosphate synthase and aspartate transcarbamylase, is monomeric and has no oligomeric structure as studied by chemical cross-linking with dimethyl suberimidate. The rate of cyclization of L-CA by the C. fasciculata enzyme was relatively high at acidic pH, decreasing to a very low rate at alkaline pH. In contrast, the rate of ring cleavage of L-DHO was very low at acidic pH and increased to a higher rate at alkaline pH. These pH-activity profiles gave an intersection at pH 6.6. The Km and kcat for L-CA were 0.846 +/- 0.017 mM and 39.2 +/- 6.4 min-1, respectively; for L-DHO, they were 25.85 +/- 2.67 microM and 258.6 +/- 28.5 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-purification of galactosyltransferases from chick-embryo liver.

Two galactosyltransferases with nearly identical Mr values were purified 5000-7000-fold from microsomal membranes of chick-embryo livers by using several affinity columns. One enzyme transfers galactose from UDP-galactose to form a beta-(1----4)-linkage to GlcNAc (N-acetylglucosamine) or AsAgAGP [asialo-agalacto-(alpha 1-acid glycoprotein)]. The other enzyme forms a beta-(1----3)-linkage to AsOSM [asialo-(ovine submaxillary mucin)]. Both enzymes were solubilized (85%) from a microsomal pellet by using 1% Triton X-100 in 0.1 M-NaCl. The supernatant activities were subjected to DEAE-Sepharose chromatography and four affinity columns: UDP-hexanolamine-Sepharose, alpha-lactalbumin-Sepharose, GlcNAc-Sepharose and either AsAgAGP-Sepharose or AsOSM-Sepharose. The AsAgAGP enzyme [(1----4)-transferase] and the AsOSM enzyme [(1----3)-transferase] behave identically on the DEAE-Sepharose and UDP-hexanolamine-Sepharose columns, and similarly on the alpha-lactalbumin-Sepharose column. Final separation of the two enzymes, however, could only be achieved on affinity columns of their immobilized respective acceptors. Both purified enzymes migrate as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after silver staining, and both have an apparent Mr of 68 000. The enzymes were radioiodinated and again subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Radioautographic analyses showed only one, intensely radioactive, band. Activity stains performed for both transferases after cellulose acetate electrophoresis indicate that, with this system too, both activities have identical mobilities, and co-migrate, as well, with the major, silver-stained, protein band. Kinetic studies with the purified enzymes show that the Km value for GlcNAc, for the (1----4)-transferase, is 4mM; for the (1----3)-transferase the Km value for AsOSM is 5mM, in terms of GalNAc (N-acetylgalactosamine) equivalents. Both enzymes have a Km value of 25 microM for UDP-galactose.     

The lower molecular weight acid phosphatase from the frog liver: isolation of homogeneous AcPase III and IV representing glycoforms with different bioactivity

1. AcPase III and AcPase IV, the major enzyme forms of the LMW AcPase of the frog (Rana esculenta) liver were resolved and purified to homogeneity. 2. AcPase III and IV showed a single protein band on SDS-PAGE corresponding to a mol. wt (Mr) of about 35,000. The Mr of the native enzyme forms were 33,200 (gel electrophoresis) and 38,200 +/- 5000 (gel filtration). This indicates that they are monomeric proteins sharing the same protein molecule. 3. AcPase III and IV differ essentially in thermostability and the activating effect of ConA binding. 4. AcPase III and IV are considerably activated with DTT but they differed markedly by the extent of this activation and the accompanying changes of their pH-activity curves. 5. It is suggested that the frog liver LMW AcPase represents a set of glycoforms whose different bioactivity is determined by the redox states of their essential cysteine residues.     

Insulin-like growth factor I/somatomedin-C: a rapid isolation procedure with FPLC

Insulin-like growth factor I (IGF I)/somatomedin-C (SM-C) was purified from lyophilized human serum by acid-ethanol extraction. The extract was precipitated with acetone-ethanol. The precipitate was purified by Sephadex G-50 chromatography. The protein peak within a molecular weight range of 5000-10 000 was further purified with FPLC-reversed phase chromatography using a Pep RPC HR 5/5 column (Pharmacia) with a solvent system of acetonitrile (CH3CN) and 0.1% trifluoroacetic acid (TFA) in water. The purification of IGF I was monitored by radioimmunoassay for SM-C. Purity was established by analytical isoelectric focusing and by SDS polyacrylamide gel electrophoresis. Analytical isoelectric focusing showed one single protein band with an apparent pI of 8.3 +/- 0.1. SDS polyacrylamide gel electrophoresis showed also one single protein band with an apparent molecular weight of 7000. Biological activity was demonstrated by measuring the (3H)thymidine incorporation into DNA of cultured arterial smooth muscle cells.     

