A new esteroproteinase (proteinase F) from the submandibular glands of female mice

One of the esteroproteinases present in the submandibular glands of female mice was purified and characterized. The enzyme, designated proteinase F in this report, had a pI value of 4.6 and a molecular weight of 27 600, being comprised of two subunits of 10 000 and 18 000 daltons. The amino acid composition of proteinase F resembled that of the epidermal growth factor-binding protein, but antiserum against proteinase F only reacted weakly against the binding protein. Proteinase F had an optimum pH at around 9.0 and was strongly inhibited by Cu²⁺ and Hg²⁺ (42 and 76% inhibition, respectively, at a concentration of 4·10^?6 M). It was also inhibited by aprotinin, phenylmethylsulfonylfluoride, iodoacetamide, leupeptin, antipain, and benzamidine but neither by trypsin inhibitors from pancreas, soybean or ovomucoid, nor by TLCK, TPCK, and ?-amino-n-caproic acid. Although its actual physiological function has yet to be determined, these properties indicate that proteinase F is a new enzyme, being distinguished from known proteinases, kallikrein, plasmin, trypsin, chymotrypsin, tonin, angiotensin-converting enzyme, proteinase A (β-nerve growth factor endopeptidase), proteinase D (epidermal growth factor-binding protein), P-esterase, renin A, and renin C. Proteinase F was present in the submandibular glands of female mice more abundantly than in those of males, but it increased in males following castration. Thus, proteinase F appears to be affected by male hormones in vivo.     

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.     

Regulation of proteolytic enzyme activities in muscles practice and hypothesis.

Two weeks of immobilization in shortened state caused 50% decrease in muscle mass and an increase in lysosomal proteinase activities. In this study causes of elevated protease activities in muscle are studied. Many factors could play a role in these elevated proteinase activities. We have found that redox state, ATP content, fuel supply and glucocorticoid receptor number were important in this period. Testosterone, insulin or proteinase inhibitors were not proved to play role in elevated proteinase activities. These practical results are explained by the results achieved in other types of cells. We conclude that changes in redox potential and/or oxygen free radical content of muscle elements can cause a post-translational covalent modification of cysteine proteinases and -SH dependent metalloproteinases, leading thereby to their activation. Lysosomal cysteine proteinases can activate procathepsin D that can damage lysosomal cysteine proteinase inhibitors and in another path it activates procathepsin B, L and H reversely. This feed-back regulation and the activation of cysteine proteinases by metalloproteinases might accelerate the proteinase activities in skeletal muscles.     

Heat shock induction of a 65 kDa ATP—binding proteinase in rat C6 glioma cells

The 45,55,65 and 100kDa ATP-binding proteinases(ATP-BPases) of the heat-shocked (44℃ for 30 min,recovery for 12h) rat C6 glioma cells were purified by DEAE-ionexchange and ATP-affinity chromatography.Their molecular masses,isoelectric points (pI),pH-optima and other properties were analyzed by native proteinase gels.It was shown that the 65 kDa ATP-BPase is specifically induced by heat shock and not detectable in control cells.Its N-terminal 1-9amino acid sequence was determined by Edman degradation,but no homologies to other proteins in the protein data bases were found.30 and 31kDa proteinases can be cleaved from the 45,55 and 65 kDa proteinases to which they are linked.A possible relationship of the heat-induced 65 kDa ATP-BPase with the ATP-dependent proteinases (ATP-DPases) in prokaryotes and eukaryotes is discussed.     

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.     

Purification of a new extracellular 90-kDa serine proteinase with isoelectric point of 3.9 from Bacillus subtilis (natto) and elucidation of its distinct mode of action.

A new extracellular 90-kDa serine proteinase with an isoelectric point (pI) of 3.9 was purified from Bicillus subtilis (natto). Microheterogeneity was detected in the 50-kDa protease of bacillopeptidase F with pI 4.4 reported previously by Wu et al. and the sequence for the first 25 amino acids in the internal region of the enzyme was analyzed: ATDGVEWNVDQIDAPKAWALGYDGA. The cleavage sites in the oxidized B-chain of insulin by the proteinase were CySO3H7-Gly8, Val12-Glu13, Try16-Leu17, and Phe25-Tyr26. The activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin, while the activity was not inhibited by proteinaceous Streptomyces subtilisin inhibitor (SSI) or alpha 2-macroglubulin.     

Monoclonal antibodies to the two most basic papaya proteinases

The proteinases fromCarica papaya include papain, isoenzymes of chymopapain and two proteinases A and B distinguished by their unusually high pI. The identity of one of the most basic proteinases has been questioned. The present report describes the preparation and characterisation of two monoclonal antibodies that react specifically with papaya proteinases A and B respectively and a third that identifies a common structural feature found in papain and proteinase A.     

