Secretion of apolipoproteins in very low density and high density lipoproteins by perfused rat liver

The incorporation of labeled amino acids into the peptides of very low density lipoproteins (VLDL) and high density lipoproteins (HDL) secreted by perfused rat liver was studied using a Ringer-albumin solution in the perfusate in place of serum to diminish exchange of peptides between VLDL and HDL. Among the lipoproteins, the greatest release of protein, greatest incorporation of amino acid, and highest specific activity were found in VLDL. After separation of the delipidated peptides by electrophoresis on polyacrylamide gel, the incorporation into VLDL peptides was found to be 5-10 times as great as into HDL peptides. There was virtually no incorporation into the peptides of low density lipoproteins (LDL). Approximately 25% of the radioactivity incorporated into perfusate VLDL failed to enter the 13% polyacrylamide gel. The remaining radioactivity was distributed primarily among three peptide bands; one, found in the upper portion of the gel, contained 45% of the total, most of the remainder being found in two rapidly migrating bands. These three peptides appear to approximate those of human apo-C in relative electrophoretic mobility. Most of the HDL peptide radioactivity entering the running gel was found in a band that migrates slightly faster than the main VLDL band. A portion of the radioactivity of this major HDL band did not enter the running gel unless beta-mercaptoethanol was present. Greater separation of these two bands by polyacrylamide gel electrophoresis for 24 hr confirmed that the major bands in VLDL and in HDL were different. The rapidly moving peptides of HDL were found to contain very little radioactivity. Determination of the intensity of staining of carrier-free perfusate VLDL and HDL peptides produced a pattern similar to the incorporation of labeled amino acids. It is concluded that the rapidly moving peptides, which may contain activators of lipoprotein lipase, are only secreted as part of the VLDL.     

The N alpha-acetylenkephalin carboxypeptidase activity of N-acetyltyrosine deacetylase from monkey kidney. Purification, characterization and substrate specificity.

N alpha-Acetylenkephalin carboxypeptidase was co-purified with N-acetyltyrosine deacetylase from monkey kidney. Almost 90% of the activity from the homogenate was recovered in a high-speed supernatant without the use of detergents. The crucial steps in the purification were Cibacron Blue F3GA--Sepharose chromatography (involving negative and positive binding sequentially) and metal chelate affinity chromatography. The purified enzyme showed three bands on gel electrophoresis under non-denaturing conditions. All the three bands exhibited both N-acetyltyrosine deacetylase and N-acetylenkephalin carboxypeptidase activity, indicating their co-migration, Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence and absence of 2-mercaptoethanol gave a single protein band of mol.wt. 34 000. The native enzyme was a dimer of mol.wt. 66 000 as observed on Bio-Gel P-300 gel filtration. The carboxypeptidase removed two amino acids from the C-terminal end of either N-acetyl[Met5]- or N-acetyl[Leu5]-enkephalin. Non-acetylated enkephalins were less active as substrates. Peptides with their carboxy end blocked were inactive as substrates. Models suggested for carboxypeptidase A [Hartsuck & Lipscomb (1971) Enzymes 3, 1-56] support the idea that the kidney N-acetylated aromatic amino acid deacetylase or acylase III [Endo (1978) Biochim. Biophys. Acta 523, 207-217] can act as a carboxypeptidase on peptides having hydrophobic amino acids at the C-terminal end.     

杨尺蠖核型多角体病毒蛋白质的特性

本文研究了杨尺蠖核型多角体病毒(AciNPV)蛋白质特性,用反复调等电点法,从AciNPV的多角体蛋白中分离到A,B两种蛋白,Sepharose 6B柱层析和超离心沉降分析表明,两者均为一个峰纯,沉降系数(S20w)分别为4.9和8.9,对A蛋白进行了16种氨基酸组成分析,Glu、Asp和Leu含量高于其它氨基酸,不含Cys,用SDS-聚丙烯酰胺凝胶电泳和免疫双扩散法分析表明,用两倍体积饱和硫酸铵沉淀法制备的多角体蛋白,与提纯的多角体A、B蛋白之间无差异,多角体蛋白结构多肽由6种组成,分子量在12500—54000道尔顿范围内,其中32000是多角体蛋白的主要多肽,病毒粒子结构多肽和核衣壳蛋白分别由19和7个多肽组成,分子量范围分别在89000～13000和34000～13000之间,间接试验结果表明,在AciNPV中有碱性蛋白酶活性存在。     