Purification and properties of an alpha-D-galactoside galactohydrolase from the seeds of Trifolium repens (white clover).

Five alpha-D-galactosidases (alpha-D-galactoside galactohydrolase; EC 3.2.1.22) have been identified by chromatography and polyacrylamide-disc-gel electrophoresis in the germinated seeds of Trifolium repens (white clover). alpha-Galactosidase I has been purified to homogeneity with an approx. 2000-fold increase in specific activity. The enzyme was purified by a procedure which included precipitation by dialysis against citrate/phosphate buffer, pH3.5; (NH4)2SO4 precipitation; hydroxyapatite, DEAE-cellulose and ECTEOLA-cellulose column chromatography. Each stage of purification was controlled by polyacrylamide-disc-gel electrophoresis; the purified enzyme showed a single protein band that corresponded to the alpha-D-galactosidic activity. The pH optimum was found to be between pH 3.8 and 4.2; the enzyme is highly thermolabile. Hydrolysis of oligosaccharides and galactomannans has been examined, and it has been found that alpha-galactosidase I exhibits two enzymic activities, namely alpha-D-galactoside galactohydrolase and galactosyltransferase. By the polyacrylamide-gel-electrophoresis method of Hendrick & Smith (1968), and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the mol.wt. has been estimated to be 43 000 and 41 000 respectively. These results indicate that alpha-galactosidase I is a monomeric protein and that both enzymic activities associated with the enzyme reside on the same polypeptide chain.     

Glycosidases induced in Aspergillus tamarii. Secreted alpha-D-galactosidase and beta-D-mannanase.

An alpha-D-galactosidase (EC 3.2.1.22) and a beta-D-mannanase (EC 3.2.1.78), which were secreted into the growth medium when Aspergillus tamarii was cultivated in the presence of galactomannan, were purified by a procedure including chromatography on hydroxyapatite and DEAE-cellulose columns. Each of these enzymes showed a single protein band, corresponding to their respective activities, on polyacrylamide-gel electrophoresis. Both enzymes were shown to be glycoproteins containing N-acetylglucosamine, mannose and galactose, with molar proportions of 1:6:1.5 for alpha-D-galactosidase and 1:13:8 for beta-D-mannanase. Mr values as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and by the electrophoretic method of Hedrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164] were 56000 and 53000 respectively. The alpha-D-galactosidase differed markedly from the mycelial forms I and II studied in the preceding paper [Civas, Eberhard, Le Dizet & Petek (1984) Biochem. J. 219, 849-855] with regard to both its kinetic and structural properties.     

Purification and characterization of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells

The bombesin/gastrin-releasing peptide (GRP) receptor was solubilized from Swiss mouse 3T3 cell membranes in an active form and was purified about 90,000-fold to near homogeneity by a combination of wheat germ agglutinin-agarose and ligand affinity chromatography. The purified receptor displayed a single diffuse band with a Mr of 75,000-100,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After treatment of the receptor with N-glycanase, removing N-linked oligosaccharide moieties, the protein yielded a Mr = 38,000 band. These results agree with the Mr value estimated for the GRP receptor that was labeled on Swiss 3T3 cells by cross-linking to 125I-GRP1-27. GRP1-27 bound to the purified receptor with a Kd of 0.038 +/- 0.019 nM. By comparison, the soluble receptor in unfractionated extracts and intact membranes displayed a Kd for GRP1-27 of 0.036 +/- 0.003 nM and 0.13 +/- 0.04 nM, respectively. The relative potencies of a series of GRP analogs for the soluble receptor and intact membranes indicated that the extraction procedure did not significantly alter the receptor's ligand binding specificity. However coupling of the receptor to its guanyl nucleotide regulatory protein was not maintained in the soluble extract, and a G-protein did not co-purify with the receptor. Physiological concentrations of NaCl greatly inhibited the binding of some GRP analogs to the receptor, while the binding of other analogs was not affected. A domain on the GRP molecule involving Lys-13 or Arg-17 was identified which promoted binding to the GRP receptor under conditions of low ionic strength. These findings aided the development of an effective ligand affinity resin for the purification of the GRP receptor.     

