Five alpha-D-galactopyranosyl-binding isolectins from Bandeiraea simplicifolia seeds.

The alpha-D-galactopyranosyl-binding lectin previously purified from Bandeiraea simplicifolia seeds (Hayes, C.H., and Goldstein, I.J. (1974) J. Biol. Chem. 249, 1904) is shown to consist of five isolectins separable on polyacrylamide gel electrophoresis at pH 9.5. The isolectins are tetrameric structures composed of various combinations of two different glycoprotein subunits designated A and B. The A and B subunits appear to be immunochemically indistinguishable against rabbit antisera prepared from the isolectin mixture. The A subunit contains no methionine, whereas the B subunit contains 1 residue. The subunits migrate differently on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and, although each subunit contains 1 residue of cysteine, they react differently toward 5,5'-dithiobis(2-nitrobenzoic acid). The carbohydrate binding specificity of the two subunits differs significantly: the A subunit exhibits a primary specificity for alpha-D-GalNAcp but also reacts with alpha-D-Galp units, whereas the B subunit shows a sharp specificity toward alpha-D-Galp residues. The differences in carbohydrate binding specificity were exploited in separating the isolectins. B. simplicifolia I isolectins (A4) and (A3B) were purified on a Bio-Gel melibionate column, and (A2B2), (AB3), and (B4) were separated on a column of insolubilized blood group A substance.     

Immunological comparison of purified DNA polymerase alpha from embryos of Drosophila melanogaster with forms of the enzyme present in vivo

Specific antisera to purified DNA polymerase alpha from embryos of Drosophila melanogaster and to two of the four constituent subunits (alpha, beta, gamma, and delta) were prepared. These antibodies have revealed the following features of the enzyme. (i) The Mr = 148,000 alpha subunit is very likely derived by in vitro proteolysis from polypeptides with molecular weights of 185,000 and 166,000 that are present in vivo. (ii) The Mr = 60,000 beta subunit occurs in rapidly replicating embryos as both an 85,000- and a 60,000-dalton form, but predominantly as a 60,000-dalton form in more slowly replicating cultured cells. (iii) There is no detectable immunologic cross-reactivity between the four subunits. (iv) There is an abundance of antigenic material in embryos that co-migrates with the delta subunit of the purified enzyme during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.     

Proteolysis of ribulose-1,5-bisphosphate carboxylase/oxygenase in Citrus leaf extracts

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase, EC 4.1.1.39) has been purified from orange [ Citrus sinensis (L.) Osbeck cv. Washington Navel] leaves using sucrose gradient centrifugation in a fixed angle rotor. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two major bands corresponding to the two subunits of RuBP carboxylase were found. The large subunit coincided with the polypeptide band that has been previously reported to be preferentially mobilized during the spring and summer flush periods.
The degradation of RuBP carboxylase during autodigestion of Citrus leaf extracts, investigated by SDS-PAGE, occurred mainly at acidic (2.5-5.5) pH. The two subunits showed differences in the rate of degradation, the smaller being more rapidly hydrolyzed than the larger. At least four proteolytic activities were identified by means of inhibitor experiments: 1) a pepstatin A-sensitive activity that acts on both RuBP carboxylase subunits, 2) a mercurial ( p -hydroxymercuribenzoate and p -chloromercuriphenylsulfonate)-sensitive activity that degrades only the small subunit, 3) an EDTA-sensitive activity that hydrolyzes both the large and small subunits, and 4) a mercurial-stimulated activity that acts only on the large subunit. It is suggested that the last two proteases may be responsible for the degradation of RuBP carboxylase observed in vivo during the periods of mobilization of leaf protein in Citrus .     

Purification and properties of different forms of modeccin, the toxin of Adenia digitata. Separation of subunits with inhibitory and lectin activity.

