Purification and characterization of fish surface mucin

Fish surface mucin from Pampus argenteus was extracted with different organic solvents and the residue passed through Sephadex G-200. The major peak was purified by DEAE-Cellulose chromatography and five fractions were obtained. Carbohydrate and protein contents showed that major peak is a glycoprotein. Rechromatography of this component on the Sephadex G-200 column gave a single peak, with an estimated minimal molecular weight of 6.9 X 10(5). Analysis of individual sugar components revealed the presence of galactose, glucose, mannose, arabinose, N-acetyl glucosamine, N-acetyl galactosamine and sialic acid. The most represented amino acids are threonine, serine, proline, glutamic acid and glycine. The N-terminal amino acid end was blocked. Nearly 47% of sulphate was acid labile. Sialic acid and fucose were released rapidly by mild acid hydrolysis. The presence of blood group-A activity suggests that some kind of terminal alpha-Gal-NAC may be present.     

Endopeptidase in human cerebrospinal fluid which cleaves proenkephalin B opioid peptides at consecutive basic amino acids

An endopeptidase releasing the common N-terminal hexapeptide, (Leu)-enkephalin-Arg6, from dynorphins A and B, and alpha-neoendorphin was purified from human cerebrospinal fluid. Purification involved ion-exchange chromatography (DEAE-Sepharose CL-6B), hydrophobic interaction chromatography (phenyl-Sepharose CL-4B) and molecular sieving (Sephadex G-100). The enzyme showed molecular heterogeneity. A major fraction had an apparent molecular weight of about 40,000. It had an optimum activity in the pH range of 6-8. The conversion of dynorphin A was not affected by EDTA or iodoacetate but strongly reduced in the presence of phenylmethyl-sulphonyl fluoride, suggesting the enzyme is a serine protease.     

Reconstitution of rapeseed high molecular weight protein from isolated subunits

The high molecular weight protein was isolated from rapeseed and characterised. Six subunits were isolated in SDS (0.01%) solution on polyacrylamide-gel electrophoresis and by gel filtration on Sephadex G-100. Reassociation by removing SDS by co-dialysis, against 10 mM sodium phosphate buffer (pH 7.9) was done and the yield was about 90%. The reconstituted protein was indistinguishable from the intact protein in all respects. The subunits isolated from the native protein and the reconstituted protein were found to have identical molecular weights and N-terminal amino acids. No disulphide bonds were observed in the subunit association. Amino acid analysis of the proteins and the six subunits was performed and the number of each amino acid residue calculated.     

Isolation and characterization of some of the proteins that shuttle between cytoplasm and nucleus in Amoeba proteus

The Rapidly Migrating Proteins (RMP) which shuttle nonrandomly between nucleus and cytoplasm and equilibrate in approximately equal amounts in each compartment, were isolated from Amoeba proteus by implanting ³H-protein containing nuclei into unlabeled cells and some time later extracting the labeled material from the cytoplasms of such cells. The labeled material was subsequently fractionated by gel filtration in Sephadex G-100 columns. The RMP are soluble in dilute salt solutions and appear as a heterogenous group of molecules, one component of which seems to be a single species of protein accounting for ca. one-third of the RMP fraction. Because of its distinctness this component, called the LR fraction, received the major attention in this study. LR was found to comprise ca. 17% of the aqueous-soluble proteins of the nucleus and ca. 3–4% of the total cell protein. LR has a very low molecular weight as determined, e.g., by its elution from a Sephadex G-100 column. Because of its low molecular weight, LR could be purified by taking advantage of the fact that LR is (1) soluble in a saturated Solution of ammonium sulfate and (2) insoluble in butanol, diethyl ether, and 10% trichloroacetic acid. LR migrates toward the anode as a single band when subjected to electrophoresis on “standard disc” and SDS polyacrylamide gels. It does not enter a gel designed to separate basic proteins (at pH 4.0). When subjected to Sephadex G-25 gel filtration LR migrates through the gel as a single band and elutes from the gel at a position in the middle of the linear separation range that indicates its molecular weight is ca. 2300. The only N-terminal amino acid found in the LR fraction is proline. Evidence is presented to show that LR is not the product of a non-specific breakdown of protein produced during its isolation, but the possibility that it results from the cleavage of a single chemical bond of a larger polypeptide, has not been eliminated. When injected into non-labeled amebae, purified radioactive LR concentrates in the nucleus — just as radioactive RMP concentrates in a recipient cell nucleus when an amino acid-labeled nucleus is implanted into an unlabeled cell.     

