Porcine high molecular weight kininogen: its purification and properties as a thiol proteinase inhibitor as compared to human high molecular weight kininogen

1. High mol. wt kininogen (HMW kininogen) was purified to a homogeneous state from porcine plasma. 2. The protein exhibited a strong inhibitory activity for thiol proteinases such as ficin, papain and calpain I, whereas it did not inhibit serine proteinases, trypsin and chymotrypsin. 3. The mol. wt, isoelectric point, amino acid and carbohydrate compositions, stabilities to temperature and pH, kinetic constants, and immunological properties of the porcine HMW kininogen were determined and compared with those of human HMW kininogen.     

Isolation and some molecular parameters of elastase-like normal proteinases from horse blood leucocytes.

Cytoplasmic granules were isolated from horse blood polymorphonuclear leucocytes by the heparin method and extracted with 0.9% NaCl by repeated freezing. Soluble proteins were separated on a column of Sephadex G-75 followed by chromatography on a column of CM-Sephadex with a NaCl gradient. Gel filtration, density-gradient centrifugation, isoelectric focusing and 0.1% sodium dodecyl sulphate/polyacrylamide-gel electrophoresis at pH 7.0 and at pH 4.5 were used to determine molecular parameters of proteinases. Three enzymes hydrolysing both casein and N-benzyloxycarbonyl-L-alanine nitrophenyl ester were found in the granule extract: proteinase 1, mol.wt. 38000, pI5.3; proteinase 2A, mol.wt. 24500, pI8.8; and proteinase 2B, mol.wt. 20500, pI above 10. The latter two elastase-like proteinases were purified to apparent homogeneity.     

Detection and characterization of epidermal proteinases by polyacrylamide gel electrophoresis

Electrophoresis of cornified cell extracts of 2-day-old rats, using SDS polyacrylamide gels copolymerized with alpha-casein or gelatin with or without plasminogen, was performed. Both Tris-HCl buffer soluble protein and KSCN solubilized protein contained a number of hydrolases for alpha-casein and/or gelatin, but PA (mol. wts 57 and 50K) was found only in the KSCN extract. The pH dependency, substrate specificity and mol. wt of plasminogen-independent proteinases were comparatively determined and DFP inhibition tested. This simple technique helped to identify the presence of several proteinases and to characterize them in partially purified epidermal extracts.     

Enzymatic digestion of human plasma inter-alpha-trypsin inhibitor by snake venom metalloproteinases

Incubation of human plasma inter-alpha-trypsin inhibitor with crotalid, viperid, colubrid or elapid venoms resulted in random cleavage of the intact inhibitor (200,000 mol. wt) and formation of inhibitor of 130,000, 77,000, 58,000, and 38,000 mol. wt, along with several minor products. The overall patterns of digestion varied among the venoms studied. However, a 77,000 mol. wt inhibitor cleavage product was formed by all venoms tested, and this fragment was resistant to proteolysis even after a 24 hr incubation with the venoms. Venom pre-treated with phenylmethylsulfonyl fluoride digested inter-alpha-trypsin inhibitor; however, pre-treatment with EDTA completely stopped the reaction, indicating that venom metalloproteinases were responsible for the inhibitor digestion. The inhibitor cleavage products retained the ability to inhibit trypsin, but had no inhibitory activity against venom proteinases.     

Purification of an NADH-(dichlorophenol-indophenol) oxidoreductase from Bacillus stearothermophilus.

The crystals of the entomocidal protein of Bacillus thuringiensis are admixed with proteinases that in the course of their dissolution cause gradual degradation of the "genuine" crystal-forming protein components (i.e. the primary biosynthetic products) to products of lower molecular weight. This phenomenon might explain at least partially the contradictory data on the molecular parameters of the crystal-forming proteins. Preliminary inactivation of the proteinases adsorbed on the crystals allowed us to eliminate this source of the artefacts and to gain more reliable data on the protein composition of the crystals formed by various strains of B. thuringiensis. It has been shown that the crystals formed by all serotypes of B. thuringiensis, with the exception of the serotype V, contain only one protein with a mol. wt. of 145000, 135000 or 130000, depending on the strain. The majority of the strains that belong to the serotype V form crystals consisting of two proteins with mol. wts. of 135000 and 130000, but some of them also have a third component with a mol. wt. of 65000.     