1. The subunits were isolated of modeccin (subsequently referred to as modeccin 4B), the toxin purified from the roots of Adenia digitata by affinity chromatography on Sepharose 4B [Gasperi-Campani, Barbieri, Lorenzoni, Montanaro, Sperti, Bonetti & Stirpe (1978) Biochem J. 174, 491-496]. They are an A subunit (mol.wt. 26 000), which inhibits protein synthesis, and a B subunit (mol.wt. 31 000), which binds to cells. Both sununits, as well as intact modeccin, gave single bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, but showed some heterogeneity on isoelectric focusing and on polyacrylamide-gel electrophoresis at pH 9.5. 2. A second form of modeccin, not retained by Sepharose 4B, was purified by affinity chromatography on acid-treated Sepharose 6B: this form is subsequently termed modeccin 6B 3. Modeccin 6B has a molecular weight indistinguishable from that of modeccin 4B, and consists of two subunits of mol.wts. 27 000 and 31 000, joined by a disulphide bond. The subunits were not isolated because of their high insolubility in the absence of sodium dodecyl sulphate. 4. As compared with modeccin 4B, modeccin 6B is slightly less toxic to animals, does not agglutinate erythrocytes, and is a more potent inhibitor of protein synthesis in a lysate of rabbit reticulocytes, giving 50% inhibition at the concentration of 0.31 microgram/ml.     

Subunit structure of anthranilate synthetase from Neurospora crassa.

Freshly purified preparations of anthranilate synthetase complex from Neurospora crassa appeared to be homogeneous on polyacrylamide disc gels and were composed of two distinct subunits, 94,000 and 70,000 daltons, respectively, as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Carboxymethylation of the complex or treatment with guanidine hydrochloride and urea before sodium dodecyl sulfate treatment did not alter the subunit pattern. When the purified complex was iodinated with 125I- or methylated with [14C]dimethylsulfate, no labeled components other than the two subunits stained with Coomassie blue were detected after electrophoresis in the presence of sodium dodecyl sulfate. Although some purified preparations were stable, most were unstable upon storage. Analysis of the unstable preparations on nondenaturing and sodium dodecyl sulfate polyacrylamide disc gels revealed that the complex in these preparations was progressively fragmented to smaller components and subunits upon repeated freeze-thaw treatment or prolonged incubation at or above 4 degrees. Distinct fragments were generated ranging in size down to 25,000 daltons, and some fragments retained some of the activities associated with the anthranilate synthetase complex. On the basis of these and earlier studies, we conclude that anthranilate synthetase from Neurospora crassa is composed of two distinct subunits in an alpha2beta2 structure; one subunit is a trifunctional peptide which contains the catalytic sites for the phosphoribosylanthranilate isomerase and indoleglycerol phosphate synthetase reactions, and associates with the second subunit to form glutamine-dependent anthranilate synthetase. The smaller subunits and components previously reported for this complex are apparently due to protease activity present in purified preparations.     

Isolation and characterization of lectins from Vicia villosa. Two distinct carbohydrate binding activities are present in seed extracts

An uncharacterized lectin from Vicia villosa seeds has been reported to bind specifically to mouse cytotoxic T lymphocytes (Kimura, A., Wigzell, H., Holmquist, G., Ersson, B., and Carlsson, P., (1979) J. Exp. Med. 149, 473-484). We have found that V. villosa seeds contain at least three lectins which we have purified by affinity chromatography on a column of immobilized porcine blood group substances eluted with varying concentrations of N-acetylgalactosamine and by anion exchange chromatography. The three lectins are composed of two different subunits with Mr = 35,900 (subunit B) and 33,600 (subunit A), estimated from their mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sedimentation equilibrium analysis suggests that the purified lectins are tetramers. They have been designated B4, A4, and A2B2 to indicate their apparent subunit compositions. The purified B4 and A4 lectins contain 6.7-9.8% carbohydrate by weight; in addition, both are rich in the acidic and hydroxylic amino acids and lack cysteine and methionine. The A4 lectin agglutinates A erythrocytes specifically and binds to A1 erythrocytes (273,000 sites/cell) with an association constant of 1.8 X 10(7) M-1. Although a blood group A agglutinating activity was recognized in the original preparation of V. villosa lectins, lectins with this activity were obtained in relatively small amounts from seed extracts. The predominant lectin in V. villosa seeds, B4, does not agglutinate A, B, or O erythrocytes.     

Purification and characterization of a novel proline-directed protein kinase from bovine brain.

