Serine proteinase from the archaebacterium Halobacterium mediterranei--an analog of eubacterium subtilisin]

A homogeneous serine proteinase was isolated from cultural filtrates of the extreme halophilic bacteria Halobacterium mediterranei 1538 using affinity chromatography on bacitracin-Sepharose, ultrafiltration and gel filtration on Sephadex G-75, with a 48% yield and 260-fold purification. The enzyme was completely inactivated by specific inhibitors of serine proteinases, PMSF and DFP, as well as by Hg2+ and PCMB. The enzyme activity was strongly dependent of NaCl concentration, the enzyme being inactivated below 0.75 M NaCl. Inactivation of the enzyme was also seen in the presence of 2-7% organic solvents. The pH optimum for Glp-Ala-Ala-Leu-pNA hydrolysis is 8.0-8.5; Km is 0.14 mM, kcat is 36.9 s-1. The stability optimum lies at pH 5.5-8.0, temperature optimum is at 55 degrees C. The enzyme molecular weight is 41,000 Da; pI is 7.5. The substrate specificity of the enzyme is comparable to that of secretory subtilisins; the extent of protein substrate hydrolysis is similar to that of proteinase K. The N-terminal sequence of Halobacterium mediterranei serine proteinase, Asp-Thr-Ala-Asn-Asp-Pro-Lys-Tyr-Gly-Ser-Gln-Tyr-Ala-Pro-Gln-Lys-Val-Asn- Ala- Asp-, reveals a 50% homology with the aminoterminal sequence of Thermoactinomyces vulgaris serine proteinase. Hence, the serine proteinase secreted by halophilic bacteria may be considered as a structural and functional analog of eubacterial enzymes.     

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.     

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.     

Aspartyl proteinase from cucumber (Cucumis sativus) seeds. Preparation and characteristics

Aspartyl proteinase (EC 3.4.23) from cucumber seeds was purified by ammonium sulphate fractionation, chromatography on immobilized pepstatin and gel filtration on Sephacryl S-200. The preparation obtained, homogeneous on polyacrylamide-gel electrophoresis in acidic and alkaline media, has a molecular mass of 42,000, pI of 5.2, and shows the highest activity with denatured haemoglobin at pH 3.2. The proteinase is stable in slightly alkaline medium, whereas it is inactivated in acidic medium, especially in the presence of NaCl. The enzyme activity is affected neither by the inhibitors of serine proteinases, sulfhydryl-proteinases and metalloproteinases, nor by divalent metal ions, whereas the enzyme is inactivated by the inhibitors of aspartyl proteinases: 1,2,3-epoxy(p-nitrophenoxy)propane, diazoacetyl-DL-norleucine and pepstatin.     

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.     

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.     

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.     

The inactivation of native enzymes by a neutral proteinase from rat intestinal muscle.

1. The solubilization and partial purification of a proteinase from the intestinal smooth muscle of rats fed on protein-free diets are described. 2. It has a mol.wt. of about 33000 and it is stable over a narrow pH range. 3. From its susceptibility to known modifers of proteolytic enzymes, it appears to be a serine proteinase of a trypsin-like nature. Active-site titration with soya-bean trypsin inhibitor shows that the concentration of proteinase was about 3 microgram/g wet wt. of intestinal smooth muscle. However, the muscle proteinase demonstrates a marked ability for inactivating enzymes in their native conformation at neutral pH. It is about 100 times more efficient than pancreatic trypsin when the inactivating activities are compared on an approximately equimolar basis. 4. Inactivation of the substrate enzymes is accompanied by limited proteolysis, as demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 5. An endogenous inhibitor was separated from the proteinase by fractionation with (NH4)2SO4. 6. Contamination of the muscle tissue by lumen, mucosal or blood proteinases and inhibitors is shown to be unlikely. 7. A role for the neutral trypsin-like proteinase in initiating the degradation of intracellular enzymes is considered.     