Spectrophotometric assay, solubilization and purification of brain 2'':3''-cyclic nucleotide 3''-phosphodiesterase.

1. A spectrophotometric assay of 2':3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37) based on the use of an acid-base indicator and a buffer having identical pKa values is described. The assay is simple and rapid; it was particularly convenient for monitoring the enzyme activity at various stages of purification. 2. Several proteinases were examined for their ability to solubilize 2':3'-cyclic nucleotide 3'-phosphodiesterase from delipidated brain white matter. Trypsin (EC 3.4.21.4) and elastase (EC 3.4.21.11) appeared to be more effective than the other proteinases examined. Trypsin, however, caused inactivation; elastase was therefore chosen to solubilize 2':3'-cyclic nucleotide 3'-phosphodiesterase. When a partially purified preparation of 2':3'-cyclic nucleotide 3'-phosphodiesterase was treated with elastase, 2':3'-cyclic nucleotide 3'-phosphodiesterase was solubilized nearly quantitatively. Elastatinal, a specific inhibitor of elastase, specifically inhibited the solubilization with elastase. 3. 2':3'-cyclic nucleotide 3'-phosphodiesterase was purified from bovine brain white matter by: (i) delipidation; (ii) solubilization with hexadecyltrimethylammonium bromide; (iii) gel chromatography on Sepharose; (iv) ethanol precipitation and resolubilization by digestion with elastase; (v) chromatography on DEAE-Sephadex; (vi) affinity chromatography on 8-(6-aminohexyl)amino-2'-AMP-Sepharose. 4. The purified enzyme migrated as a single protein band on polyacrylamide-gel electrophoresis at pH 4.3 and on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the estimated mol.wt. in the latter electrophoresis was 27000-31000. Gel filtration of the purified enzyme through Sephadex G-150 indicated a mol.wt. of 31000. Therefore the purified enzyme is a monomer protein with a mol.wt. of approx. 30000.     

Isolation of two hemorrhagic toxins from Crotalus basiliscus basiliscus (Mexican west coast rattlesnake) venom and their effect on blood clotting and complement

1. Two hemorrhagic toxins of mol. wt 27,000 (B1) and 27,500 (B2) and pI 9.8 and 5.2 respectively were isolated from Crotalus basiliscus venom. 2. The two proteinases did not cross-react antigenically. 3. Both toxins caused hemorrhage in mice and each was capable of hydrolyzing hide power azure, casein, collagen and fibrin. 4. B1 hydrolyzed the A alpha, B beta and gamma chains of fibrinogen. B2 hydrolyzed the A alpha and B beta chains of fibrinogen, but not the gamma chain. 5. Both proteinases inactivated guinea pig complement.     

Dissolved organic matter of a low-coloured stream

SUMMARY. The dissolved organic matter (DOM) of White Clay Creek (Pennsylvania, U.S.A.), a low-coloured stream, was characterized by gel permeation chromatography, humic acid determination, compound classification analysis and gas chromatography.
Large polymers (approximate mol.wt above 5000) were virtually absent. The majority of the DOM consisted of fulvic acid-like material, of probable mol.wt less than 3000. Highly coloured material represented only a small portion of the total. Substances classifiable as phenols, carbohydrates, lipids, amino acids or proteins made up only a small fraction of the DOM. Sixteen low mol. wt compounds (carboxylic acids, amino acids, carbohydrates and an amide) were tentatively identified.
DOM concentration had a mean value of 6.4 mgC/l and showed annual fluctuations, with maxima in autumn and in late winter.     

Amino acid sequence of the Bb fragment from complement Factor B. Sequence of the major cyanogen bromide-cleavage peptide (CB-II) and completion of the sequence of the Bb fragment.