A novel protein kinase which phosphorylates a synthetic peptide substrate (RRPDAHRTPNRAF) has been purified approximately 200,000-fold from bovine brain. This peptide contains the consensus sequence for phosphorylation by the p34cdc2 kinase. The purification procedure took advantage of the phenomenon that this novel brain kinase, in partially purified extracts, chromatographed on a gel filtration column as a high molecular weight complex which dissociated in buffer containing 1 M NaCl. The purified native enzyme was estimated to be approximately 63,000, and displayed two bands of M(r) = 33,000 and 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On Western immunoblot, the M(r) = 33,000 peptide reacted strongly with antibodies specific for a conserved amino-terminal sequence, weakly with antibodies to the conserved PSTAIRE sequence, and not at all with antibodies to the carboxyl terminus, of HeLa cell p34cdc2. The brain kinase and p34cdc2 were similar in displaying good activity toward the parent peptide substrate, but no activity toward peptide analogues in which the -T-P- motif was substituted with either -T-G- or -T-A-. Both kinases showed marked preference in phosphorylating a peptide derived from H1 histone (KTPKKAKKPKTPKKAKKL), and both kinases could be phosphorylated by the src-family tyrosine kinase, p56lyn, purified from bovine spleen. However, the brain kinase did not co-purify with a subunit having a molecular weight corresponding to known cyclins, nor did it undergo specific interaction with p13suc1 beads, suggesting that this enzyme is distinct from p34cdc2.     

Analysis of thyrotropin receptors by photoaffinity labelling. Orientation of receptor subunits in the cell membrane.

Porcine thyrotropin (TSH) receptors have been purified by Sepharose-TSH affinity chromatography and crosslinked to a 125I-labelled photoactive derivative (N-hydroxysuccinimidyl 4-azidobenzoate; HSAB) of TSH (125I-HSAB-TSH). Purification of the crosslinked complexes on Sephacryl S-300 followed by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate showed that the receptor contained two subunits. One subunit (A) with Mr 45 000 was crosslinked to TSH and the other (B) subunit, Mr 25 000, was linked to the A subunit by a disulphide bridge(s). Other, as yet unidentified, subunits may have been non-covalently associated with the A and B subunits. Analysis of reduced and non-reduced crosslinked TSH receptor-125I-HSAB-TSH on Sephacryl S-300 in the presence and absence of detergent indicated that the A subunit was a hydrophilic peptide. This was confirmed in studies of the release into aqueous solution by reducing agent treatment of 125I-HSAB-TSH crosslinked to the TSH receptor A subunit in thyroid membranes. Similar results were obtained with TSH receptors in human thyroid and guinea pig fat cell membranes. These studies suggest that the hydrophilic A subunit of the receptor forms a binding site for TSH on the outside surface of the cell membrane and that the A subunit is linked to the cell membrane by way of a disulphide bridge to the receptor B subunit.     

Further analysis of the polypeptide subunits of yeast cytochrome c oxidase. Isolation and characterization of subunits III, V, and VII

By using a modified purification procedure in which we have substituted detergent exchange gel filtration for DEAE-cellulose or hydroxylapatite chromatography (Mason, T. L., Poyton, R. O., Wharton, D. C., and Schatz, G. (1973) J. Biol. Chem. 248, 1346-1354), we have isolated yeast cytochrome c oxidase preparations which are low in contaminating polypeptides and which have been successfully used for the large scale purification of subunits. Subunits have been purified from this preparation by a simple two-step procedure which involves: 1) the release of subunits IV and VI from an "insoluble" core composed of subunits I, II, III, V, and VII; and 2) gel filtration of the "core" subunits in the presence of sodium dodecyl sulfate. Molecular weights of the isolated subunits, obtained from sodium dodecyl sulfate gel retardation coefficients (KR) derived from Ferguson plots, were: I, 54,000; II, 31,000; III, 29,500; IV, 14,500; V, 12,500; VI, 9,500; VII, 4,500. In their purified state all subunits, except for subunit V, exhibited electrophoretic behavior similar to that exhibited by unpurified subunits in sodium dodecyl sulfate-dissociated holoenzyme preparations. As purified, subunit V exhibits a slightly smaller apparent molecular weight than its counterpart in the holoenzyme. Amino acid analysis of the isolated subunits revealed that subunit III, a mitochondrial translation product, contained 41.9% polar amino acids, whereas subunits V and VII, cytoplasmic translation products, each contained 47.7% polar amino acids. These results extend and support our previous finding that the mitochondrially translated subunits of yeast cytochrome c oxidase are more hydrophobic than the cytoplasmically translated subunits.     

Escherichia coli nitrate reductase subunit A: its role as the catalytic site and evidence for its modification.