Properties of the thioesterase component obtained by limited trypsinization of the fatty acid synthetase multienzyme complex.

The fatty acid synthetase from lactating rat mammary gland is shown to consist of two polyfunctional polypeptides of similar molecular weight (about 220,000); a 4'-phosphopantetheine residue is covalently bound to one, or both subunits. Limited trypsinization of the fatty acid synthetase releases on enzymatically active thioesterase component which has been purified and its properties studied. The thioesterase sediments in the ultracentrifuge as a single component of molecular weight 32,000; its sedimentation coefficient is 2.9 x 10-(13) s its diffusion coefficient 5.0 x 10-(7) cm2 s-(1). The thioesterase also elutes from a column of Sephadex G-75 as a single, symmetrical peak of constant specific activity. However, electrophoresis of the denatured thioesterase in the presence of sodium dodecyl sulfate reveals that the enzyme has been partially nicked during isolation. The kinetic data of the enzyme reaction were studied using palmityl-CoA as a model substrate. Solvent pH was found to affect both Vmax and Km (Km = 0.5 micron at pH 6.6, 2.5 micron at pH 8.0) wereas solvent ionic strength affected Vmax but no Km. The thioesterases from the fatty acid synthetases of rat liver and lactating mammary gland have identical physical properties, identical amino acid compositions, and are immunologically indistinguishable. Both thioesterases hydrolyze long chain, in preference to short chain, thioesters of CoA, an observation consistent with their role in regulation of the chain-terminating step in fatty acid synthesis by the parent multienzyme complexes.     

The structure and mechanism of stem bromelain. Evaluation of the homogeneity of purified stem bromelain, determination of the molecular weight and kinetic analysis of the bromelain-catalysed hydrolysis of N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester

1. Purified stem bromelain (EC 3.4.22.4) was eluted from Sephadex G-100 as a single peak. The specific activity across the elution peak was approximately constant towards p-nitrophenyl hippurate but increased with elution volume with N(2)-benzoyl-l-arginine ethyl ester as substrate. 2. The apparent molecular weight, determined by elution analysis on Sephadex G-100, is 22500+/-1500, an anomalously low value. 3. Purified stem bromelain was eluted from CM-cellulose CM-32 as a single peak and behaved as a single species during column electrophoresis on Sephadex G-100. 4. Purified stem bromelain migrates as a single band during polyacrylamide-gel electrophoresis under a wide variety of conditions. 5. The molecular weight determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate is 28500+/-1000. 6. Sedimentation-velocity and equilibrium-ultracentrifugation experiments, under a variety of conditions, indicate that bromelain is an apparently homogeneous single peptide chain of mol.wt. 28400+/-1400. 7. The N-terminal amino acid composition is 0.64+/-0.04mol of valine and 0.36+/-0.04mol of alanine per mol of enzyme of mol.wt. 28500. (The amino acid recovery of the cyanate N-terminal amino acid analysis was standardized by inclusion of carbamoyl-norleucine at the cyclization stage.) 8. The pH-dependence of the Michaelis parameters of the bromelain-catalysed hydrolysis of N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester was determined. 9. The magnitude and pH-dependence of the Michaelis parameters have been interpreted in terms of the mechanism of the enzyme. 10. The enzyme is able to bind N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester relatively strongly but seems unable to make use of the binding energy to promote catalysis.     

一种新阿片肽的分离纯化

报道了一种新阿片肽OP1的分离纯化。将重组毕赤酵母经适宜的生长和表达培养后,所得的发酵液经离心得无细胞上清液,上清液经超滤后过Sephadex G-10柱。将经Sephadex G-10柱所得具有阿片活性的粗组分用HPLC-MS分析,根据阿片肽N-端均有一个酪氨酸残基,且在肽链的第三或第四位上有一个芳香族氨基酸残基这一性质,依据分子量确定活性组分中可能存在的所有阿片肽,然后根据这些阿片肽的等电点,利用AKTA Purifier 100快速纯化系统的DEAE-阴离子交换纤维素柱将其进一步分离,活性组分再用Sephasil peptide C18反相高压液相柱分离得到活性组分OP1肽,鉴定纯度后测定其氨基酸组成。最后确定该肽的一级序列为YPFPGPIRYG,该阿片肽序列目前尚未见报道。     