The identification and characterization of two separate carboxylesterases in guinea-pig serum.

The esterase activity of guinea-pig serum was investigated. A 3-fold purification was achieved by removing the serum albumin by Blue Sepharose CL-6B affinity chromatography. The partially purified enzyme preparation had carboxylesterase and cholinesterase activities of 1.0 and 0.22 mumol of substrate/min per mg of protein respectively. The esterases were labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated electrophoretically on sodium dodecyl sulphate/polyacrylamide gels. Two main labelled bands were detected: band I had Mr 80 000 and bound 18-19 pmol of [3H]DiPF/mg of protein, and band II had Mr 58 000 and bound 7 pmol of [3H]DiPF/mg of protein. Bis-p-nitrophenyl phosphate (a selective inhibitor of carboxylesterase) inhibited most of the labelling of bands I and II. The residual labelling (8%) of band I but not band II (4%) was removed by preincubation of partially purified enzyme preparation with neostigmine (a selective inhibitor of cholinesterase). Paraoxon totally prevented the [3H]DiPF labelling of the partially purified enzyme preparation. Isoelectrofocusing of [3H]DiPF-labelled and uninhibited partially purified enzyme preparation revealed that there were at least two separate carboxylesterases, which had pI3.9 and pI6.2, a cholinesterase enzyme (pI4.3) and an unidentified protein that reacts with [3H]DiPF and has a pI5.0. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these enzymes showed that the carboxylesterase enzymes at pI3.9 and pI6.2 corresponded to the 80 000-Mr subunit (band I) and 58 000-Mr subunit (band II). The cholinesterase enzyme was also composed of 80 000-Mr subunits (i.e. the residual labelling in band I after bis-p-nitrophenyl phosphate treatment). The unidentified protein at pI5.0 corresponded to the residual labelling in band II (Mr 58 000), which was insensitive to neostigmine and bis-p-nitrophenyl phosphate. These studies show that the carboxylesterase activity of guinea-pig serum is the result of at least two separate and distinct enzymes.     

Purification of vasoactive intestinal peptide receptor from porcine liver by a newly designed one-step affinity chromatography

Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membrane using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The solubilized VIP receptor has been purified approximately 50,000-fold to apparent homogeneity by a one-step affinity chromatography using a newly designed VIP-polyacrylamide resin. The purified receptor bound 125I-VIP with a Kd of 22.3 +/- 0.7 nM and retained its peptide specificity toward VIP-related peptides. The specific activity of the purified receptor (16,400 pmol/mg of protein) was very close to the theoretical value (18,900 pmol/mg of protein) calculated assuming one binding site/protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified receptor revealed a single band with an Mr of 53,000 after either silver staining or radioiodination. Affinity labeling of the purified receptor with 125I-VIP using dithiobis(succinimidyl propionate) gave a single radioactive band, the labeling of which was completely inhibited by an excess of unlabeled VIP. In conclusion, an Mr 53,000 protein containing the VIP-binding site was purified to homogeneity by a one-step affinity chromatography using immobilized VIP.     

Purification to homogeneity of GD3 synthase and partial purification of GM3 synthase from rat brain

A CMP-sialic acid: GM3 sialyltransferase (GD3 synthase) and a CMP-sialic acid: LacCer sialyltransferase (GM3 synthase) have been purified 10,000- and 3,000-fold, respectively, from the Triton X-100 extract of rat brain. The two enzymes were purified and resolved by affinity chromatography on two successive CDP-Sepharose columns by NaCl gradient elution. Final purification of GD3 synthase was achieved by specific elution from a 'GM3 acid'-Sepharose column with buffer containing GM3. Sodium dodecylsulfate-gel electrophoresis of GD3 synthase revealed a single major protein band with an apparent molecular weight of 55,000.     