Subunits A and B were isolated from purified nitrate reductase by preparative electrophoresis in low levels of sodium dodecyl sulfate. Nonheme iron and low levels of molybdenum were associated with isolated subunit A but not with isolated subunit B. After dialysis against a source of molybdenum cofactor, subunit A regained tightly bound molybdenum and concomitantly regained enzyme activity and reactivity with anti-nitrate reductase antiserum. Subunit B neither bound cofactor nor regained activity or reactivity with antiserum. These data indicate that subunit A contains the active site of the enzyme. Subunit A was also found to be modified posttranslationally in a similar fashion as is subunit B. This was determined by comparison of partial proteolytic digests and amino acid analyses of A subunits from precursor and membrane-bound forms of nitrate reductase.     

Evolution of allantoinase and allantoicase involved in urate degradation in liver peroxisomes. A rapid purification of amphibian allantoinase and allantoicase complex, its subunit locations of the two enzymes, and its comparison with fish allantoinase and allantoicase

Allantoinase and allantoicase are located in the same protein molecule in amphibian liver, whereas the two enzymes are different proteins in marine fish and invertebrate liver (Takada, Y., and Noguchi, T. (1983) J. Biol. Chem. 258, 4762-4764). The amphibian enzyme was rapidly purified from frog liver by using its following characteristics. 1) The enzyme binds to the intracellular membranes in the hypotonic solution. 2) The membrane-bound enzyme is not solubilized by the detergent. 3) The membrane-bound enzyme is solubilized by oxaloacetate. The electrophoresis of the purified enzyme gave a single protein band in the absence of sodium dodecyl sulfate, and gave two protein bands with molecular weights of 48,000 and 54,000, respectively, in the presence of sodium dodecyl sulfate. With a specific antibody raised against each subunit, allantoinase activity was found to be from the large subunit, and allantoicase activity to be from the small subunit. This amphibian allantoinase and allantoicase complex was compared with allantoinase and allantoicase purified from fish liver. Fish allantoinase was a single peptide and fish allantoicase was composed of two identical subunits. Fish allantoinase had an identical molecular weight with amphibian large (allantoinase) subunit and the subunit of fish allantoicase with amphibian small (allantoicase) subunit. These results suggest that the evolution of fish to amphibian resulted in the dissociation of allantoicase into subunits and in the association of allantoinase with allantoicase. The two enzymes are lost by further evolution.     

Purification and characterization of two ribonucleases from developing tomato fruit

Two neutral ribonucleases have been purified from developing tomato fruit. Their activity is maximal 5 days after anthesis, declines during maturation, and then increases slightly in the mature green through breaker stages. The ribonucleases Tf1 and Tf2 have molecular weights of 59 and 29 K, respectively, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and are glycoproteins. The reduced and denatured Tf1 is composed of two subunits, 30 and 29 K, of which only the 30-K subunit displays ribonuclease activity after renaturation. Reduced and denatured Tf2 is a single 29-K polypeptide that is renaturable to an active ribonuclease. Only the 30-K, active subunit of Tf1 is immunologically cross-reactive with Tf2. Both ribonucleases are cyclyzing endoribonucleases with a strong preference for cleavage at pyrimidine residues, thus generating oligonucleotide products ending with pyrimidine 2',3'-cyclic phosphate. These tomato fruit ribonucleases share a number of properties in common with the S-glycoprotein ribonucleases that are involved in self-incompatibility reactions in some solanaceous plants.     

Cytochrome b from Escherichia coli nitrate reductase. Its properties and association with the enzyme complex

Membrane-bound nitrate reductase purified from Escherichia coli was resolved into two separate forms. The majority of the enzyme complex had a subunit composition of 2A:2B:4C, exhibited cytochrome b spectra, and was found to be stable after purification. A second form of nitrate reductase activity was a modified complex with a subunit composition of 2A:2B and lacked cytochrome. The subunit B from this complex was altered in its mobility on sodium dodecyl sulfate-polyacrylamide gels. The cytochrome-containing enzyme had 28 +/- 2 atoms of iron and 1.35 atoms of molybdenum whereas iron and molybdenum in cytochromeless enzyme were 24 +/- 2 atoms and 1.18 atoms/molecule, respectively. Besides cytochrome-containing nitrate reductase, two other cytochrome b-containing fractions were also resolved. These were cytochrome b associated with formate dehydrogenase and a novel cytochrome b with reduced absorption maxima at 430, 529.5, and 560 nm. Nitrate reductase cytochrome b (subunit C) was isolated from subunits A and B as a partially denatured form and its renaturation was accomplished by dialyzing against hemin. The renatured cytochrome yielded absorption spectra similar to the holoenzyme. The pure cytochrome aggregated upon heating, even in the presence of sodium dodecyl sulfate. It had a high isoelectric point (pH greater than 9.5) and had 45% hydrophobic amino acids.     