The amino acid sequence of peptide CB-II, the major product (mol.wt. 30 000) of CNBr cleavage of fragment Bb from human complement Factor B, is given. The sequence was obtained from peptides derived by trypsin cleavage of peptide CB-II and clostripain digestion of fragment Bb. Cleavage of two Asn-Gly bonds in peptide CB-II was also found useful. These results, along with those presented in the preceding paper [Gagnon & Christie (1983) Biochem. J. 209, 51-60], yield the complete sequence of the 505 amino acid residues of fragment Bb. The C-terminal half of the molecule shows strong homology of sequence with serine proteinases. Factor B has a catalytic chain (fragment Bb) with a molecular weight twice that of proteinases previously described, suggesting that it is a novel type of serine proteinase, probably with a different activation mechanism.     

Maturation of vacuolar (lysosomal) enzymes in yeast: proteinase yscA and proteinase yscB are catalysts of the processing and activation event of carboxypeptidase yscY.

Studies were performed to unravel the activation and maturation mechanism of vacuolar (lysosomal) proteinases in Saccharomyces cerevisiae. In vivo and in vitro studies show that proteinase yscA and proteinase yscB are involved in the activation and processing event of pro-carboxypeptidase yscY. Processing and activation of pro-carboxypeptidase yscY by proteinase yscA depends on an additional factor contained in the vacuolar fraction. Comparable activation can be mimicked by sodium polyphosphate. Optimum pH for processing by this proteinase yscA-triggered event is 5. The proteinase yscA-triggered maturation process of pro-carboxypeptidase yscY leads to an intermediate mol. wt form of the enzyme which is, however, fully active. Proteinase yscB transfers the intermediate mol. wt form of the original precursor to the apparently authentic, mature and active carboxypeptidase yscY. An activation and maturation scheme is devised.     

Clonorchis sinensis: purification and characterization of a cysteine proteinase from adult worms

1. Adult Clonorchis sinensis, the Chinese liver fluke, is known to migrate to the bile ducts of its mammalian host and cause significant pathology. 2. An acidic, thiol-dependent proteinase with a native mol. wt of approximately 18,500 was purified to homogeneity using ion-exchange chromatography and gel filtration chromatography. By SDS-polyacrylamide gel electrophoresis, the mol. wt of the enzyme was estimated to be 15,000. 3. The enzyme was similar to cathepsin B-like cysteine proteinases based on pH optimum, substrate specificity, and inhibitor sensitivity. 4. Antisera from human clonorchiasis and C. sinensis-infected rabbits reacted in immunoblots with the partially purified proteinase. The C. sinensis proteinase may be useful for serodiagnosis of clonorchiasis.     

Chemical variation and defense of Encelia farinosa

Chemical analysis of leaves from 12 different localities of Encelia farinosa (including var. phenicodonta and var. radians) collected on the peninsula of Baja California (Mexico) revealed the presence of various chemotypes that differed with regard to the concentrations of chromenes and sesquiterpene lactones. Localities of E. farinosa collected in the northern part of Baja California were characterized by high concentrations of the chromene encecalin (up to 252 μmol g⁻¹ dry wt.), whereas the sesquiterpene lactone farinosin was not detected. Localities of E. farinosa collected at the southern tip of the peninsula lacked encecalin, but were shown to accumulate farinosin (up to 85 μmol g⁻¹ dry wt.) instead. On the mainland of Mexico, as well as in Arizona (U.S.A.), farinosin concentrations varied from 18 to 44 μmol g⁻¹ dry wt. for 10 different localities analyzed. Chromenes were not detected or present only in minor amounts (up to 13 μmol g⁻¹ dry wt.), when compared to the samples from northern Baja California. Chemical variation within localities was small when compared to variation between different localities. Accumulation of encecalin and aridity seem to coincide at least on the peninsula of Baja California, as localities of E. farinosa that receive the least amount of rainfall contained the largest amounts of encecalin in their leaves. Leaves of E. farinosa that contained sufficiently large amounts of either encecalin or farinosin were both detrimental to neonate larvae of the polyphagous pest insect Spodoptera littoralis as shown by addition of the respective crude leaf extracts to artificial diet. Possible advantages of the observed intraspecific chemical variability of E. farinosa with regard to adaptation by generalist insect herbivores are discussed.     