Zinc binding in cow''s milk and human milk

In both cow's milk and human milk, zinc was associated with proteins of high molecular weight (greater than 100 000), as judged by analysis with Sephadex G-75. Precipitation of the casein at pH 4.6 and filtration of the resultant acid whey on Sephadex G-25 led, however, to the recovery of about 90% of the zinc as a compound of low molecular weight, which was tentatively identified as zinc citrate. Over 95% of the zinc of cow's milk was sedimented with the casein micelles on ultracentrifugation. Filtration of these micellar caseins on Sephadex G-150 gave two peaks containing zinc, which corresponded to aggregates of alpha-casein-kappa-casein and of alpha-casein-beta-casein. Ultracentrifugation of human milk sedimented only approx. 40% of total zinc. Analysis of sediment and supernatant on Sephadex G-150, however, indicated that about 85% of the zinc was associated with a protein complex of molecular weight greater than 150 000. The major protein of this complex was identified as lactoferrin. A minor zinc-binding component of average molecular weight 30 000 was also observed in the supernatant. The results indicated that zinc is bound to different macromolecules in cow's and human milk. This may be a factor affecting the bioavailability to the human infant of zinc from the two milks, and it is suggested that in human milk lactoferrin may be involved in the uptake of zinc.     

Studies on epsilon-prototoxin of Clostridium perfringens type D. Physicochemical and chemical properties of epsilon-prototoxin.

epsilon-Prototoxin of clostridium perfringens type D consists of one polypeptide chain of 311 amino acids with the following composition: Asp52 Thr31 Ser25 Glu28 Pro12 Gly17 Ala14 Val28 Met5 Ile15 Leu18 Tyr17 Phe8 Lys31 His2 Arg5 Tyr2. It has no free cysteine but contains one blocked cysteine. The N-terminal as well as the C-terminal residue is lysine. The ultracentrifuge pattern gave one single boundary having S020,w = 2.15 S and Do20,w = 5.56-10(-7) cm2/s. Calculation of the molecular weight from D020,w and S020,w gave a value of 34 250. The molecular weight determined from sedimentation equilibrium using ultraviolet optics gave a value of 33 000 +/- 1000. On the other hand molecular weights calculated from a calibrated Sephadex G-100 column in borate buffer was 25 000 and that in sodium phosphate, ionic strength 0.2, was 27 500. This discrepancy between values obtained in the ultracentrifuge and by gel filtration is attributed to adsorption of epsilon-prototoxin by Sephadex.     

Multiple forms of barley root acid phosphatase: purification and some characteristics of the major cytoplasmic isoenzyme

The major acid phosphatase form (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) was purified from the soluble extract of barley roots. The enzyme is homogeneous on polyacrylamide gel electrophoresis and moves as a single band of Mr approximately 38,000 in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme was Mr 77,600 and 79,000 as determined, respectively, by gel filtration on a Sephadex G-100 column and by density gradient ultracentrifugation. The isoelectric point was about 6.28. The enzyme is competitively inhibited by molybdate (Ki = 9 x 10(-7) M). NaF, Ag(+), Hg(2+), Pb(2+) and Zn(2+) are also inhibitors, while other cations showed no effect. The enzyme hydrolyzes a wide variety of natural and synthetic phosphate esters. In particular, the enzyme seems to be active on ATP, o-phosphotyrosine, o-phosphoserine and glucose 1-phosphate. The pH dependence studies between pH 4-8 using p-nitrophenylphosphate as substrate and diethylpyrocarbonate inactivation indicate the presence of essential histidine residue at the active site.     

蓖麻β-半乳糖苷酶的纯化及其基本性质

蓖麻籽黄化苗中存在高活性β-半乳糖苷酶。经硫酸铵分级分离、DEAE-纤维素离子交換层析、Sephadex G-100、CM-Sephadex和DEAE-Sephadex层析纯化。活性收率为6.4％,纯化倍数达107倍。纯化了的酶经聚丙烯酰胺凝胶电泳显示单一蛋白带,SDS-PAGE显示两条蛋白带,其相应分子量分别为3.25×10~4和2.94×10~4。用Sephadex G-200分子筛层析法测得分子量为6.7×10~4。综合上述结果推测该酶是由两个不同的亚基构成。以邻硝基苯酚-β-半乳糖苷为底物测得该酶的表观Km为5.9×10~(-3)mol/L。最适pH和最适温度分别为4.5和50℃。酸碱稳定区域在pH4.6—7.5之间。不同浓度缓冲液以及不同种类缓冲液、不同金属离子对酶活性影响均进行了讨论。     

Characterization of the multiple forms and main component of dextransucrase from Leuconostoc mesenteroides NRRL B-512F