Purification and properties of pyruvate dehydrogenase kinase from bovine kidney

Pyruvate dehydrogenase kinase was purified about 2,700-fold to apparent homogeneity from extracts of bovine kidney mitochondria. The kinase consists of two subunits (alpha beta) with molecular weights of 48,000 (alpha) and 45,000 (beta) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinase activity resides in the alpha subunit. The alpha subunit is sensitive to proteolysis by chymotrypsin, whereas the beta subunit is selectively modified by trypsin. These observations, together with the results of peptide mapping, indicate that the two subunits are distinctly different proteins. It is proposed that the beta subunit is a regulatory subunit.     

Synthesis and characterization of a sulfated and a non-sulfated cyclic CCK8 analogue functionalized with a chelating group for metal labelling.

Two cyclic peptides, cyclo29,34[Dpr29, Lys34(DTPA-Glu)]-CCK8 (1) and cyclo29,34[Tyr27(SO3H), Dpr29, Lys34(DTPA-Glu)]-CCK8 (2), bearing the chelating moiety DTPA-Glu covalently bound to the Lys side chain have been synthesized by solid-phase methodology. The presence in compound 2 of many acidic functions characteristic of the chelating agent increases the lability of the sulfate group on the Tyr side chain. This finding suggests that prolonged acid treatments should be avoided during the preparation of such peptides. Sulfation of cyclo29,34[Dpr29, Lys34(DTPA-Glu)]-CCK8 was performed using a pyridine-SO3 complex as reagent. This reaction has been found to be the most suitable synthetic strategy for obtaining compound 2 in good yield. Cyclo29,34[Tyr27(SO3H), Dpr29, Lys34(DTPA-Glu)]-CCK8 is a new promising CCK8 analogue, able to coordinate radioactive isotopes of metal ions such as 111In(III), and to bind, in a selective way, the CCKA-R receptor.     

Oligomeric structure and catalytic activity of G type casein kinase. Isolation of the two subunits and renaturation experiments

Casein kinase G purified from bovine tissue is an oligomeric cyclic nucleotide-independent protein kinase made of two different monomers, namely an alpha (Mr = 38 kilodaltons) and a self-phosphorylatable beta (Mr = 27 kilodaltons) subunit. Treatment of the native enzyme under denaturing conditions (0.5 M NaCl, 4 M LiCl, and 20 to 35% formamide) resulted in a progressive selective removal of the beta subunit following gel filtration and a correlated loss of activity of the corresponding renatured enzyme. Mild digestion with papain resulted in a proteolytic alteration limited to the beta monomer with a concomitant partial loss of the enzyme activity. Isolation of the alpha and beta casein kinase G subunits was achieved by preparative reversed polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Renaturation of the proteins following sodium dodecyl sulfate removal by acetone and/or Triton X-100 treatment allowed reconstitution of a functional casein kinase G. Whereas the isolated alpha subunit was found to exhibit a weak catalytic activity, addition of the beta subunit was required for recovery of a maximal casein kinase activity. The process was dose-dependent and reached a plateau for an alpha:beta subunit molar ratio of approximately 1 to 1. These data suggest that while the casein kinase G alpha subunit bears the catalytic site, stoichiometric combination with the beta subunit is required for optimal enzymatic activity. A possible role of the beta subunit as a regulatory component of casein kinase G activity in the intact cell remains to be examined.     