Mitochondrial gene expression and cytoplasmic male sterility in sorghum

Variation in sorghum mitochondrial translation products has enabled fertile (Kafir) cytoplasm to be distinguished from Milo cytoplasmic male sterile cytoplasm and from three alternative sources of cytoplasmic male sterile cytoplasm. Mitochondria from Milo cytoplasm synthesised a 65 000 mol. wt. polypeptide which was not synthesised by those from Kafir cytoplasm. In the cytoplasmic male sterile combination of Kafir nucleus in Milo cytoplasm synthesis of this polypeptide was dramatically increased. Mitochondria from two cytoplasmic male sterile lines (Kafir nucleus in IS1112 cytoplasm and Yellow Feterita nucleus in M35-1 cytoplasm) did not synthesise the 65 000 mol. wt. polypeptide but synthesised additional high molecular weight polypeptides (from 54 000 to 82 000 mol. wt.), the major one being 82 000. Mitochondria from cytoplasm IS1112 were also distinguished by synthesis of an additional 12 000 mol. wt. polypeptide. Mitochondria from the cytoplasmic male sterile line Martin nucleus in 9E cytoplasm synthesised an additional 42 000 mol. wt. polypeptide but did not synthesise a 38 000 mol. wt. polypeptide detected in all other cytoplasms. Immunoprecipitation of mitochondrial translation products with antiserum raised against subunit I of yeast cytochrome oxidase tentatively identified the 38 000 mol. wt. polypeptide as subunit I of sorghum cytochrome oxidase. The 42 000 mol. wt. polypeptide was also immuno-precipitated by this antiserum and thus is probably an altered form of cytochrome oxidase subunit I.Analysis of native mitochondrial DNA by agarose gel electrophoresis revealed the presence of two plasmid-like DNA species of molecular weight 5.3 and 5.7 kb in the cytoplasmic male sterile lines Kafir nucleus in cytoplasm IS1112 and Yellow Feterita nucleus in M35-1 cytoplasm. Thus there is a positive correlation between the synthesis of the 82 000 mol. wt. polypeptide and the presence of the additional DNA species.     

Purification and characterization of proteolytic enzymes of Leishmania mexicana mexicana amastigotes and promastigotes

Leishmania mexicana mexicana amastigote and promastigote soluble proteinases were purified using gel filtration and ion exchange chromatography. For the amastigotes, two main proteinase activity peaks were separated with both methods. These accounted for approximately 10% and 90% of the total activity. Characterization of the two activities for substrate specificity and sensitivity to inhibitors indicated that the major peak from both column methods contained enzymes with the characteristics of cysteine proteinases. SDS-polyacrylamide gel electrophoresis of the enzyme from the major peak purified by gel filtration revealed one polypeptide with a molecular weight in the region of 31 000. In contrast, the activity of the minor peak eluted from the columns was of higher molecular weight (67 000) and was similar to metalloproteinases. Purification of the soluble proteinases in the promastigote of L. m. mexicana produced only one activity peak from both column techniques. This activity (mol. wt 67 000) corresponded to the high molecular weight proteinase of the amastigote. The purified proteinases were active on 4-nitroanilide and 7-amino-4-methylcoumarin derivatives of various small peptides. The high molecular weight proteinases of both amastigotes and promastigotes were similarly active against most of the peptides, suggesting a low specificity of the enzymes. In contrast, the low molecular weight amastigote proteinases were particularly active against two of the substrates, namely BZ-Pro-Phe-Arg-Nan and Z-Phe-Arg-MCA. These results indicate that a highly active, substrate-specific, soluble proteinase, with characteristics of a cysteine proteinase, is produced upon transformation of the L. m. mexicana promastigote to amastigote. The discovery and characterization of this enzyme offers opportunities for the development of new antileishmanial agents.     

Purification and characterization of a metallo-endoproteinase from mouse kidney.