N5-(L-1-carboxyethyl)-L-ornithine:NADP+ oxidoreductase from Streptococcus lactis. Purification and partial characterization

N5-(L-1-Carboxyethyl)-L-ornithine:NADP+ oxidoreductase (EC 1.5.1.-) from Streptococcus lactis K1 has been purified 8,000-fold to homogeneity. The NADPH-dependent enzyme mediates the reductive condensation between pyruvic acid and the delta- or epsilon-amino groups of L-ornithine and L-lysine to form N5-(L-1-carboxyethyl)-L-ornithine and N6-(L-1-carboxyethyl)-L-lysine, respectively. The five-step purification procedure involves ion-exchange (DE52 and phosphocellulose P-11), gel filtration (Ultrogel AcA 44), and affinity chromatography (2',5'-ADP-Sepharose 4B). Approximately 100-200 micrograms of purified enzyme of specific activity 40 units/mg were obtained from 60 g of cells, wet weight. Anionic polyacrylamide gel electrophoresis revealed a single enzymatically active protein band, whereas three species (pI 4.8-5.1) were detected by analytical electrofocusing. The purified enzyme is active over a broad pH range of 6.5-9.0 and is stable to heating at 50 degrees C for 10 min. Substrate Km values were determined to be: NADPH, 6.6 microM; pyruvate, 150 microM; ornithine, 3.3 mM; and lysine, 18.2 mM. The oxidoreductase has a relative molecular mass (Mr = 150,000) as estimated by high pressure liquid chromatography exclusion chromatography and by polyacrylamide gradient gel electrophoresis. Conventional gel filtration indicated an Mr = 78,000, and a single protein band of Mr = 38,000 was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is composed of identical subunits of Mr = 38,000, which may associate to yield both dimeric and tetrameric forms. Polyclonal antibody to the purified protein inhibited enzyme activity. The amino acid composition of the enzyme is reported, and the sequence of the first 37 amino acids from the NH2 terminus has been determined by stepwise Edman degradation.     

Purification, characterization and induction of L-phenylalanine ammonia-lyase in Phaseolus vulgaris

The enzyme L-phenylalanine ammonia-lyase was purified from leaves of Phaseolus vulgaris by Sephacryl S-200 gel filtration and Sepharose-4-B--succinyl-aminoethyl-L-phenylalanine affinity chromatography. L-Phenylalanine ammonia-lyase was specifically eluted from the affinity matrix with its substrate L-phenylalanine at 20-25 degrees C. The purified enzyme was shown to be homogeneous by gel electrophoresis both in presence and absence of SDS. Its Mr, determined by gel filtration and non-denaturing gel electrophoresis, was 320,000 +/- 9000 and 330,000 +/- 4000 respectively. After SDS electrophoresis only one band of Mr 83,000 +/- 4000 was detected, indicating that the enzyme is an oligomer containing four subunits. The pH optimum of enzyme activity was 8.8-9.2. Ampholyte isoelectrofocusing in polyacrylamide demonstrated the presence of a single charged species at pH 4.2. The homogeneous enzyme catalyzed the deamination of L-phenylalanine to trans-cinnamate but did not catalyze the transamination of L-phenylalanine to L-phenylpyruvate. The enzyme showed Km 1.25 mM for L-phenylalanine. Antibodies to homogeneous L-phenylalanine ammonia-lyase recognised specific epitopes on L-phenylalanine aminotransferase as demonstrated by immunoaffinity purification and immunoblotting. The induction of L-phenylalanine ammonia-lyase activity during phaseollin biosynthesis in the Phaseolus vulgaris--Colletotrichum lindemuthianum interaction was regulated by an increase in enzyme concentration resulting from an increase in de novo synthesis of L-phenylalanine ammonia-lyase protein.     