Hemocyanins in spiders, IV[1]. Subunit heterogeneity of Eurypelma (Dugesiella) hemocyanin, and separation of polypeptide chains.

The hemocyanin of the North American tarantula Eurypelma californicum (Dugesiella californica) is dissociated at pH 9.6 into monomers (Mr about 70 000) and dimers (Mr about 140 000), which were separated by gel filtration. The monomer peak was resolved by preparative polyacrylamide gel electrophoresis and yielded 4 protein bands, three of which (1, 3 and 4M) are apparently homogeneous. Band 2 contains two sub-fractions (2I and 2II). The dimer peak contains two dimers (bands 4D and 5). Upon treatment with 5mM cysteine the dimer band 5 is dissociated, yielding only one type of monomer identical with band 3. The other dimer, which was only partially dissociated by 10mM EDTA, is most probably a heterodimer, one component being electrophoretically indistinguishable from band 2II. After treatment of the native hemocyanin with sodium dodecylsulfate and analysis in gradient gel slabs, 6 polypeptide chains were observed (labeled a - f). They correspond to the products of alkaline dissociation as follows: band 1 = e, band 2I = a, band 2II = c, band 3 = f, band 4M = d, band 4D = b plus c, band 5 = f. The molecular weights were determined by dodecylsulfate gel electrophoresis in gradient gels, and by sedimentation equilibrium analysis and found to range between 67 000 and 76 000. The sedimentation coefficients are between 4.4 and 4.7 S for the monomers and 6.6 and 6.7 for the dimers. The isoelectric points range from pH 4.5 to pH 5.4. The findings are discussed with respect to the limitations of molecular weight determination by conventional dodecylsulfate gel electrophoresis, to the structure of the hemocyanin oligomers and to possible biological significance.     

Purification of human erythrocyte proteolytic enzyme responsible for degradation of oxidant-damaged hemoglobin. Evidence for identifying as a member of the multicatalytic proteinase family

Exposure of human red cells to oxidants such as phenylhydrazine, 2,4-dimethylphenylhydrazine and 4-hydrazinobenzoic acid stimulates the proteolysis of hemoglobin as evidenced by the increase in the rate of the free alanine and acid soluble amino groups released. An enzyme responsible for proteolytic degradation of oxidized hemoglobin, was purified from cytosolic fraction of erythrocytes by a DEAE-batch procedure followed by gel-filtration and ion-exchange chromatography. The final enzyme preparation produces a single band in non-denaturing polyacrylamide gel electrophoresis, and eight different bands of 23-32 kDa when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The native enzyme has a molecular mass of about 700 kDa as estimated by gel filtration. The enzyme, unable to hydrolyze native hemoglobin, cleaves phenylhydrazine-treated hemoglobin into small peptides without free amino acid release. In addition, the enzyme shows an endopeptidase activity towards synthetic peptides having a tyrosine or an arginine in the P1 position, whereas it does not hydrolyze shorter peptides and those with a proline in the P1 or P2 position. The proteolytic activity of the enzyme against oxidized hemoglobin is inhibited by chymostatin and p-chloromercuribenzoate, while it is stimulated by N-ethylmaleimide and epoxysuccinylleucylamido-(4-guanidino)butane (E-64). The peptidase activity assayed on succinyl-Leu-Leu-Val-Tyr-MCA is inhibited by chymostatin, hemin, N-ethylmaleimide and p-chloromercuribenzoate. The results obtained show that in human erythrocytes oxidized hemoglobin is cleaved into peptides by a high molecular mass proteinase identified as a member of the multicatalytic proteinase family. It is also suggested that the complete degradation of oxidized hemoglobin to free amino acids requires the involvement of a further proteolytic enzyme(s) which remain(s) to be identified.     