A metallo-endoproteinase was purified from mouse kidney. The enzyme was solubilized from the 100 000 g sediment of kidney homogenates with toluene and trypsin, and further purified by fractionation with (NH4)2SO4. DEAE-cellulose chromatography and gel filtration. The molecular weight of the metalloproteinase was estimated by gel filtration on Sepharose 6B to be 270 000--320 000. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence of 2-mercaptoethanol, a single major protein with a mol.wt. of 81 000 was observed. Thus the active enzyme is an oligomer, probably a tetramer. It is a glycoprotein and has an apparent isoelectric point of 4.3. Kidney homogenates and purified preparations of the metalloproteinase degraded azocasein optimally at pH 9.5 and at I 0.15--0.2. The activity was not affected by inhibitors of serine proteinases (di-isopropyl phosphorofluoridate, phenylmethanesulphonyl fluoride), cysteine proteinases (4-hydroxymercuribenzoate, iodoacetate), aspartic proteinases (pepstatin) or several other proteinase inhibitors from actinomycetes (leupeptin, antipain and phosphoramidon). Inhibition of the enzyme was observed with metal chelators (EDTA, EGTA, 1,10-phenanthroline), and thiol compounds (cysteine, glutathione, dithioerythritol, 2-mercaptoethanol). The metalloproteinase degraded azocasein, azocoll, casein, haemoglobulin and aldolase, but showed little or no activity against the synthetic substrates benzoylarginine 2-naphthylamide, benzoylglycylarginine, benzyloxycarbonylglutamyltyrosine or acetylphenylalanyl 2-naphthyl ester. This metalloproteinase from mouse kidney appears to be distinct from previously described kidney proteinases.     

Proteoglycan-degrading enzymes of rabbit fibroblasts and granulocytes.

A neutral proteinase secreted by rabbit synovial fibroblasts in parallel with specific collagenase was partially purified by ion-exchange chromatography. At pH 7.6 this proteinase degraded 35S-labelled bovine nasal proteoglycan and azo-casein. The enzymic activity was inhibited by EDTA, 1,10-phenanthroline and serum, whereas di-isopropyl phosphorofluoridate and soya-bean trypsin inhibitor had little effect. By gel filtration the apparent mol.wt. of the enzyme was 25000. The fibroblast neutral proteinase was compared with the proteoglycan-degrading neutral proteinases of rabbit polymorphonuclear-leucocyte granules. Two distinct activities were found in the granules: one was inhibited by soya-bean trypsin inhibitor and the other by EDTA. The proteoglycan-degrading proteinases of rabbit fibroblasts and polymorphonuclear leucocytes at acid pH also were examined. Both cathepsin D and a thiol-dependent proteinase contributed to the degradation of proteoglycan at pH 4.5.     

Effect of seaweed extracts on the growth and biochemical constituents of Vigna sinensis

The effect of seaweed liquid fertilizers (SLF) of Sargassum wightii and Caulerpa chemnitzia on growth and biochemical constituents of Vigna sinensis was studied. The seeds soaked with aqueous extract of seaweeds performed better when compared to the water soaked controls. Hundred per cent germination was recorded both in aqueous extract soaked and water soaked treatments. The low concentration (20%) of aqueous extracts of S. wightii and C. chemnitzia promoted the seedling growth including the parameters of shoot length (15.87, 14.13 cm/seedling), root length (6.42, 5.38 cm/seedling), fresh weight (4.017, 4.012 g/seedling) and dry weight (0.878, 0.865 g/seedling), chlorophyll (1.599, 1.491 mg g-1 fr. wt.), carotenoids (0.899, 0.875 mg g-1 fr. wt.), protein content of shoot (3.956, 3.474 mg g-1 fr. wt.) and root (2.926, 2.890 mg g-1 fr. wt.), amino acid content of shoot (1.447, 1.429 mg g-1 fr. wt.) and root (0.698, 0.680 mg g-1 fr. wt.), reducing sugar content of shoot (6.426, 6.233 mg g-1 fr. wt.) and root (5.118, 5.103 mg g-1 fr. wt.), total sugar content of shoot (11.846, 11.350 mg g-1 fr. wt.) and root (10.368, 10.102 mg g-1 fr. wt.), alpha-amylase (1.927, 1.819 microg min-1 mg-1 protein) and beta-amylase (1.730, 1.617 microg min-1 mg-1 protein) activities in V. sinensis. Among the two seaweeds tested, S. wightii exhibited better responses.     