Substrate Specificity and Salt Inhibition of Five Proteinases Isolated from the Pyloric Caeca and Stomach of Sardine

To elucidate the mechanism of hydrolysis of fish muscle proteins by fish proteinases in fish sauce production, each pure preparation of three alkaline proteinases and two acid proteinases from sardine was tested for its ability to hydrolyze various proteins and its stability in the presence of 0 to 25% of NaCl. Each of the alkaline proteinases hydrolyzed casein more rapidly than other proteins. A major alkaline proteinase (III) hydrolyzed sarcoplasmic protein from sardine 5-times faster than other alkaline proteinases. Each of two acid proteinases hydrolyzed hemoglobin and myoglobin more rapidly than the other proteins. After preincubation with 25% NaCl, an alkaline proteinase (III) and an acid proteinase (II) were stable although the other proteinases became unstable. The two proteinases, alkaline proteinase III and acid proteinase II, were also stable for three months after the beginning of fish sauce production. The proteolytic activity of each of alkaline and the acid proteinases was strongly inhibited by more than 15% NaCl; however, minimum inhibition was observed when sardine muscle proteins were used as the substrate.     

Purification and characterization of an intracellular aspartyl acid proteinase (pumAi) from Ustilago maydis

The intracellular proteinase pumAi in Ustilago maydis has been associated with yeast-mycelium dimorphic transition. The proteinase was purified from a cell-free extract by ammonium sulfate fractionation and chromatographic steps including hydrophobic interactions on a Phenyl Superose column, ion exchange on a Mono Q column, and gel filtration on Superose 12 columns. The enzyme has a mass of 35.3-36.6 kDa, a pH and temperature optimum of 4.0 and 40 degrees C, respectively, and a pI of 5.5. The enzyme degraded hemoglobin, gelatin, albumin, and casein, but not collagen, and the enzymatic activity was strongly inhibited by pepstatin A, an aspartyl proteinase-specific inhibitor. The biochemical characteristics of pumAi are similar to other fungal intracellular aspartyl proteinases, however, this is the first biochemical characterization of a basidiomycete proteinase probably associated with dimorphic yeast-mycelium transition.     

Thiol-dependent serine proteinase from Streptomyces thermovulgaris

The cultural filtrates of S. thermovulgaris contain a proteinase which is active towards the chromogenic subtilisin substrate, Z-Ala-Ala-Leu-pNa, and azocasein. Pure enzyme preparations were obtained by affinity chromatography on bacitracin-Sepharose with subsequent rechromatography on the same adsorbent. The proteinase was completely inactivated by PMSF and DFP, the specific inhibitors for serine proteinase, by thiol reagents (HgCl2, PCMB) and by the protein inhibitor from S. jantinus. The pH activity optimum for the enzyme is 7.8-8.2, temperature optimum is 55 degrees C. The enzyme is stable at pH 6-9, has a pI of 5.0 and a molecular mass of 32 kDa. When tested against the peptide substrate, the enzyme shows a specificity characteristic for subtilisins. The N-terminal sequence of the enzyme, Tyr-Thr-Pro-Asn-Asp-Pro-Tyr-Phe-Ser-Ser-Arg-Gln-Tyr-Gly, shows a 100% homology with that of terminase, a thiol-dependent serine proteinase. On the basis of the above considerations the enzyme may be related to the subfamily of thiol-dependent serine proteinases.     