Photorhabdus luminescens W-14 insecticidal activity consists of at least two similar but distinct proteins. Purification and characterization of toxin A and toxin B

Both the bacterium Photorhabdus luminescens alone and its symbiotic Photorhabdus-nematode complex are known to be highly pathogenic to insects. The nature of the insecticidal activity of Photorhabdus bacteria was investigated for its potential application as an insect control agent. It was found that in the fermentation broth of P. luminescens strain W-14, at least two proteins, toxin A and toxin B, independently contributed to the oral insecticidal activity against Southern corn rootworm. Purified toxin A and toxin B exhibited single bands on native polyacrylamide gel electrophoresis and two peptides of 208 and 63 kDa on SDS-polyacrylamide gel electrophoresis. The native molecular weight of both the toxin A and toxin B was determined to be approximately 860 kDa, suggesting that they are tetrameric. NH2-terminal amino acid sequencing and Western analysis using monospecific antibodies to each toxin demonstrated that the two toxins were distinct but homologous. The oral potency (LD50) of toxin A and toxin B against Southern corn rootworm larvae was determined to be similar to that observed with highly potent Bt toxins against lepidopteran pests. In addition, it was found that the two peptides present in toxin B could be processed in vitro from a 281-kDa protoxin by endogenous P. luminescens proteases. Proteolytic processing was shown to enhance insecticidal activity.     

Multiple forms of rat dentin phosphoproteins

Previous studies have shown that the phosphoprotein from rat dentin is heterogenous and can be partially separated into two fractions by ion-exchange chromatography. These proteins were further characterized by polyacrylamide gel electrophoresis, gel chromatography, and amino acid and phosphate analysis, after chromatographic separations on ion-exchange columns. On 5-15% gradient gels, the phosphoproteins extracted from rat dentin and precipitated by CaCl2 gave three Alcian blue-staining bands with apparent molecular weights in the 90-95,000 range. The two slower-moving bands corresponded to highly phosphorylated proteins (HP) that had phosphoserine contents of greater than 400 residues per thousand and contained little or no valine, leucine, phenylalanine, or arginine. The faster-moving band corresponded to a moderately phosphorylated protein that contained about 250 residues per thousand of phosphoserine and greater quantities of glutamic acid, proline, and several other amino acids than HP. The nature of the phosphoproteins in HP was further studied after total removal of the phosphate with an insoluble form of bovine intestinal alkaline phosphatase. The dephosphorylated product (dP-HP) gave a single major band on gel electrophoresis but showed evidence for two closely related NH2-terminal sequences, Asp-Asp-Asp-Asn and Asp-Asp-Pro-Asn. The dephosphorylated material was separated into two components (dP-HP1 and dP-HP2) by chromatography on QAE-Sephadex A-25. The amino acid compositions of the two components showed that they differed in their primary structures. This conclusion was verified by the finding of the proline-containing sequence in dP-HP2. In addition to these two groups of phosphoproteins, a third class, LP, contains low levels of phosphoserine and high amounts of glutamic acid (W.T. Butler, M. Bhown, M.T. DiMuzio, and A. Linde, (1981) Coll. Res. 1, 187-199).     