Purification and structural studies on the complement-system control protein beta 1H (Factor H).

An efficient procedure for the isolation of the complement-system control protein beta 1H (Factor H) from human plasma was developed. The chemical composition and physical characteristics of the protein were studied, and a sequence of 17 amino acid residues at the N-terminus was determined. Factor H is a single-polypeptide-chain glycoprotein of mol.wt. 155 000 containing 9.3% carbohydrate. Factor H is cleaved by plasma proteinases to a two-chain form. This cleavage can be mimicked by trypsin, and the two-chain form retains fully the C3b-inactivator cofactor activity of Factor H. The proteolytic fragments of Factor H are compared with those of other proteins (C4b-binding protein and erythrocyte C3b-receptor) that act as cofactors for C3b-inactivator.     

Evidence for a thiol ester in duck ovostatin (ovomacroglobulin)

The structure and the mechanism for proteinase inhibition of the egg white protein ovostatin (ovomacroglobulin) are similar to those of plasma alpha 2-macroglobulin, but previous studies have shown that chicken ovostatin lacks a reactive thiol ester (Nagase, H., and Harris, E. D., Jr. (1983) J. Biol. Chem. 258, 7490-7498). Here we show that duck ovostatin has conserved such a thiol ester and is capable of inhibiting both metallo- and serine proteinases stoichiometrically. Evidence for thiol esters was established by the following results with duck ovostatin: 1) autolysis into fragments of Mr = 123,000 and 60,000 occurred by heating in sodium dodecyl sulfate, but was prevented by treatment with CH3NH2; 2) covalent linkages were formed with proteinases on complex formation; 3) reaction with CH3NH2 generated 3.6 SH groups/mol, and 3.9 mol of [14C]CH3NH2 were incorporated per mol of protein; and 4) saturation with a proteinase liberated 3.8 SH groups/mol of the inhibitor. Conformational rearrangement of duck ovostatin upon reacting with CH3NH2 or proteinases was demonstrated by an increased mobility of the protein in polyacrylamide gel electrophoresis. CH3NH2-treated duck ovostatin was able to bind and inhibit proteinases without forming covalent bonds, but, unlike unmodified ovostatin, its inhibitory activity was destroyed by freezing and thawing. Complexes formed between CH3NH2-treated duck ovostatin and a proteinase were not dissociable except under denaturing conditions. These results and other evidence indicate that covalent bond formation through reaction with a thiol ester is a separate process from the trapping and inhibition of proteinases by this family of proteins.     

Fractionation of the proteinases present in the endosperm of germinating seed of Scots pine, Pinus sylvestris

When fresh extracts of endosperms separated from germinating seeds of Scots pine were dialysed at 5°C, proteinase activity on haemoglobin at pH 3.7 showed only a small initial increase, proteinase activities on casein at pH 5.4 and at pH 7.0 increased several-fold, and all the corresponding inhibitor activities disappeared (Salmia and Mikola 1980, Physiol. Plant. 48: 126–130). To find out what happens during dialysis, both fresh and dialysed extracts were fractionated by gel chromatography on Sephacryl S-200. – The fresh extracts had a major proteinase peak (mol. wt. 42,000) with high activity at pH 3.7 and moderate activities at pH 5.4 and 7.0 (pine proteinase I) and a smaller peak (mol. wt. 30,000) with high activity at pH 5.4 and 7.0 and smaller activity at pH 3.7 (pine proteinase II). In dialysed extracts the situation was reversed: the peak of proteinase I was very small while the peak of proteinase II was very high. Apparently, proteinase I is largely inactivated during dialysis while the activity of proteinase II increases, at least partly due to destruction of inhibitors. – The two enzymes were -SH proteinases, as they were completely inhibited by p -hydroxymercuribenzoate; both of them were also inhibited by the endogenous proteinase inhibitors of resting pine seeds. Besides these enzymes, the endosperm extracts contained pepsin-like acid proteinase activity, which is not affected by the endogenous inhibitors. This enzyme activity was largely inactivated during dialysis.