Subcellular distribution of enkephalin precursor proteins in rat dental pulp and the usefulness as a substrate for enkephalin-producing enzymes

The subcellular distribution of enkephalin (EK) precursor proteins was investigated to clarify the intracellular site of biosynthesis of EK in rat dental pulp tissue. The contents of met-EK-like peptides in nuclear, microsomal, and supernatant fractions of the pulp tissue were markedly increased after sequential digestion with trypsin and carboxypeptidase B, indicating the enrichment of the precursors in these fractions. Sephadex G-100 gel filtration showed a common peak of the precursor proteins in the homogenate and its microsomal and supernatant fractions, and the molecular weight was determined to be about 58,000 by SDS polyacrylamide gel electrophoresis. Both the partially purified precursor protein from the supernatant fraction and N alpha-benzoyl-DL-arginine-beta-naphthylamide (BANA) were used as substrates for a lysosomal enzyme separated by Sephadex G-75 gel filtration. The major peak of EK-producing activity of the enzyme was identical with that of BANA-degrading activity of the enzyme. These results demonstrate the EK-producing activity of lysosomal proteinase, and also indicate the usefulness of the two substances as substrates for the enzyme.     

Affinity labelling of the allosteric site of the L-lactate dehydrogenase of Lactobacillus casei

We have recently described the addition of 2-keto-3-butynoic acid to flavin-free flavocytochrome b2, a reaction which leads to the loss of flavin-binding capacity ('inactivation') [D. Pompon and F. Lederer (1982) Eur. J. Biochem. 129, 143-137]. For total inactivation, the extrapolated incorporation value was 0.9 mol reagent/mol subunit. In this work we report the results of sequence studies which elucidate the nature of the modification. The modified protein was cleaved with cyanogen bromide and the peptides separated on Sephadex G-100 and SP-Sephadex C-25. 14C-labeled peptides were digested with trypsin and chymotrypsin and smaller labeled fragments purified by chromatography on Sephadex G-50 and thin-layer fingerprinting. It is shown that three cysteine residues are fractionally labeled with nearly complete mutual exclusion. Furthermore, a fraction of the modified peptides is found under the form of cross-linked fragments, where two cysteines have added to the same ketobutynoate molecule. Only two of the possible cross-links were found. These results show that the three cysteines are close to one another in space in the flavin-free enzyme and hence probably also in the holoenzyme. These results, combined with those obtained in the affinity labeling reaction of holoenzyme with bromopyruvate [Alliel et al. (1982) Eur. J. Biochem. 122, 553-558], show that the three residues are located in or close to the active site. Their possible role is discussed.     

Characterization and partial purification of the sodium-potassium-ATPase inhibitor released from cultured rat hypothalamic cells

An inhibitor of sodium-potassium-ATPase has been partially purified from the culture medium obtained from hypothalamic cells maintained in a capillary membrane perfusion system, and some of the properties of this inhibitory factor have been investigated. Gel filtration (Sephadex G-25 Superfine) of heat-treated medium (80 degrees C for 10 min) resulted in elution of inhibitory activity in the post-salt fraction. These fractions inhibited active (i.e. sodium-potassium-ATPase-mediated) sodium transport in intact human erythrocytes, displaced [3H]ouabain from its binding site, and directly inhibited canine kidney sodium-potassium-ATPase as measured by NADH oxidation. High-performance liquid chromatography (on Hypersil ODS) of these fractions after desalting yielded one region which showed inhibitory activity on all three assays. Inhibition of sodium-potassium-ATPase was dose-related and filtered through an Amicon UM10 membrane. Incubation of this material with dispase, carboxypeptidase A, chymotrypsin, and prolidase destroyed inhibitory activity, whereas trypsin and leucine aminopeptidase were ineffective. These studies show that hypothalamic neurones release a low molecular weight heat-stable peptide which inhibits active sodium transport, ouabain binding, and sodium-potassium-ATPase.     