Zein of maize grain. Preparation and characterization]

A laboratory procedure for isolation and purification of zein from grains of 4 varieties of Maize was described. The preparations were characterized by their physicochemical properties. Upon polyacrylamide gel electrophoresis in sodium dodecyl sulphate (SDS), native zein (from INRA 260 hybrid) was resolved into 2 major classes with average molecular weights of 45,000 and 22,000. After reduction with mercaptoethanol zein contained only two subunits of 22,000 and 24,000 daltons. Upon starch gel electrophoresis in 6 M urea at pH 3.5, native zein exhibited five major or medium intensity bands and several minor ones. The latter, under reducing conditions, disappeared to reinforce the major bands or to yield some new minor bands. Amino acid analysis revealed a very low content of lysine. The NH2-terminal amino acids were determined to be threonine and phenylalanine with a preponderance of the former. Zeins isolated from the varieties studied appeared tohave the same NH2-terminal residues and similar amino acid compositions with an arginine/histidine ratio ranging from 1.1 to 1.2. They differed in relative importance of components, detected by electrophoresis in the presence of SDS or urea. Changes in zein characteristics with the grain genotype allow one to conclude that the components of molecular weights of 22,000 and 24,000 consist of several subunits differing in charge and amino acid content.     

Structural changes in the glutamine-chargeable Escherichia coli transfer RNA-Trp produced by chemical modification with sodium bisulfite

Glutamine-mischargeable tRNA produced by sodium bisulfite-treated Escherichia coli tRNA-Trp was isolated by dihydroxyboryl-cellulose affinity column chromatography. This tRNA was shown to have dual specificity tryptophan and glutamine, and, when charged with either amino acid, bound to ribosomes in response to the non-sense codon UAG but not in response to the tryptophan codon UGG. The results were consistent with the reported properties of Su+7 tRNA. The bisulfite-treated tRNA-Trp migrated as two bands during polyacrylamide gel electrophoresis. The faster moving band (band I) coincided with that of untreated tRNA-Trp. The slower moving band (band II) coincided with the glutamine-chargeable tRNA-Trp. Su+7 tRNA behaved like band II tRNA upon gel electrophoresis. Nucleotide sequence analysis showed that a cytidine-uridine transition occurred at the 1st or the 2n position of the anitcodon of band II tRNA. Band I and band II tRNAs differed from each other in their thermal melting profiles. It is suggested that the single base change in the anticodon is responsible for the altered conformation of band II tRNA.     

Cockroach larval-specific protein, a tyrosine-rich serum protein

Larval-specific protein (LSP) is the most abundant protein in the hemolymph of cockroaches shortly before molting, but is rapidly cleared from the hemolymph during the molt (Kunkel, J. G., and Lawler, D. M. (1974) Comp. Biochem. Physiol. 47B, 697-710). Blatta orientalis LSP was purified by sedimentation in preparative sucrose gradients followed by 2-hydroxypropylamino-cellulose anion-exchange chromatography and gel filtration on a column of Bio-Gel A-1.5m. The amino acid composition of LSP includes 16.3 mol % tyrosine and 4.9 mol % phenylalanine, but virtually no cysteine and little methionine. The following physical properties were determined for LSP: R8 = 68.3 A, 8(20),w = 17.8, and V = 0.723. From these values an Mr = 507,900 was calculated. In electron micrographs, LSP appears as rectangular particles of 121 by 134 A. In disc polyacrylamide gel electrophoresis, native LSP exhibits a single band, but in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, LSP is resolved into a doublet of closely spaced bands of Mr = 88,100 and 84,400 present in a ratio of 1.38:1. These data indicate that native B. orientalis LSP is a hexamer of subunits averaging approximately Mr = 86,000. Crossed immunoelectrophoresis of Blattella germanica larval serum indicates that LSP in that species is a hexamer composed of a random assortment of two subunits of different charge in the ratio 1.25:1. The amino acid composition and physical properties of LSP suggest that LSP may be the hemimetabolous analogue of the tyrosine- and phenylalanine-rich storage proteins of holometabolous insects.     