Gene expression patterns of Helicoverpa armigera gut proteases

Relative quantification of reported gut proteinase cDNAs from Helicoverpa armigera larvae fed on various host plants (cotton, chickpea, pigeonpea, tomato and okra), non-host plant PIs (winged bean, bitter gourd, ground nut, and capsicum) and during larval development has been carried out using semi-quantitative RT-PCR. Five trypsin-like and three chymotrypsin-like proteinases were categorized as insensitive or sensitive to most of the proteinase inhibitors (PIs) and insensitive/sensitive to specific PIs based on their expression analysis. These results were supported by amino acid sequence analysis, where diverged amino acids were observed in the regions, which are reported to be involved in typical trypsin-trypsin inhibitor interactions and critical for proteinase inhibitor resistance. Among exopeptidases (five aminopeptidase and three carboxypeptidase), HaAmi4 and HaAmi5 of aminopeptidase and HaCar1 of carboxypeptidase exhibited considerable differential expression. Elastase and cathepsin B-like proteinases were expressed at negligible levels. The proteases identified in the study would be ideal candidates for further interactions studies with protease inhibitors to understand the structural reasons of protease inhibitor insensitivity.     

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.     

Structural determinants in the stability of the serpin/proteinase complex

Serpins inhibit serine proteinases through formation of stable 1:1 complexes. In this study we have evaluated the effects of PAI-1 neutralizing antibodies (MA) on the stability of PAI-1/proteinase complexes, partially destabilized through prolongation of the reactive center loop. MA-8H9D4, reacting with residues Arg(300), Gln(303), and Asp(305), had no effect on the stability. In contrast, MA-33H1F7 and MA-55F4C12, reacting with alpha-helix F and the turn connecting hF with s3A, affected significantly and proteinase-dependently formed PAI-1/proteinase complexes. That is, MA-33H1F7 increased the stability of both PAI-1/t-PA and u-PA complexes (7- and 3-fold, respectively) whereas MA-55F4C12 stabilized PAI-1/t-PA complexes (3-fold) but destabilized PAI-1/u-PA complexes (2-fold). It is concluded that interference with the docking site of the cognate proteinase in the preformed serpin/proteinase complex may affect the intrinsic stability. We hypothesize that this is the consequence of a decreased or increased torsion of the RCL on the catalytic triad in the proteinase.     

Regulation of k influx in barley : effects of low temperature

The proteinases present in dark-germinated flax seeds (Linum usitatissimum) were studied as a function of germination at 25°C. A majority of activity was present in basic proteinases with an acidic pH optimum and a temperature optimum of 45°C in the digestion of hemoglobin. Electrophoresis in a sodium dodecyl sulfate-polyacrylamide mixture which had been polymerized with gelatin was used to separate proteins in extracts of seedlings. Subsequent activation of proteinases with Triton X-100 and resultant digestion of gelatin proved to be very reproducible and afforded detection and good quantification of various proteinase zones. An ethylenediaminetetraacetate-sensitive proteinase zone, P4 (about 60,000 daltons), appeared at day 3 after imbibition and attained maximum activity at day 4. This correlates with a rapid loss in vivo of the glyoxysomal enzyme, isocitrate lyase (EC 4.1.3.1). Ethylenediaminetetraacetate also slowed the loss of isocitrate lyase activity in extracts of 4-day seedlings in a dose-dependent manner. The addition of leupeptin, α-tolylsulfonyl fluoride, Pepstatin A, p-chloromercuribenzoate, or 1,10-phenanthroline prior to, during, or after exchange of Triton X-100 for sodium dodecyl sulfate had almost no inhibitory effect upon proteinases in 4-day seedlings.     

Two-dimensional analysis of proteinase activity

A method was developed to separate proteinases in a complex mixture in two dimensions followed by activity detection using class specific substrates. Using this method, serine proteinase activity was evaluated in gut extracts from a stored-product pest, Plodia interpunctella. With the substrate n-alpha-benzoyl-l-arginine rho-nitroanilide, three major groups of at least six trypsin-like activities were identified, consisting of proteinases with estimated molecular masses of 25-27, 40-41, and 289 kDa, and all with an acidic pI of 4.7-5.5. With the substrate, n-succinyl-ala-ala-pro-phenylalanine rho-nitroanilide, two groups of at least five chymotrypsin-like activities were detected, with estimated molecular masses of 28 and 192 kDa and pI values ranging from 6.1 to 7.3. Using the 2-DE activity blot method, information was obtained on the relative number and physical properties of serine proteinases in a mixture of insect gut proteinases without prior fractionation.     