Inhibition of Growth of L5178Y Leukemia Cells by a Fungal Folate-deaminating Enzyme

Reactive sites of adzuki bean proteinase inhibitor II were determined by limited hydrolyses with catalytic amounts of trypsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1] at pH 3.0. Treatment of the trypsin-modified inhibitor with carboxypeptidase B [EC 3.4.12.3] released lysine from the inhibitor and led to complete loss of the activity for trypsin, virtually, without affecting the chymotrypsin-inhibitory activity. Limited hydrolysis with chymotrypsin resulted in a selective cleavage of a single tyrosyi peptide bond in the inhibitor, and treatment of this modified inhibitor with carboxypeptidase A [EC 3.4.12.2] abolished the chymotrypsininhibitory activity, having no effect on the trypsin-inhibitory activity. After reduction and S-carboxymethylation, the trypsin- and the chymotrypsin-modified inhibitors both could be separated into two components by gel-filtration on Sephadex G–50 and DEAE-cellulose chromatography. Amino acid and end group analyses of these components indicated that the reactive sites of inhibitor II are the Lys²⁷-Ser²⁸ bond against trypsin and the Tyr⁵⁴-Ser⁵⁵ against chymotrypsin.

Chemical modification of inhibitor II with cyanogen bromide had a fatal effect on the inhibitory activity against trypsin but no effect against chymotrypsin.     

Isolation and partial characterization of the pancreatic secretory trypsin inhibitor in the rat

Isolation in a 55% yield of the low molecular weight pancreatic secretory trypsin inhibitor was achieved by gel filtration of an acid extract of whole inactive rat pancreas juice on Sephadex G-50 at pH 2.5 followed by desalting and ion-exchange chromatography on SP Sephadex C-50 at pH 4.5. Two distinct chromatographic fractions were obtained, labeled fraction 1 and 2. Fractions 1 and 2 showed three, respectively two, distinct closely migrating cationic bands on gel electrophoresis in barbital buffer, pH 8.6. Each fraction demonstrated one band on polyacrylamide disc electrophoresis at pH 4.6. The inhibitor is homogenous on gel filtration and on the basis of its stoichiometry with active site titrated rat anionic trypsin. Its molecular weight is approx. 6024. The amino acid composition is included. Rat pancreatic secretory trypsin inhibitor is trypsin-specific and interacts on a 1:1 molar basis with rat trypsin. It is good inhibitor of bovine trypsin but a poor inhibitor of human cationic trypsin and its binding to trypsin is reversible by acidification. Like other inhibitors of this sort, it is present in about 0.1–0.2% of the total protein content of the juice, and normally exists in its free form. A simple procedure for the production of antiserum to the inhibitor which is a poor antigen is also described.     

Differential interactions of rabbit plasma alpha-1-antiproteinases S and F with porcine trypsin

Two major forms of rabbit plasma alpha-1-antiproteinase, S and F, were separated by affinity chromatography on Red Sepharose, and their modes of interaction with porcine trypsin were studied. The S form interacted with trypsin much more slowly than the F form, and the resulting complex partially retained the amidolytic and proteolytic activities towards benzoyl-L-arginine p-nitroanilide and remazol brilliant blue hide powder, respectively. This S form-trypsin complex also prevented the inactivation of bound trypsin by soybean trypsin inhibitor. In marked contrast, an equimolar complex of trypsin and the F form retained neither amidolytic nor proteolytic activity. These results suggest that the F form blocks the active site of trypsin while the S form does not bind directly to the active site, thereby preserving the catalytic potential of trypsin. No similar interaction was observed, however, between the S form and either bovine chymotrypsin or porcine pancreatic elastase. Both the S and F forms inactivated these proteinases in a stoichiometric manner with differing inhibitor/proteinase binding ratios. The S form showed about twofold greater capacity to inhibit elastase than the F form, whereas the reverse was the case for chymotrypsin.     

Endogenous thiol protease inhibitor from rat liver

A thiol protease inhibitor was purified from rat liver by a rapid procedure involving heat treatment of the post-lysosomal fraction, affinity chromatography on papain-Sepharose 4B and Sephadex G-75. The purified inhibitor appeared homogeneous on sodium dodecyl sulfate electrophoresis. The inhibitor had a molecular weight of about 11,500 and consisted of three forms (pI 4.9, 5.2 and 5.6). The preparation inhibited thiol proteases, such as papain, cathepsin H, cathepsin B and cathepsin L, but not serine proteases (trypsin, chymotrypsin, mast cell protease and cathepsin A) or cathepsin D.     