Chemical and physicochemical characterization of the sialic acid-specific lectin from Cepaea hortensis

A sialic acid-specific lectin was isolated from the albumin glands of the garden snail Cepaea hortensis by affinity chromatography on fetuin-Sepharose following gel filtration on Superdex 200. The purified native lectin showed a molecular mass of about 95 kDa by gel filtration and 100 kDa by SDS electrophoresis. It was cleaved by boiling in buffer containing SDS in three serological identical bands corresponding to molecular masses of about 24, 20 and 16 kDa, respectively. From these three fragments, only the 24- and the 20-kDa bands were found to be glycosylated. Only the three sugars mannose, galactose and N-acetylglucosamine could be detected in a molar ratio of 3:8.6:2. The oligosaccharide moieties seem to be N- and partially O-glycosidic bound. Isoelectric focusing (IEF) of the purified lectin revealed a heterogeneous pattern with bands in the pH range of 4.3-5.0. Isolated bands of different isoelectric points showed in SDS electrophoresis the same three fragments with molecular masses of 24, 20 or 16 kDa. The heterogeneity of the lectin was revealed either by IEF or amino acid sequencing of internal tryptic peptides.     

Gel electrophoretic investigations of prolamins in eu- and alloplasmatic octoploid primary triticale forms

 Unreduced prolamin fractions of eu- and alloplasmatic octoploid triticale forms were investigated by means of gel electrophoresis. The electrophoretic separation of the prolamin fractions was carried out by using one-dimensional, horizontal, native acidic polyacrylamide gel electrophoresis (APAGE) and gradient gels. A comparison of the electropherogram patterns of triticale forms with those of the genome donors showed that most of the parent prolamins can be found again in the corresponding triticale. However, the bands of the triticale forms exhibited lower intensities than those of the genome donors. On average the gliadin bands of the triticale forms showed equal reductions in intensities, thereby preserving the relationships of the band intensities in most cases. On the other hand, secalin bands which came from the rye parent showed varying reductions in intensities. A comparative analysis of the band patterns revealed that differences could often be found between triticale forms with the same genomic constitution. Often it concerned differences in intensity. In some cases the bands showed changes in mobility or a splitting up into two sub-bands. Starting with the plant material presented here we developed a new nomenclature of the prolamin band patterns of the triticale forms. Received: 15 July 1997 / Accepted: 23 July 1997     

Isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone

A phosphorylated glycoprotein was purified from the mixture of proteins extracted by demineralization of rat bone with 0.5 M EDTA in 4 M guanidinium chloride. A high level of purity for the preparation was indicated by a single band on sodium dodecyl sulfate (SDS)-gradient gel electrophoresis, sedimentation equilibrium ultracentrifugal data, and by automated Edman degradation results. The molecular weight of the phosphoprotein was shown to be about 44,000 by sedimentation equilibrium analyses in 4 M guanidinium chloride, even though an Mr of 75,000 was obtained by 5-15% SDS-polyacrylamide gel electrophoresis. Subsequent analysis by 15% SDS-polyacrylamide gel electrophoresis gave an Mr of 45,000. Analytical data showed that the protein contained 16.6% carbohydrate, possibly including 1 N-linked oligosaccharide and 5-6 O-linked oligosaccharides. The aspartic acid- and glutamic acid-rich protein contained about 300 amino acid residues including 1 phosphothreonine and 12 phosphoserine residues. Alkaline beta-elimination/NaBH4 reduction data showed that the phosphate obtained by complete acid hydrolysis prior to amino acid analysis was equivalent to the phosphate subject to alkaline beta-elimination. In this experiment, the losses of serine plus threonine exceeded the amount of phosphate liberated by 5-6 residues/protein. These serine and threonine residues probably represent O-linked oligosaccharides, since the protein contained about this number of N-acetyl-galactosamine residues. That the phosphoprotein is synthesized and secreted by osteoblast-like cells was shown with cultures of clonal rat osteosarcoma cells. After pulsing with 32PO4 the proteins secreted into the medium were precipitated with trichloroacetic acid and the radiolabeled proteins were immunoadsorbed. A protein migrating in the same position, on 5-15% SDS-polyacrylamide gel electrophoresis (i.e. with an Mr = 75,000) and on 15% gels (Mr = 45,000), as the phosphoprotein obtained from bone could be specifically immunoprecipitated.     

Subunit structure and amino acid composition of xylose isomerase from Streptomyces albus.