Analysis of where and which types of proteinases participate in lysosomal proteinase processing using bafilomycin A1 and Helicobacter pylori Vac A toxin

Lysosomal proteinases are translated as preproforms, transported through the Golgi apparatus as proforms, and localized in lysosomes as mature forms. In this study, we analyzed which subclass of proteinases participates in the processing of lysosomal proteinases using Bafilomycin A1, a vacuolar ATPase inhibitor. Bafilomycin A1 raises lysosomal pH resulting in the degradation of lysosomal proteinases such as cathepsins B, D, and L. Twenty-four hours after the withdrawal of Bafilomycin A1, NIH3T3 cells possess these proteinases in amounts and activities similar to those in cells cultured in DMEM and 5% BCS. In the presence of various proteinase inhibitors, procathepsin processing is disturbed by E-64-d, resulting in abnormal processing of cathepsins D and L, but not by APMSF, Pepstatin A, or CA-074. In the presence of Helicobacter pylori Vac A toxin, which prevents vesicular transport from late endosomes to lysosomes, the processing of procathepsins B and D occurs, while that of procathepsin L does not. Thus, procathepsins B and D are converted to their mature forms in late endosomes, while procathepsin L is processed to the mature form after its arrival in lysosomes by some cysteine proteinase other than cathepsin B.     

Purification and properties of a fibrinogenase from the venom of Naja nigricollis

A fibrinogenolytic proteinase from the venom of Naja nigricollis was purified by chromatography on Bio-Rex 70 and Phenyl-Sepharose. The purified enzyme, designated proteinase F1, was homogeneous by the criterion of SDS-polyacrylamide gel electrophoresis, and consisted of a single chain with a molecular weight of 58 000. Purified proteinase F1 had approximately 15-fold more proteinase activity than the crude venom, based on its ability to inactive α₂-macroglobulin. The enzyme acted on only the Aα-chain of fibrinogen and left the Bβ- and γ-chains intact. The pH optimum for this fibrinogenolytic activity was in the range of pH 8 to 10. In addition to its activity on fibrinogen, proteinase F1 was active on α₂-macroglobulin and fibronectin, but did not degrade casein, hemoglobin or bovine serum albumin. The enzyme was not inhibited by inhibitors of serine proteinases, cysteine proteinases or acid proteinases, but only by the metalloproteinase inhibitor, EDTA. The inhibition by EDTA could be prevented by Zn²⁺, but not by Ca²⁺ or Mg²⁺.     

Expression of the yeast aspartyl protease, proteinase A. Phosphorylation and binding to the mannose 6-phosphate receptor are altered by addition of cathepsin D sequences

Proteinase A, a yeast aspartyl protease that is highly homologous to the mammalian lysosomal aspartyl protease, cathepsin D, was expressed in Xenopus oocytes and its biosynthesis and post-translational modifications were characterized. While 29-45% of the proteinase A was secreted from oocytes, approximately 37% of the cell-associated proteinase A underwent proteolytic cleavage, characteristic of delivery to a lysosomal organelle. Although proteinase A is not targeted to the yeast vacuole by a mannose 6-phosphate receptor-dependent pathway, 2-5% of the proteinase A molecules expressed in oocytes bound to a Man-6-P receptor column. However, analysis of its [2-3H]mannose-labeled oligosaccharides revealed that 14-23% of these units contain phosphomannosyl residues. A hybrid molecule (H6), in which the propiece and first 12 amino acids of proteinase A were changed to the cathepsin D sequence, was also expressed in oocytes. The binding of H6 to the Man-6-P receptor was approximately 12-fold greater than observed for proteinase A. This increased level of receptor binding could be accounted for by three factors: 1) a small increase in the total amount of phosphorylated oligosaccharides, 2) an increase in the number of oligosaccharides which acquire two phosphomonoesters, and 3) the presence of a greater percentage of oligosaccharides with one phosphomonoester which exhibit high affinity binding to the Man-6-P receptor. These results demonstrate that proteinase A is recognized by UDP-GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase. However, this interaction is altered by the addition of cathepsin D sequences, resulting in the generation of a higher affinity ligand for binding to the Man-6-P receptor.     

Purification and characterization of the mature,membrane-associated cell-envelope proteinase of Lactobacillus helveticus L89

Lactobacillus helveticus L89 possesses a cell-envelope proteinase (Lb-CEP) that is biochemically and genetically related to that of the lactococci (Lc-CEP). The in-situ proteinase is resistant to autoproteolysis and remains associated with the membrane during lysozyme treatment of cells and subsequent mechanical disruption of the treated cells. The proteinase was purified from isolated membranes by a procedure that preserves the complete in-situ proteinase (mature proteinase) assumed to be the N-terminally processed translation product including the membrane anchor: its monomer molecular mass is approximately 180 kDa. The purified enzyme appeared to be more stable towards heat than hitherto known related, but C-terminally truncated cell-envelope proteinases of lactobacilli and lactococci, which were released from the cells by autoproteolysis. On the basis of its specificity towards caseins, towards the _sl-casein-(1-23)-fragment and towards two differently charged chromophoric peptides, the proteinase was recognized as an (Lb-)CEP_I/III mixed-type variant different from those identified so far among the lactococcal proteinases. Correspondence to: F. A. Exterkate     