Purification to homogeneity of extracellular polygalacturonase and isoenzymes of pectate lyase of Erwinia carotovora subsp. atroseptica by column chromatography

Extracellular polygalacturonase (PG) and two pectate lyase isoenzymes (PLI and PLII) produced by a 48 h culture of Erwinia carotovora subsp. atroseptica in pectate-based medium were purified 2027, 2036 and 2374-fold respectively to homogeneity with corresponding 59%, 61% and 32% recovery. This was achieved first by ion exchange chromatography on a S-Sepharose fast flow column with 20 mmol/1 Tris at pH 8.0 followed by elution of bound proteins with a 1 mol/1 NaCl gradient which separated PG from PL. The two enzymes were then further purified to homogeneity (assessed by SDS-PAGE) by selective adsorption chromatography on a hydroxyapatite column equilibrated with distilled water; PG was eluted with a 3 mol/1 KCl gradient and PLI with a 3 mol/1 KCl gradient followed by a 1.2 mol/1 PO₄ buffer pH 6.5 gradient to elute PLII. The Mr of the three enzymes determined by SDS-PAGE was 39 kDa and the pI values for PG, PLI and PII were 10.3, 10.3 and 10.0 respectively as determined by isoelectric focusing (IEF)-gel electrophoresis followed by activity staining.     

New subtilisin-like collagenase from leaves of common plantain

A new subtilisin-like proteinase hydrolyzing chromogenic peptide substrate Glp-Ala-Ala-Leu-p-nitroanilide optimally at pH 8.1 was found in common plantain leaves. The protease named plantagolisin was isolated by ammonium sulfate precipitation of the leaves' extract followed by affinity chromatography on bacitracin-Sepharose and ion-exchange chromatography on Mono Q in FPLC regime. Its molecular mass is 19000 Da and pI 5.0. pH-stability range is 7-10 in the presence of 2 mM Ca(2+), temperature optimum is 40 degrees C. The substrate specificity of subtilase towards synthetic peptides and insulin B-chain is comparable with that of two other subtilisin-like serine proteinases: proteinase from leaves of the sunflower and taraxalisin. Besides, the proteinase is able to hydrolyze substrates with Pro in P(1) position. The enzyme hydrolyzes collagen. alpha and beta chains are hydrolyzed simultaneously in parallel; there are only low-molecular-mass hydrolysis products in the sample after 2 h of incubation. Pure serine proteinase was inactivated by specific serine proteinases inhibitors: diisopropylfluorophosphate, phenylmethylsulfonyl fluoride and Hg(2+). The plantagolisin N-terminal sequence ESNSEQETQTESGPGTAFL-, traced for 19 residues, revealed 37% homology with that of subtilisin from yeast Schizosaccharomyces pombe.     

Serine proteinase from the higher basidiomycetes of Coprinus genus

A thiol-dependent serine proteinase has been isolated for the first time from a higher basidiomycete Coprinus 7N culture filtrate by affinity chromatography on bacitracin-Sepharose combined with ion-exchange chromatography on DEAE-Sepharose. This procedure resulted in a homogeneous enzyme with 32-fold purification and 55% yield. The enzyme has a molecular mass of 33,000 Da and pI of 8.5; its amino acid composition appears as follows: Lys7, His7, Arg10, Asx29, Thr24, Ser30, Glx19, Pro13, Gly39, Ala40, Cys2-3, Val23, Met1-2, Ile14, Leu13, Tyr6, Phe7. The enzyme shows the optimal activity towards Z-Ala-Ala-Leu-pNA at 8.5 and is stable at pH 6-9. The temperature optimum of the enzyme activity lies at 37 degrees C. The proteinase is completely inactivated by the specific inhibitors of serine proteinases, diisopropylfluorophosphate and phenylmethylsulfonylfluoride, as well as by the SH-group reagent, p-chloromercuribenzoate. The Coprinus 7N proteinase hydrolyzes, azocasein, azoalbumin, hemoglobin, fibrin and synthetic chromogenic peptide substrates, e. g., Z-Ala-Ala-Leu-pNA, Z-Gly-gly-Leu-pNA. Some properties of the Coprinus 7N proteinase are very similar to those of thiol-dependent serine proteinases from bacilli, actinomycetes, fungi and plants which form a subfamily of thiol-dependent serine proteinases within the family of subtilisins.     