The subunit structure and amino acid composition of xylose isomerase from Streptomyces albus have been examined. A native molecular weight of 165,000 determined by sedimentation equilibrium was reduced to 43,000 when the protein was treated with 6 M guanidine hydrochloride. No further reduction in molecular weight was observed when potential disulfide bridges of xylose isomerase were reduced and alkylated, indicating that the protein was devoid of interchain disulfide bonds. NH2-terminal analysis using [3H]dansyl chloride showed 0.86 residues of methionine per Mr equals 41,500 unit. Analysis of the native protein with an automated protein sequenator revealed the presence of only one degradable polypeptide chain. Fractionation of the soluble tryptic peptides of S-[14C]carboxymethyl xylose isomerase by ion exchange chromatography and one-dimensional paper electrophoresis yielded 37 to 43 peptides. When the acid-insoluble tryptic peptides were dissolved and analyzed using gel filtration techniques, and additional four peptides were found. A unique radioactive tryptic peptide containing S-carboxymethylcysteine was found among the soluble peptides, confirming cysteine as the limiting amino acid residue in the amino acid composition of xylose isomerase. On the basis of its lysine and arginine content, the number of tryptic peptides is consistent with the hypothesis that the native xylose isomerase is a tetramer of four very similar or identical subunits of Mr equals 41,500, associated by noncovalent bonds.     

Antigenic structure of rabies virus glycoprotein: ordering and immunological characterization of the large CNBr cleavage fragments.

After rabies virus glycoprotein was treated with CNBr, the peptide mixture was fractionated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CNBr-cleaved peptide fragments were resolved into seven peptide bands under reducing conditions and six peptide bands under nonreducing conditions. The isolated nonreduced polypeptides were further analyzed by electrophoresis under reducing conditions. The N-terminal amino acid sequences were determined for the peptides in each of the isolated bands. The sequence data identified eight CNBr peptides and allowed the peptide fragments to be ordered within the deduced amino acid sequence of the glycoprotein. Analysis of the nonreduced CNBr peptides revealed two conformations of the glycoprotein. Two CNBr peptide fragments were specifically immunoprecipitated with a hyperimmune anti-rabies glycoprotein serum. These two and one other CNBr peptide induced the production of rabies virus-neutralizing antibodies, indicating the existence of at least three distinct antigenic sites on the rabies virus glycoprotein.     

Restriction and modification in Bacillus subtilis: two DNA methyltransferases with BsuRI specificity. I. Purification and physical properties

Two S-adenosyl-L-methionine:DNA (cytosine 5)-methyltransferases, termed M.BsuRIa and M.BsuRIb, were purified 3,000- and 4,000-fold, respectively, from Bacillus subtilis strain OG3R (r+m+) by successive column chromatography. The molecular weights determined by gel filtration were 37,000 for M.BsuRIa and 40,000 for M.BsuRIb. The sedimentation coefficients s20,w were 3.55 for both enzymes as determined by glycerol gradient centrifugation, corresponding to molecular weights of 43,000. Analysis of the two methyltransferases by agarose gel electrophoresis under native conditions, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed correspondence of the M.BsuRIa activity with one protein band at a molecular weight of 41,000, whereas M.BsuRIb activity was associated with two protein bands with molecular weights of 42,000 and 39,000, respectively.     

Subunit structure of canavalin

The subunit structure of canavalin was studied. The native protein has a sedimentation coefficient of 6.4 S and a MW of 91 000. SDS gel electrophoresis of the fully dissociated protein gave a single band, corresponding to a polypeptide chain with a MW of 22700. A minimal MW of 20700 was estimated from the amino acid composition. On a MW basis the native molecule consists of 4 chains. Support for the tetrameric structure of canavalin is provided by the electrophoretic pattern of partially dissociated protein.     