Purification and Properties of Neutral Proteinase I from Aspergillus oryzaePurification and Properties of Neutral Proteinase II from Aspergillus oryzae

Neutral proteinase I (the first peak in DEAE-cellulose chromatogrraphy) was purified from the Amberlite IRC-50 adsorbed fraction by chromatography on DEAE-cellulose and gel filtration through Sephadex G-100. It shows an optimum pH of 7.0 for milk casein. The enzyme was found to be stable in the pH range of 5.5 to 12.0. The molecular weight of the enzyme was estimated to be about 41,000 by gel filtration. The enzyme had neither aminopeptidase nor carboxypeptidase activity, but degraded carbobenzoxy-glycyl-phenyl-alanine amide, poly-l-lysine and poly-l,α-glutamic acid. The enzyme was inhibited by ethylenediaminetetraacetate, but not inhibited by diisopropylphosphorofluoridate and potato inhibitor.     

The covalent structure of calf skin type III collagen. V. The amino acid sequence of the cyanogen bromide peptide alpha 1(III)CB9A (position 789 to 927).

The cyanogen bromide peptide alpha 1(III)CB9A is 139 amino acid residues in length and occupies positions 789--927 along the alpha 1(III) chain. Peptides necessary for the complete sequence analysis were obtained after fragmentation of alpha 1(III)CB9B with trypsin, protease from Staphylococcus aureus V8, hydroxylamine and chymotrypsin. They were separated mainly by chromatography on Sephadex G-50 and phosphocellulose and subsequently sequenced using the automated Edman degradation procedure.     

Identification and partial purification of a cation-sensitive neutral endopeptidase from bovine pituitaries.

An apparently new endopeptidase with a pH optimum between 7.0 and 7.5 was purified 600 fold from bovine pituitaries. The enzyme hydrolyzed synthetic substrates such as Cbz-Gly-Gly-Leu-pNA and Cbz-Gly-Gly-Tyr-Leu-pNA by splitting the bond between the leucine residue and p-nitroaniline. Replacement of the leucine residue by alanine, greatly diminished the rate of reaction. Simple model trypsin and chymotrypsin substrates such as Bz-DL-Arg-2NA and N-succinyl-Phe-2NA were not attacked. The enzyme was also inactive toward aminopeptidase and carboxypeptidase substrates. Strong inhibition of the enzyme was observed at relatively low concentrations of sodium and potassium ions. Leupeptin, pepstatin and phenylmethylsulfonylfluoride had no effect on enzyme activity, however inhibition was obtained with p-mercuribenzoate. Preliminary experiments with filtration through Sephadex G-100 columns suggest a molecular weight in excess of 100,000.     

Measurement of Arginine Carboxypeptidase-Generating Activity of Adult Plasma

Arginine carboxypeptidase (CPR) is a novel carboxypeptidase which was first described by Campbell and Okada. CPR is generated from a stable precursor of CPR (proCPR) during coagulation or under other circumstances and is promptly inactivated at 37 C. Therefore, it is not easy to determine CPR in blood samples. Since proCPR can be separated from the other basic carboxypeptidase (carboxypeptidase N; CPN) by passing plasma through DEAE gel, we have established a method to determine the amount of proCPR after converting it to active CPR by trypsin treatment. We first separated the proCPR from CPN using a filter cup tube (FC tube) packed with DEAE Sephadex, and measured activity after conversion of the enzyme to its active form using trypsin. With this method, no significant decrease in proCPR was noted in the plasma of patients including those with rheumatoid arthritis (RA), although CPR activity in fresh sera has been reported to be decreased. This discrepancy suggests that proCPR is not depleted in most patient sera, but that the level of activity of the enzyme which converts proCPR into active CPR may be compromised in RA patients.     