Production of extracellular proteinases—protein C activators of blood plasma—by the micromycete Aspergillus ochraceus during submerged and solid-state fermentation

The conditions for the submerged and solid-state fermentation of the micromycete Aspergillus ochraceus VKM F-4104D, producing extracellular proteinases that activate protein C of human blood plasma, were optimized. It is shown that the protein C-activating activity of the micromycete in a solid-state culture was 1.5-3.5 times higher than in a submerged culture (as calculated per milliliter of culture medium). Among the extracellular proteins secreted by A. ochraceus VKM F-4104D during submerged and solid-state fermentation, a protein C-activating proteinase with a pI of 6.0–6.3 was identified.     

The effect of dilution rate and pH on biomass and proteinase production by Micrococcus sedentarius grown in continuous culture

K.T. HOLLAND, J. MARSHALL AND D. TAYLOR. 1992. Micrococcus sedentarius , an organism associated with pitted keratolysis, produced two proteinases in culture supernatant fluids, as shown by non-denaturing PAGE with overlaying with a casein substrate. A mixture had optimal activity at pH 10 with azocasein substrate. At pH 7.1 and 8.1 in continuous culture with varying dilution rates high proteinase production occurred at relative specific growth rates (μ_rels) 0.39 and 0.77 and biomass concentrations decreased with increasing dilution rate. One proteinase was constitutive and varied little in production with different growth rates. The other proteinase was under control with high production at low growth rates and no production at high growth rates. With varying pH at μ_rels, 0.39 and 0.77 maximum biomass concentration and proteinase production occurred between pH 8.0 and 9.0 as did the highest specific growth rate. These results support the hypothesis that Mic. sedentarus produces pitting in the stratum corneum when the skin is hydrated and the pH rises above neutrality.     

Predicting functional residues of the Solanum lycopersicum aspartic protease inhibitor (SLAPI) by combining sequence and structural analysis with molecular docking

The Solanum lycopersicum aspartic protease inhibitor (SLAPI), which belongs to the STI-Kunitz family, is an effective inhibitor of the aspartic proteases human cathepsin D and Saccharomyces proteinase A. However, in contrast with the large number of studies on the inhibition mechanism of the serine proteases by the STI-Kunitz inhibitors, the structural aspects of the inhibition mechanism of aspartic proteases from this family of inhibitors are poorly understood. In the present study, we have combined sequence and structural analysis methods with protein-protein docking to gain a better understanding of the SLAPI inhibition mechanism of the proteinase A. The results suggest that: i) SLAPI loop L9 may be involved in the inhibitor interaction with the proteinase A′s active site, and ii) the residues I144, V148, L149, P151, F152 and R154 are implicated in the difference in the potency shown previously by SLAPI and another STI-Kunitz inhibitor isolated from Solanum tuberosum to inhibit proteinase A. These results will be useful in the design of site directed mutagenesis experiments to understand more thoroughly the aspartic protease inhibition mechanism of SLAPI and other related STI-Kunitz inhibitors.     

Kraft Pulp Bleaching and Delignification by Dikaryons and Monokaryons of Trametes versicolor

The ability of 10 dikaryotic and 20 monokaryotic strains of Trametes (Coriolus) versicolor to bleach and delignify hardwood and softwood kraft pulps was assessed. A dikaryon (52P) and two of its mating-compatible monokaryons (52J and 52D) derived via protoplasting were compared. All three regularly bleached hardwood kraft pulp more than 20 brightness points (International Standards Organization) in 5 days and softwood kraft pulp the same amount in 12 days. Delignification (kappa number reduction) by the dikaryon and the monokaryons was similar, but the growth of the monokaryons was slower. Insoluble dark pigments were commonly found in the mycelium, medium, and pulp of the dikaryon only. Laccase and manganese peroxidase (MnP) but not lignin peroxidase activities were secreted during bleaching by all three strains. Their laccase and MnP isozyme patterns were compared on native gels. No segregation of isozyme bands between the monokaryons was found. Hardwood kraft pulp appeared to adsorb several laccase isozyme bands. One MnP isozyme (pI, 3.2) was secreted in the presence of pulp by all three strains, but a second (pI, 4.9) was produced only by 52P. A lower level of soluble MnP activity in one monokaryon (52D) was associated with reduced bleaching ability and a lower level of methanol production. Since monokaryon 52J bleached pulp better than its parent dikaryon 52P, especially per unit of biomass, this genetically simpler monokaryon will be the preferred subject for further genetic manipulation and improvement of fungal pulp biological bleaching.