Multiple forms of dextransucrase (sucrose:1.6-alpha-D-glucan 6-alpha-D-glucosyltransferae EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F strain were shown by gel filtraton and electrophoretic analyses. Two components of enzyme, having different affinities for dextran gel, were separated by a column of Sephadex G-100. The major component voided from the Sephadex column was treated with dextranase and purified to an electrophoretically homogeneous state. The ]urified enzyme had a molecular weight of 64 000-65 000, pI value of 4.1, and 17% of carbohydrate in a molecule. EDTA showed a characteristic inhibition on the enzyme while stimulative effects were observed by the addition of exogenous dextran to the incubation mixture. The enzyme activity was stimulated by various dextrans and its Km value was decreased with increasing concentration of dextran. The purified enzyme showed no affinity for a Sephadex G-100 gel, and readily aggregated after the preservation at 4 degrees C in a concentrated solution.     

常山凝集素的分离纯化及其性质研究

 本文采用分子筛层析和离子交换技术,从植物常山(Dichroa febrifuga Lour)的鲜叶中,分离纯化出一种新的凝集素,定名为常山凝集素(DFL)。并对其理化性质进行了鉴定:测得其亚基分子量为37,000道尔顿;等电点为4.2。DFL是一种糖蛋白,糖含量为2.8％。DNS-CI法测得其肽链的N-末端为L-缬氨酸。本文还对其糖专一性、不同动物红细胞的凝集专一性,以及对猪精子和某些肿瘤细胞的凝集活性等生物学性质进行了研究。     

The purification and properties of microsomal palmitoyl-coenzyme A synthetase

1. Cell-free culture filtrates of the fungus Fusarium solani were examined for homogeneity with respect to beta-d-glucosidase and C(x) activities. 2. o-Nitrophenyl beta-d-glucoside and cellobiose were both used as substrates for beta-d-glucosidase activity. 3. No evidence for the non-identity of nitrophenyl beta-d-glucosidase and cellobiase activities could be found, either by heat treatment, gel filtration on Sephadex G-100 or by isoelectric focusing. 4. The beta-d-glucosidase component was also a feeble exo-beta-glucanase: it had a molecular weight of approx. 400000. 5. The fall in viscosity of a solution of CM-cellulose, the formation of reducing sugars in a solution of CM-cellulose and the solubilization of phosphoric acid-swollen cellulose (Walseth cellulose), were all used for the measurement of C(x) activity. 6. The ratio of the two types of CM-cellulase activity was not changed after gel filtration on Sephadex G-100 or after chromatography on DEAE-Sephadex. 7. Three peaks of C(x) activity were obtained after electrofocusing, but all three possessed the same ratio of the two types of CM-cellulase activity as well as the same CM-cellulase/Walseth activity ratio, as the unfractionated enzyme; all three isoenzymes (isoelectric points, 4.75, 4.80-4.85 and 5.15) acted in synergism with a mixture of the C(1) and the beta-d-glucosidase components to the same extent in the solubilization of cotton fibre. 8. The molecular weight of the C(x) component was approx. 37000.     

粘质赛氏菌胞外蛋白酶的提取、纯化及部分性质研究

经过硫酸铵３０％～５０％分级沉淀、二步柱层析可获聚丙烯酰胺凝胶电泳均一的粘质赛氏菌胞外蛋白酶制品,收率可达５３％,并制备了酶的结晶,该酶以ＳｅｐｈａｄｅｘＧ１００柱层析及ＳＤＳ－ＰＡＧＥ测得分子量约为８１０００,该酶的最适ｐＨ为７．０,最适温度为４５℃,Ｚｎ２＋、Ｍｎ２＋、Ｆｅ２＋、Ｃｕ２＋、Ｃｏ２＋等重金属离子不同程度地抑制酶活性。     

Structural features of naegeli amylodextrin as indicated by enzymic degradation

Naegeli amylodextrin is the insoluble residue remaining after prolonged treatment of native starch granules with strong aqueous acid. The Naegeli amylodextrin from waxy maize starch was separated by gel chromatography on Sephadex G-50 into three fractions. Although the fractions were heterogeneous, their average structures were examined by enzymic degradation with porcine-pancreactic alpha amylase, beta-amylase, and pullulanase. The results show that Fraction I (highest molecular weight) has complex branching, Fraction II (major component, d.p. ～25) contains about one branch per molecule, and Fraction III (d.p. ～12) is mostly linear. Formation of these acid-resistant fractions may be explained as arising from a cluster model of amylopectin in which the outer chains are in a double-helical, crystalline arrangement.     