去B链羧端三肽人胰岛素的分离纯化及其性质研究

将去Ｂ链羧端三肽人胰岛素原基因克隆到表达质粒ｐＢＶ２２０上,在大肠杆菌系统中经温度诱导表达,表达产物占细胞总蛋白量的１２％,表达产物经ＳｅｐｈａｄｅｘＧ－５０柱层析分离以及胰蛋白酶和羧肽酶Ｂ的酶促转化等步骤,可得到去Ｂ链羧端三肽人胰岛素,其纯度达９３％,其氨基酸组成与预期值相符,但其受体结合活性仅是标准猪胰岛素的４５％．     

Partial purification and characterization of Gigantocotyle explanatum somatic antigens

Soluble extracts of Gigantocotyle explanatum, isolated from the liver of buffalo Bubalus bubalis were fractionated on Sephadex G-200 columns. Nine major fractions referred to as F1, F2, F3, F4, F5, F6, F7, F8 and F9 were separated. Each fraction was tested by ELISA for antigenicity using sera from G. explanatum-infected field buffaloes. Fractions F1 and F2 were highly antigenic, F3, F4, F6 and F7 were moderately antigenic and F5, F8 and F9 were poorly antigenic. Analyses by SDS-PAGE revealed that each fraction comprised several polypeptide(s) in the molecular weight range of <29 to >205 kDa. Results of Western blotting indicated that not all polypeptides which appeared in the SDS-PAGE were antigenic. The antigenic molecules of each fraction were mostly in the low molecular weight range of <14 to >94 kDa with the polypeptides in the range of >14, 14, 18, 21-25 and 34-36 kDa.     

Stimulation of phagocytic function by factors inhibiting leukocyte and macrophage migration in humans

Fractions containing macrophage migration inhibition factor (MIF) and leucocyte migration inhibition factor (LIF) were obtained using Sephadex G-200 filtration from supernatant fluids of human lymphocyte cultures stimulated by PHA. The fractions were tested for the ability to affect migration and phagocytic activity of target cells. Peripheral blood leucocyte migration capacity was inhibited by the fraction with the molecular mass of 60,000-70,000 D (LIF), while migration activity of mouse peritoneal exudate cells was suppressed by the fraction with the molecular mass of 20,000-30,000 D (MIF). MIF- and LIF-containing fractions increased almost three-fold Fc-receptor-mediated phagocytic activity of neutrophils.     

Separation of the Proteolytic and Streptolytic Components of a Streptomyces albus Enzyme Preparation and Their Effect on Streptococcal Cells

The complex of enzymes elaborated by a strain of Streptomyces albus was separated by Sephadex G-75 column chromatography. Two major and one minor proteolytic peaks and one streptolytic peak were obtained. The streptolytic fraction alone did not cause lysis of the cells, but was highly effective in the presence of small amounts of trypsin or of the proteolytic components of the enzyme complex. Treatment of cells with the purified streptolytic fraction effected release of group carbohydrate and of a trypsin and pepsin-resistant protein antigen which reacted with type-specific antisera.     

Structure of shigella antigens. Heterogeneity of specific polysaccharides of 2 Shigella flexneri strains and 2 Sh. flexneri/Escherichia coli hybrids]

The S-specific polysaccharide from 2 Sh. flexneri wild strains (with serological var. X- and var. Y-specificity, respectively) and 2 Sh. flexneri E. coli hybrids (with the same specificities) can be separated by means of gel chromatography on Sephadex G-200 and G-50 into altogether 6 fractions per strain. Fraction G-200/1 (molecular weight greater than 10(6)D) represents a polymer consisting nearly exclusively of glucose and is present mainly in the two Y-type strains, much less in the two X-type strains. Fractions G-200/2 and G-200/3 (molecular weight approximately 10(5)D and approximately 2 - 10(4)D, respectively) seem to consist mainly of the S-specific side chains while fraction G-50/2 (molecular weight approximately 2000 D) presumably contains an SR-polysaccharide (core with one repeating unit.) Fraction G-50/3 (molecular weight approximately 100 D) contains the core polysaccharide and fraction G-50/4 splitting products (mainly KDO). No significant differences in chromatographical behaviour and quantitative composition could be found between the polysaccharides of the wild strains and the hybrid strains. Because of the well-known stability of the glucosaminyl linkages the sugar analysis was not only performed after acidic hydrolysis. In some cases the acid hydrolysate was reacted with HNO2 to cleave the glucosaminyl linkages. In most cases the values obtaines now were higher than those obtained directly.     