聚丙烯酰胺凝胶电泳配合荧光增白剂显色检测木霉几丁质酶同工酶谱

为提高木霉几丁质酶检测方法的准确性和灵敏度,建立一种快速检测几丁质酶同工酶的方法。采用活性凝胶电泳、变性凝胶电泳、原位显色凝胶电泳结合荧光增白剂（Calcofluor white M2R）显色从绿色木霉LTR-2发酵产物中检测几丁质酶同工酶。活性凝胶电泳在粗酶液浓缩5倍时显示两条活性谱带,变性凝胶电泳在浓缩10倍时显示一条活性谱带,原位显色凝胶电泳在浓缩20倍时显示两条不清晰的活性谱带,SDS-PAGE显示这两条活性谱带的分子量分别为65kDa和42kDa。结果表明活性聚丙烯酰胺凝胶电泳和Calcofluor white M2R显色相结合的方法在几丁质酶上样量为0.47U时具有较好的分辨能力,是检测木霉几丁质酶同工酶的有效的方法。     

Subunit structure and properties of the glycogen-bound phosphoprotein phosphatase from skeletal muscle

A high molecular weight phosphoprotein phosphatase was purified approximately 11,000-fold from the glycogen-protein complex of rabbit skeletal muscle. Polyacrylamide gel electrophoresis of the preparation in the absence of sodium dodecyl sulfate showed a major protein band which contained the activity of the enzyme. Gel electrophoresis in the presence of sodium dodecyl sulfate also showed a major protein band migrating at 38,000 daltons. The sedimentation coefficient, Stokes radius, and frictional ratio of the enzyme were determined to be 4.4 S, 4.4 nm, and 1.53, respectively. Based on these values the molecular weight of the enzyme was calculated to be 83,000. The high molecular weight phosphatase was dissociated upon chromatography on a reactive red-120 agarose column. The sedimentation coefficient, Stokes radius, and frictional ratio of the dissociated enzyme (termed monomer) were determined to be 4.1 S, 2.4 nm, and 1.05, respectively. The molecular weight of the monomer enzyme was determined to be 38,000 by polyacrylamide gel electrophoresis. Incubation of the high molecular weight phosphatase with a cleavable cross-linking reagent, 3,3'-dithiobis(sulfosuccinimidyl propionate), showed the formation of a cross-linked complex. The molecular weight of the cross-linked complex was determined to be 85,000 and a second dimension gel electrophoresis of the cleaved cross-linked complex showed that the latter contained only 38,000-dalton bands. Limited trypsinization of the enzyme released a approximately 4,000-dalton peptide from the monomers and dissociated the high molecular weight phosphatase into 34,000-dalton monomers. It is proposed that the catalytic activity of the native glycogen-bound phosphatase resides in a dimer of 38,000-dalton subunits.     

Active streptokinase from the cloned gene in Streptococcus sanguis is without the carboxyl-terminal 32 residues

The streptokinase expressed by the cloned gene in Streptococcus sanguis has a molecular weight of about 44 000 [Malke, H., Gerlach, D., Kohler, W., & Ferretti, J.J. (1984) MGG, Mol. Gen. Genet. 196, 360-365] while the molecular weight of the native streptokinase is 47 000. The structural and activity differences of the cloned streptokinase (cSK) as expressed by S. sanguis and the native streptokinase (nSK) were investigated. From a partially purified cSK, two active fractions were obtained by reversed-phase HPLC. The minor fraction cSKL was nearly as active as SK in plasminogen activation. The major fraction cSKs had only about one-fourth of the specific activity. The structures of cSKL and cSKs were studied and compared to the known amino acid sequence of SK [Jackson, K. W., & Tang, J. (1982) Biochemistry 21, 6620-6625]. From the NH2- and COOH-terminal sequences and amino acid composition of the cyanogen bromide (CNBr) fragments, it could be deduced that cSKL and cSKs are without 31 and 32 residues, respectively, from the COOH-terminal end of SK. Since the cloned gene contained the full SK structure, the missing structures must have been due to posttranslational proteolysis. An SK fragment similar in size to cSK was observed from a chymotryptic digest of SK.