Alpha-galactoside-binding isolectins from wild jack fruit seed (Artocarpus hirsuta): purification and properties

Five isolectins with marked specificity for alpha-linked galactose were purified from the wild jack (Artocarpus hirsuta) seeds by affinity chromatography on cross-linked guar gum. They were composed of a glycosylated subunit A (Mr = 16 kDa) and a nonglycosylated subunit B (Mr = 11 kDa) in noncovalent association. The isolectins which eluted as a single peak of Mr 45 kDa on gel filtration in Biogel P-100 and in a TSK G-3000 SW high pressure column, were resolved into five peaks on electrophoresis at pH 4.5. Sodium dodecyl sulphate polyacrylamide gel electrophoreogram of the major isolectin band suggested that the isolectins may be the five possible tetrameric combinations of A and B subunits. The combined isolectins bound only two molecules of 4-methyl umbelliferyl alpha-D-galactoside with a binding constant of 4.75 x 10(4) M-1. The pH optimum of sugar binding was 7.0. The isolectins specifically bound to human IgG and IgA but not to IgM.     

Isolation of a 5.3-S calmodulin-deficient 3'':5''-cyclic nucleotide phosphodiesterase from cardiac muscle.

1. In the presence of Ca2+, a 5.3-S 3':5'-cyclic nucleotide phosphodiesterase (EC 3.1.4.17) from bovine ventricle was isolated and purified by (NH4)2SO4 precipitation and DEAE-cellulose and Affi-Gel Blue chromatography. The enzyme activity was enriched 800-fold by these procedures. 2. Sucrose-density gradient centrifugation, gel filtration and non-denaturing polyacrylamide-gel electrophoresis resolved a single enzyme species with an Mr of 89 000. 3. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the purified enzyme demonstrated a prominent protein band at Mr 59000 and a minor band of Mr 28000. Calmodulin was not detected. 4. The hydrolysis of micromolar concentrations of 3':5'-cyclic guanosine monophosphate (cyclic GMP) but not 3':5'-cyclic adenosine monophosphate (cyclic AMP) was stimulated by calmodulin. 5. Anomalous biphasic kinetics plots were observed for both the catalysis of cyclic AMP and cyclic GMP hydrolysis. Kinetic plots became linear in the presence of calmodulin. 6. After several months of storage at -20 degrees C, the 5.3-S enzyme was transformed into a 6.2-S cyclic GMP-specific enzyme and a 4.4-S non-specific form.     

Purification and studies on some properties of the 4-aminobutyrate : 2-oxoglutarate transaminase from rat brain.

Using various chromatographic procedures, 4-aminobutyrate : 2-oxoglutarate transaminase from rat brain has been purified 2400 times with respect to the initial brain homogenate. The purified protein, which has a specific activity of 10 mumol times min -1, x mg-1 gave a single band by acrylamide gel electrophoresis and isoelectric focusing. It has a molecular weight of 105000 +/- 5000 and an isoelectric point of 6.8. In the presence of 0.1% sodium dodecylsulphate, a single protein band is seen on polyacrylamide gel, corresponding to a molecular weight of 57000 +/- 5000. N-terminal analysis reveals two chains with the same N-terminal amino acid, thus the enzyme may be considered as a dimer consisting of two identical subunits. The pH optimum for enzyme activity is 8.5. Studies of the enzymic reaction show that the general mechanism is of the ping-pong bi-bi model. The Km for 2-oxoglutarate at saturating 4-aminobutyrate extrapolated to saturating 2-oxoglutarate concentration is 4 mM. 2-Oxoglutarate competitively inhibits the enzyme with respect to 4-aminobutyrate, with a Ki of 1.8 times 10(-4) M. The same phenomenon is seen for the reverse reaction where the Ki is 6.6 times 10(-4) M for succinic semi-aldehyde.     

Solubilization and Purification of an α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid Binding Protein from Bovine Brain

alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) is a selective ligand for an excitatory amino acid receptor subtype in mammalian brain. We have solubilized an AMPA binding protein from bovine brain membranes with 1% Triton X-100 in 0.5 M phosphate buffer and 20% glycerol at 37 degrees C and purified the stable binding sites using a series of chromatographic steps. Scatchard analysis of the purified preparation showed a curvilinear plot with dissociation constants of 10.6 and 323 nM and Bmax values of 670 and 1,073 pmol/mg of protein for the high- and low-affinity sites, respectively. Inhibition constants for several excitatory amino acid analogues were similar to those obtained for other membrane and solubilized preparations. Gel filtration of the soluble AMPA binding protein showed a single peak of [3H]AMPA binding activity at Mr approximately 500,000. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified AMPA binding protein showed a single major band at Mr = 110,000. Previously, we have shown that a monoclonal antibody (KAR-B1) against a frog brain kainate binding protein selectively recognizes an unknown protein in mammalian brain migrating at Mr approximately 100,000. We now show that this antibody recognizes the major component of the purified AMPA binding protein, supporting a structural similarity between the frog brain kainate binding protein and the mammalian AMPA binding protein.     