Purification and properties of arylsulphatase A from rabbit testis.

Rabbit testis arylsulphatase A was purified 140-fold with a recovery of 20% from detergent extracts of an acetone-dried powder by using DE-52 cellulose column chromatography, gel filtration on Sephadex G-200 and preparative isoelectric focusing. The purified enzyme showed one major band with one minor contaminant on electrophoresis in a 7.5% (w/v) polyacrylamide gel at pH8.3. On sodiumdodecyl sulphate/polyacrylamidegel electrophoresis, a single major band was observed with minor contaminants. The final preparation of enzyme was free from general proteolytic, esterase, hyaluronidase, beta-glucuronidase and beta-galactosidase activities. Rabbit testicular arylsulphatase A exists as a dimer of mol.wt. 110000 at pH7.1. At pH5.0 the enzyme is a tetramer of mol.wt. 220000. Arylsulphatase A appears to consist of two identical subunits of mol.wt. 55000 each. The highly purified enzyme has pI4.6. The enzyme hydrolyses p-nitrocatechol sulphate with Km and Vmax, of 4.1 mM and 80nmol/min respectively, but has no activity toward p-nitrophenyl sulphate. The pH optimum of the enzyme varies with the incubation time. By applying Sephacex G-200 chromatography and preparative isoelectric focusing, one form of enzyme was obtained. The enzyme has properites common to arylsulphatase A of other sources with respect to the anomalous time-activity relationship, pI, inhibition by PO42-, SO32- and Ag+ ions and substrate affinity to p-nitrocatechol sulphate. However, the enzyme shows the temperature optimum of arylsulphatase B of other species.     

Subtilisin-like proteinase SSPB from Streptomyces spheroides, strain 35

A serine proteinase possessing a fibrinolytic activity was isolated from a culture filtrate of Streptomyces spheroides, strain 35. A consecutive use of affinity chromatography on bacillichin-silochrome and bacitracin-sepharose and ion-exchange chromatography on anionie PAP and cationic KMT resulted in a homogeneous proteinase with 1060-fold purification and 19% yield. The enzyme has a molecular weight of 28000; its amino acid composition is Asp31, Ser28, Thr29, Glu9, Pro14, Gly35, Ala42, Val26, Ile14, Leu13, Met2, Tyr9, Phe4, Trp3, His6, Lys4, Arg10. The enzyme has a pI at pH greater than 10 and the activity optimum against Z-L-Ala-L-Ala-L-Leu-pNA at pH 10-11. The enzyme is stable within the pH range of 4-11 and in 6 M guanidinium chloride pH 8.0 in the presence of Ca2+. The enzyme is inhibited by diisopropylfluorophosphate and benzylsulfofluoride, specific inhibitors of serine proteinases as well as by potato proteinase inhibitor. The serine proteinase SSPB isolated from Str. spheroides, strain 35 can be related to subtilisin-like serine proteinase, especially to those of SGPD and SGPE of Str. griseus.     

A study in situ of the effect of metallo- and serine proteinase inhibitors on the birefringence of the secretory stage enamel organic extracellular matrix