Biochemical study of a recombinant soluble interleukin-2 receptor. Evidence for a homodimeric structure

A truncated soluble form of the human interleukin-2 receptor p55 chain (T-S-IL-2R) was expressed to high levels in RODENT (mammalian) cells and affinity-purified. Its biochemical behavior was analyzed by polyacrylamide gel electrophoresis (PAGE), gel filtration, and sucrose gradient centrifugation. It migrated as a single 40-kDa band on sodium dodecyl sulfate-PAGE (reducing or nonreducing conditions), whereas it ran as a 80-kDa component on native PAGE or as a 86-kDa component on gel filtration. The combination of gel filtration and density gradient sedimentation gave a Stokes radius of 4.0 nm and a sedimentation coefficient of 3.72 S. The deduced molecular mass was 67 kDa, and the fractional ratio was 1.516. These data therefore indicated that the T-S-IL-2R was secreted as an homodimer of two noncovalently associated 40-kDa subunits. Cross-linking experiments using bifunctional reagents enabled the materialization of the dimeric structure on sodium dodecyl sulfate-PAGE. Stoichiometric binding studies using two monoclonal antibodies (mAbs 33B3.1 and 11H2) reacting with separate epitopes on the p55 chain also strongly supported the dimeric structure. Indeed, there was one binding site for the 33B3.1 mAb (and Fab fragment) per T-S-IL-2R 40-kDa subunit, whereas the 11H2 mAb (or Fab fragment) could bind only half a site per subunit, a result which could only be explained when assuming more than one subunit for the native T-S-IL-2R. Soluble interleukin-2 receptor species were also purified from culture supernatants of either L cells transfected with the full-length p55 cDNA or a normal alloreactive T cell clone. Similar biochemical behavior and reactivities with the two mAbs were found. Finally, cell-surface p55 chains expressed either by pgL21 or 4AS cells bound the 33B3.1 and 11H2 mAbs in a 2:1 ratio, suggesting that the p55 chains are also associated as homodimers when imbedded in the membrane.     

Purification and properties of pantohenase from Pseudomonas fluorescens.

Pantothenase (EC 3.5.1.22) from Pseudomonas fluorescens UK-1 was purified to homogeneity as judged by disc-gel electrophoresis and isoelectric focusing. The purification procedure consisted of four steps: DEAE-Sephadex chromatography, (NH4)2SO4 precipitation, hydroxyapatite chromatography and preparative polyacrylamide-gel electrophoresis. Gel filtration on Ultrogel AcA 34 was used to determine the molecular weight, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to study the subunit molecular weight. The enzyme appeared to be composed of two subunits with mol.wts. of approx. 50000 each. The total mol.wt. of the enzyme was thus about 100000. The isoelectric point was 4.7 at 10 degrees C.     

The saxitoxin receptor of the sodium channel from rat brain. Evidence for two nonidentical beta subunits

The saxitoxin receptor of the sodium channel purified from rat bran contains three types of subunits: alpha with Mr approximately 270,000, beta 1 with Mr approximately 39,000, and beta 2 with Mr approximately 37,000. These are the only polypeptides which quantitatively co-migrate with the purified saxitoxin receptor during velocity sedimentation through sucrose gradients. beta 1 and beta 2 are often poorly resolved by gel electrophoresis in sodium dodecyl sulfate (SDS), but analysis of the effect of beta-mercaptoethanol on the migration is covalently attached to the alpha subunit by disulfide bonds while the beta 1 subunit is not. The alpha and beta subunits of the sodium channel were covalently labeled in situ in synaptosomes using a photoreactive derivative of scorpion toxin. Treatment of SDS-solubilized synaptosomes with beta-mercaptoethanol decreases the apparent molecular weight of the alpha subunit band without change in the amount of 125I-labeled scorpion toxin associated with either the alpha or beta subunit bands. These results indicate that the alpha and beta 1 subunits are labeled by scorpion toxin whereas beta 1 is not and that the beta 2 subunit is covalently attached to alpha by disulfide bonds in situ as well as in purified preparations.     

赤子爱胜蚓超氧化物歧化酶的纯化和部分性质研究

采用硫酸铵分级沉淀和柱层析的方法,从赤子爱胜蚓整体细胞抽提液内分离得到纯的铜锌超氧化物岐化酶（Ｃｕ,Ｚｎ－ＳＯＤ）。每１００ｇ蚯蚓得到的ＳＯＤ制品,总活力为１１１５０Ｕ,比活力为５１３８Ｕ／ｍｇ蛋白,回收率为２０％。铜锌超氧化物岐化酶呈淡蓝绿色,最大紫外吸收波长为２７０ｎｍ。测得该酶分子量为３３０００,亚基分子量为１６５００。该酶亚基由１５６个氨基酸残基组成,不含酪氨酸。