Purification and properties of a lipid acyl-hydrolase from potato tubers.

1. A pure lipid acyl-hydrolase was prepared from potato tubers by acetone precipitation, Sephadex G-100 and DEAE-Sephadex A-50 column chromatography, and by electrofocusing. 2. The purified enzyme was an acidic protein of pI 5.0 and molecular weight of about 70 000. Km values were 0.38 mM for monogalactosyldiacylglycerol and 1.7 mM for phosphatidylcholine. 3. The hydrolytic activity of the enzyme on different substrates was determined. The relative rates were acylsterylglucoside greater than monogalactosyldiacylglycerol greater than monogalactosylmonoacylglycerol greater than digalactosyldiacylglycerol greater than diagalactosylmonoacylglycerol, while the rates for phospholipids were lysophosphatidylcholine greater than phosphatidylcholine greater than lysophosphatidylethanolamine greater than phosphatidylethanolamine. 4. Analyses of enzymatic hydrolysis products suggested that a single enzyme had both galactolipase and phospholipase activities, and for the phospholipids it showed activities similar to phospholipase B and glycerylphosphorylcholine diesterase. 5. A competitive relation was found between monogalactosyldiacylglycerol and phosphatidylcholine as substrates of the enzyme, indicating that the active sites for both substrates may be the same. 6. It was suggested that histidine and probably serine residues were important to the enzymic activity, and that a tyrosine residue might be involved in the activity as an accessory component.     

Starch phosphorylase from tapioca leaves: Absence of pyridoxal phosphate

Starch phosphorylase from tapioca leaves has been purified to homogeneity, using the technique of ammonium sulfate fractionation, heat treatment, DEAE-cellulose chromatography, filtration through Sephadex G-100 and Sephadex G-200, and DEAE-Sephadex chromatography. The enzyme has a molecular weight of 450,000, as determined by gel filtration through Sephadex G-200 and contains 22 sulfhydryl groups per mole of the enzyme protein. Several types of evidence indicate the absence of pyridoxal 5′-phosphate as a prosthetic group of the enzyme. The kinetic data show a sequential type of the reaction mechanism. The enzyme activity is inhibited by tyrosine (K_i = 2.15 mm).     

The cell wall of Bacillus licheniformis N.C.T.C. 6346. Isolation of low-molecular-weight fragments from the soluble mucopeptide

1. Soluble mucopeptide was prepared by lysozyme treatment of acid-extracted walls of Bacillus licheniformis N.C.T.C. 6346 and separated into fractions differing in molecular size by chromatography on Sephadex G-25 and G-50. 2. About 16% of the weight of soluble mucopeptide has a weight-average molecular weight in excess of 20000. About one half has a weight-average molecular weight of less than 2000 and the balance of soluble mucopeptide is of intermediate size. 3. In the mucopeptide fractions isolated from Sephadex there is a correlation between the weight-average molecular weight, the number of non-reducing muramic acid residues and the proportion of diaminopimelic acid residues recovered after treatment with 1-fluoro-2,4-dinitrobenzene. 4. The extent of cross-linking between peptide side chains is relatively low, even in mucopeptide material of the large molecular size. 5. The small amount of residual phosphorus present in preparations of B. licheniformis soluble mucopeptide remains associated mainly with mucopeptide material of large molecular size. 6. The mucopeptide components of lowest molecular weight are not produced as artifacts during the preparation of soluble mucopeptide, but are apparently incorporated in the insoluble mucopeptide present in walls of exponentially growing cells. 7. Soluble mucopeptide isolated in a complex with acidic polymers after lysozyme treatment of walls of B. licheniformis N.C.T.C. 6346 and Bacillus subtilis W23 retains a high molecular weight when the covalent bonds between mucopeptide and the acidic polymers are broken. 8. Pure fragments were isolated from B. licheniformis soluble mucopeptide. A major component, C1, of the material of smallest size is made up of one residue each of N-acetylglucosamine, N-acetylmuramic acid, l-alanine, glutamic acid and diaminopimelic acid. The N-acetylglucosamine is in beta-glycosidic linkage with a reducing N-acetylmuramic acid residue. The peptide unit is probably amidated. A quantitatively minor component, C2, has amino acid and amino sugar composition identical with that of component C1, but probably lacks an amide group. Another fragment, B1, is made up of two molecules of component C1 or C2 that are joined together through a molecule of d-alanine.