Proteinases of small intestine enterocytes of swine. Purification and properties of aspartyl proteinase similar to cathepsin D

A new aspartic proteinase was isolated from porcine intestine mucosa by affinity chromatography on pepstatin-Sepharose 4B and gel filtration on Sephadex G-100. The enzyme was purified 1600-fold and appeared homogeneous upon polyacrylamide gel electrophoresis. The proteinase has a Mr 60 000 +/- 4000 Da. During sodium dodecyl sulfate polyacrylamide gel electrophoresis the enzyme produced a single protein band (Mr 30 000 +/- 3000 Da). Isoelectric focusing revealed that the enzyme has several multiple forms (pI 6.9, 7.5, 8,0). The enzyme is a glycoprotein containing 5.9% of carbohydrates; the mannose to galactose ratio is 1:3. The amino acid composition of the enzyme was studied. The proteinase splits an oxidized insulin B-chain and synthetic substrates. The pH optimum is 3.2. The enzyme is immunologically identical to porcine spleen cathepsin D.     

Purification and characterization of inositol 1,4,5-trisphosphate 3-kinase from pig aortic smooth muscle.

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase, which phosphorylates InsP3 to form inositol 1,3,4,5-tetrakisphosphate, was purified to apparent homogeneity by (NH4)2SO4 fractionation and sequential chromatographic steps on DEAE-sepharose, calmodulin-Affi-Gel and DEAE-5PW h.p.l.c. The purified enzyme had a specific activity of 24.4 nmol of inositol tetrakisphosphate formed/min per mg of protein, which represented a purification of approx. 195-fold with a 0.29% recovery, compared with the cytosol fraction of the muscle. SDS/polyacrylamide-gel electrophoresis showed a single protein-staining band of Mr 93,000. Moreover, the major protein peak, of Mr 84,000, was detected by TSK gel G3000SW gel-permeation chromatography of the purified sample. As this value was approximately consistent with the Mr determined by SDS/polyacrylamide-gel-electrophoretic analysis, the InsP3 3-kinase might be a monomeric enzyme. The purified enzyme had a Km for InsP3 of 0.4 microM, with an optimum pH range of 5.8-7.7. The enzyme was maximally activated by calmodulin, with a stoichiometry of 1:1.     

Purification and characterization of Leydig cell luteinizing hormone receptor

We have purified the testicular luteinizing hormone (LH/human choriogonadotropin (hCG)) receptor by sequential affinity chromatography on hCG-Sepharose. The purified LH/hCG receptor was identified as a single protein of Mr = 90,000 +/- 2,000 on sodium dodecyl sulfate-gel electrophoresis (SDS-PAGE), showed high affinity binding for hCG, and a binding capacity of 3.8 nmol/mg of protein. Electrophoretically blotted receptor retained the ability to bind 125I-hCG on nitrocellulose membrane, and the Mr of radioactive band was consistent with that revealed by silver staining. Autoradiography after SDS-PAGE analysis of cross-linked purified receptor-hCG complex showed Mr = 145,000 and Mr = 105,000 bands. These results are consistent with a Mr value for the receptor of 90,000 after accounting for contribution by the intact hormone or its alpha-subunit. Analysis of the free receptor by fast protein liquid chromatography on Superose 12 revealed a single peak of binding activity for 125I-hCG which eluted in the position of Mr = 200,000-240,000 in the presence of Triton X-100. Since a single protein species is observed under reducing or nonreducing conditions in SDS-PAGE, the receptor could exist in the membrane as a dimeric form composed of subunits Mr = 90,000 associated through noncovalent interactions. The pure receptor can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase (approximately 0.3 mol of phosphate/mol of receptor). This phosphorylation does not affect the binding characteristics of the receptor. The method described is simple and allows rapid purification of microgram amounts of biological active Leydig cell LH/hCG receptor for structural, functional, and immunological studies.