鹰嘴豆种子胰蛋白酶抑制剂的分离纯化与鉴定

为了寻找具有药物作用的天然胰蛋白酶抑制物,采用硫酸铵分级沉淀、离子交换层析(DEAE-纤维素52)及Sephadex G-100凝胶层析等方法, 从鹰嘴豆种子中分离出一种鹰嘴豆胰蛋白酶抑制剂（CPTI）. 研究表明：CPTI对胰蛋白酶有较强的抑制作用,抑制率达80%,而对胰凝乳蛋白酶抑制作用较弱,抑制率为32%, 对胃蛋白酶、木瓜蛋白酶及枯草杆菌蛋白酶均无抑制作用; 用SDS-PAGE测得CPTI近似分子质量为25.7 kD; CPTI具有较高的热稳定性,在100 ℃下加热60 min,对胰蛋白酶活性仍保持78%抑制率; Lineveaer-Burk作图得知该抑制剂属竞争性抑制类型. 动力学测定显示,来自鹰嘴豆中的CPTI对胰蛋白酶的抑制作用常数(Ki)为3.99×10^-7 mol/L.     

Antiproteinase activity of alpha 2-macroglobulin

Blood serum separation by the method of gel filtration on Sephadex G-200 with the subsequent immunochemical determination of the quantitative content of basic proteolysis inhibitors permitted isolating the alpha 2-macroglobulin fraction while alpha 1-antitrypsin and alpha 1-antichymotrypsin separation was a failure. The immunochemical analysis of the antienzymic activity of the isolated inhibitors showed that 32.3 +/- 3.5% of the introduced kallikrein, 18.7 +/- 0.6% of trypsin and 14.4 +/- 4.1% of chymotrypsin were bound in the zone of alpha 2-macroglobulin. The rest of antienzymic activity was localized in the zone of alpha 1-antitrypsin and alpha 1-antichymotrypsin. After a preliminary saturation of blood serum with trypsin in the amount equivalent to its antitryptic capacity (200 micrograms/ml) the ability of alpha 2-macroglobulin to bind kallikrein and chymotrypsin lowers considerably (by 69 and 72%, respectively). In the zone of alpha 1-antitrypsin and alpha 1-antichymotrypsin a decrease in the ability to bind kallikrein and chymotrypsin amounted to 44 and 12% respectively. Thus, alpha 2-macroglobulin being bound with trypsin looses considerably its ability to bind other enzymes.     

Insulin-like substance and insulin-degrading complex in hemolysates of human erythrocytes

Human erythrocyte lysate was fractionated on various gel filtration media and immunoreactive insulin, insulinase and the influence of individual fractions of the insulin-degrading activity were determined. The hemolysate was shown to contain a complex of substances including an insulin-like substance, insulinase, protease inhibitor and insulinase activator. The insulin-like substance eluted from a Sephadex G-50 column in the same manner as native insulin, and its concentration exceeded the plasma level. Insulinase (Mr 100,000) degraded insulin to the trichloroacetic acid soluble fragments but did not degrade protein or glycoprotein hormones from human pituitaries. Insulinase was inhibited by low temperature, aprotinin and by a newly discovered protease inhibitor from erythrocytes which also inhibits serine proteases--trypsin and chymotrypsin. Another newly discovered substance eluted from a Sephadex G-100 column in the region of low molecular weight substances and showed an insulinase activating activity. The elution patterns of the protease inhibitor and insulinase activator suggest the possibility of the presence of more than one inhibiting and activating factor. The experimental results suggest that the insulin-degrading complex plays a role of a regulator of plasma insulin level. The nonpancreatic origin of the insulin-like substance is also possible.     

Partial separation of the pituitary proteins of Labeo umbratus, Smith (mudfish)

L. umbratus pituitary glands were partially separated by means of preparative polyacry-lamide electrophoresis and chromatography on DEAE-cellulose and Sephadex G-50. Eight fractions were obtaìned and it was found that fraction 1 contained potent adrenocorticotropic stimulating activity, with a molecular weight in the region of 6000. Fractions 3 and 4 contained potent gonadotropic and a lesser degree of exopthalmic producing activity, with molecular weights in the region of 14 000 to 20 000. Fraction 6 displayed pigeon-crop stimulating activity, with a molecular weight above 30 000. Fractions 7 and 8 displayed M.S.H. and vasopressinoxytocic activity respectively, with a molecular weight in the region of 5500–6500. Fractions 2 and 5 could not be identified. The fractions seemed to be glycoproteins and the results are discussed in relation to previous findings.