Abstract

Dental enamel formation occurs extracellularly and establishment of an ordered enamel organic extracellular matrix (ECM) seems to be crucial for proper construction of the enamel mineral phase. Polarizing microscopy shows that the ordered supramolecular structure of the secretory stage enamel organic ECM exhibits strong birefringence. We reported earlier that this birefringence is lost in unfixed specimens, probably due to extensive proteolytic cleavage of enamel proteins. Therefore, we investigated the association between enamel proteinase activities by analyzing the effects of metallo- and serine proteinase inhibitors in situ on the birefringence of the secretory stage enamel organic ECM. Male rats were used in the present study. After sacrifice, distal 10 mm fragments of upper incisors were removed and immersed for 15 h under continuous shaking at 37°C in one of the following solutions: 1) 10 mM Tris, pH 8.0; 150 mM NaCl (negative control, n = 8); 2) 2% paraformaldehyde and 0.5% glutaraldehyde in 0.2 M phosphate-buffered saline (PBS), pH 7.2 (positive control, n = 5); 3) 10 mM Tris, pH 8.0; 150 mM NaCl; 2 mM 1,10-phenanthroline (n = 9); 4) 10 mM Tris, pH 8.0; 150 mM NaCl; 2 mM phenylmethyl-sulfonyl fluoride (PMSF) (n = 8); 5) 10 mM Tris, pH 8.0; 150 mM NaCl; 2 mM 1,10-phenanthroline; 2 mM PMSF (n = 9). Samples then were immersed in fixative solution for 24 h and processed to obtain 5 μm thick longitudinal sections of the secretory stage enamel organic ECM. The sections were immersed in 80% glycerin for 30 min and analyzed by transmitted polarizing light microscopy. 1,10-Phenanthroline (inhibitor of metalloproteinases) and 1,10-phenanthroline + PMSF (inhibitor of serine proteinases) clearly prevented a decrease in the optical retardation of birefringence brightness from the tissue. PMSF alone promoted a slight preservation of the birefringence exhibited by the secretory stage enamel organic ECM. Rapid loss of birefringence in secretory stage enamel organic ECM that is not fixed immediately is caused by enamel proteinases and the activity of metalloproteinases seems to lead to preliminary degradation of the enamel organic ECM, which in turn facilitates subsequent serine proteinase activity.     

Use of bead cellulose derivatives to isolation of bacterial alkaline proteinase by column liquid chromatography

Crude preparation of bacterial proteinase was purified by liquid chromatography. Combinations of individual ion-exchange chromatography methods and ion-exchange, hydrophobic and dye-ligand affinity chromatography, respectively, were used. The adsorbents were in all cases bead cellulose derivatives (Perloza), either commercially available (DEAE- and CM-Ostsorb) or prepared in the laboratory. Increase in column size resulted in a better separation efficiency of DEAE-Ostsorb IEC,i.e. step used in both separation protocols. The preparation of alkaline proteinase purified exclusively by this IEC method was highly active and comprised only trace amount of other proteins. This was proved by size-exclusion chromatography using the FPLC and HPLC mode. The relative molar mass of the enzyme (29.7 kDa) determined by SDS-polyacrylamide gel electrophoresis and its isoelectric point (pI 8.3) assayed by isoelectric focusing are at limit values typical for bacterial alkaline proteinases (30 kDa, pI about 9). The pH optimum of about 10.5 is typical for alkaline proteinase activity.     

IUB commission of editors of biochemical journals the citation of bibliographic references in biochemical journals recommendations (1971)

A pentose-rich acidic glycoprotein was isolated from protease digested bovine vitreous humor by fractionation on an AG1-X2 column using NaCl solution gradient.The material eluted at 0.35 M NaCl (glycoprotein) was electrophoretically heterogeneous at pH 8.6 after partial purification on Sephadex G-25. Gel filtration on G-100 resolved the glycoprotein into two fractions. These fractions differ in molecular weight; mol. wt approx. 95 000 material consisted of two components on electrophoresis and mol. wt approx. 28 000 material showed only a single component on electrophoresis. The lower molecular weight component was re-chromatographed on Sephadex G-100 yielding a single orcinol positive component which gave a homogeneous band on gel electrophoresis.Quantitative analysis of this material gave 30% protein, 7.0% pentose, 18.7% glucosamine, 9.2% galactosamine, 10.9% hexuronic acid and 16.1% hexose.Treatment with 0.5 M NaOH at 20°C for 24 h resulted in a 50% decrease in the threonine content suggesting the possible involvement of this amino acid in the protein-carbohydrate linkage group.Paper chromatography of the fraction hydrolysate demonstrated the presence of glucurone, xylose, arabinose, glucose and galactose.