Mechanism of Proteinase Release from Lactococcus lactis subsp. cremoris Wg2

The procedure generally used for the isolation of extracellular, cell-associated proteinases of Lactococcus lactis species is based on the release of the proteinases by repeated incubation and washing of the cells in a Ca²⁺-free buffer. For L. lactis subsp. cremoris Wg2, as many as five incubations for 30 min at 29°C are needed in order to liberate 95% of the proteinase. Proteinase release was not affected by chloramphenicol, which indicates that release is not the result of protein synthesis during the incubations. Ca²⁺ inhibited, while ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) stimulated, proteinase release from the cells. The pH optimum for proteinase release ranged between 6.5 and 7.5, which was higher than the optimum pH of the proteinase measured for casein hydrolysis (i.e., 6.4). Treatment of cells with the serine proteinase inhibitor phenylmethylsulfonyl fluoride prior to the incubations in Ca²⁺-free buffer reduced the release of the proteinase by 70 to 80%. The residual proteinase remained cell associated but could be removed by the addition of active L. lactis subsp. cremoris Wg2 proteinase. This suggests that proteinase release from cells of L. lactis subsp. cremoris Wg2 is the result of autoproteolytic activity. From a comparison of the N-terminal amino acid sequence of the released proteinase with the complete amino acid sequence determined from the nucleotide sequence of the proteinase gene, a protein of 180 kilodaltons would be expected. However, a proteinase with a molecular weight of 165,000 was found, which indicated that further hydrolysis had occurred at the C terminus.     

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⁺.     

Characteristics of the Chromaffin Granule Aspartic Proteinase Involved in Proenkephalin Processing

Abstract: Proteolytic processing of neuropeptide precursors is required for production of active neurotransmitters and hormones. In this study, a chromaffin granule (CG) aspartic proteinase of 70 kDa was found to contribute to enkephalin precursor cleaving activity, as assayed with recombinant ([³⁵S]Met)preproenkephalin. The 70-kDa CG aspartic proteinase was purified by concanavalin A-Sepharose, Sephacryl S-200, and pepstatin A agarose affinity chromatography. The proteinase showed optimal activity at pH 5.5. It was potently inhibited by pepstatin A, a selective aspartic proteinase inhibitor, but not by inhibitors of serine, cysteine, or metalloproteinases. Lack of inhibition by Val-d -Leu-Pro-Phe-Val-d -Leu—an inhibitor of pepsin, cathepsin D, and cathepsin E—distinguishes the CG aspartic proteinase from classical members of the aspartic proteinase family. The CG aspartic proteinase cleaved recombinant proenkephalin between the Lys¹⁷²-Arg¹⁷³ pair located at the COOH-terminus of (Met)enkephalin-Arg⁶-Gly⁷-Leu⁸, as assessed by peptide microsequencing. The importance of full-length prohormone as substrate was demonstrated by the enzyme's ability to hydrolyze ³⁵S-labeled proenkephalin and proopiomelanocortin and its inability to cleave tri- and tetrapeptide substrates containing dibasic or monobasic cleavage sites. In this study, results provide evidence for the role of an aspartic proteinase in proenkephalin and prohormone processing.     

Efficient degradationin vitro of all intermediate filament subunit proteins by the Ca2+-activated neutral thiol proteinase from Ehrlich ascites tumor cells and porcine kidney

Vimentin, desmin, glial fibrillary acidic protein, neurofilament triplet proteins, and a mixture of cytokeratins were digested with Ca²⁺-activated neutral thiol proteinase isolated from Ehrlich ascites tumor (EAT) cells and porcine kidney. All intermediate filament proteins were degraded by the proteinase, although with different rates and Ca²⁺ optima. These results are in part at variance with our previous statement that the Ca²⁺-activated proteinase from EAT cells is specific for vimentin and desmin.     

The effect of calciums on molecular motions of proteinase K

The native serine protease proteinase K binds two calcium cations. It has been reported that Ca²⁺ removal decreased the enzyme’s thermal stability and to some extent the substrate affinity, but has discrepant effects on catalytic activity of the enzyme. Molecular dynamics simulations were performed on the Ca²⁺-bound and Ca²⁺-free proteases to investigate the mechanism by which the calciums affect the structural stability, molecular motions, and catalytic activity of proteinase K. Very similar structural properties were observed between these two forms of proteinase K during simulations; and several long-lived hydrogen bonds and salt bridges common to both forms of proteinase K were found to be crucial in maintaining the local conformations around these two Ca²⁺ sites. Although Ca²⁺ removal enhanced the overall flexibility of proteinase K, the flexibility in a limited number of segments surrounding the substrate-binding pockets decreased. The largest differences in the equilibrium structures of the two simulations indicate that, upon the removal of Ca²⁺, the large concerted motion originating from the Ca1 site can transmit to the substrate-binding regions but not to the catalytic triad residues. In conjunction with the large overlap of the essential subspaces between the two simulations, these results not only provide insight into the dynamics of the underlying molecular mechanism responsible for the unchanged enzymatic activity as well as the decreased thermal stability and substrate affinity of proteinase K upon Ca²⁺ removal, but also complement the experimentally determined structural and biochemical data.     

Proteinase Produced by Chlamydia psittaci in L Cells

L cells (mouse fibroblasts) infected with Chlamydia psittaci (strain meningopneumonitis) produced a proteinase differing in solubility in ammonium sulfate from the proteinase of uninfected L cells. Synthesis of the enzyme was inhibited by chloramphenicol but not by cycloheximide, indicating that the new proteinase in infected L cells was synthesized by Chlamydia psittaci. The chlamydial proteinase had no demonstrable ion requirements and was not inhibited by a variety of inhibitors of proteinase activity. Gel filtration experiments suggested a molecular weight of approximately 250,000. The proteinase appeared in infected L cells at the time host cells began to die and the large chlamydial cells began to reorganize into small ones. Some possible functions for the chlamydial proteinase were proposed.     

Characterization of a Cell Envelope-Associated Proteinase Activity from Streptococcus thermophilus H-Strains

The production and biochemical properties of cell envelope-associated proteinases from two strains of Streptococcus thermophilus (strains CNRZ 385 and CNRZ 703) were compared. No significant difference in proteinase activity was found for strain CNRZ 385 when cells were grown in skim milk medium and M17 broth. Strain CNRZ 703 exhibited a threefold-higher proteinase activity when cells were grown in low-heat skim milk medium than when grown in M17 broth. Forty-one percent of the total activity of CNRZ 385 was localized on the cell wall. The optimum pH for enzymatic activity at 37°C was around 7.0. Serine proteinase inhibitors, such as phenylmethylsulfonyl fluoride and diisopropylfluorophosphate, inhibited the enzyme activity in both strains. The divalents cations Ca²⁺, Mg²⁺, and Mn²⁺ were activators, while Zn²⁺ and Cu²⁺ were inhibitors. β-Casein was hydrolyzed more rapidly than α_s1-casein. The results of DNA hybridization and immunoblot studies suggested that the S. thermophilus cell wall proteinase and the lactococcal proteinase are not closely related.     

alpha 1-Proteinase inhibitor, alpha 1-antichymotrypsin, and alpha 2-macroglobulin are the antiapoptotic factors of vascular smooth muscle cells

Serum depletion induces cell death. Whereas serum contains growth factors and adhesion molecules that are important for survival, serum is also likely to have antiapoptotic factor(s). We show here that the plasma proteinase inhibitors alpha1-proteinase inhibitor, alpha1-antichymotrypsin, and alpha2-macroglobulin function as critical antiapoptotic factors for human vascular smooth muscle cells. Cell survival was assured when serum-free medium was supplemented with any one or all of the above serine proteinase inhibitors. In contrast, the cells were sensitive to apoptosis when cultured in medium containing serum from which the proteinase inhibitors were removed. The antiapoptotic effect conferred by the proteinase inhibitors was proportional to proteinase inhibitory activity. Without proteinase inhibitors, the extracellular matrix was degraded, and cells could not attach to the matrix. Cell survival was dependent on the intact extracellular matrix. In the presence of the caspase inhibitor z-VAD, the cells detached but did not die. The activity of caspases was elevated without proteinase inhibitors; in contrast, caspases were not activated when medium was supplemented with one of the proteinase inhibitors. In conclusion, the plasma proteinase inhibitors prevent degradation of extracellular matrix by proteinases derived from cells. Presumably an intact cell-matrix interaction inhibits caspase activation and supports cell survival.     

Degradations of human immunoglobulins and hemoglobin by a 60 kDa cysteine proteinase of Trichomonas vaginalis

The present study was undertaken to investigate the role of cysteine proteinase of Trichomonas vaginalis in escaping from host defense mechanism. A cysteine proteinase of T. vaginalis was purified by affinity chromatography and gel filtration. Optimum pH for the purified proteinase activity was 6.0. The proteinase was inhibited by cysteine and serine proteinase inhibitors such as E-64, NEM, IAA, leupeptin, TPCK and TLCK, and also by Hg²⁺, but not affected by serine-, metallo-, and aspartic proteinase inhibitors such as PMSF, EDTA and pepstatin A. However, it was activated by the cysteine proteinase activator, DTT. The molecular weight of a purified proteinase was 62 kDa on gel filtration and 60 kDa on SDS-PAGE. Interestingly, the purified proteinase was able to degrade serum IgA, secretory IgA, and serum IgG in time- and dose-dependent manners. In addition, the enzyme also degraded hemoglobin in a dose-dependent manner. These results suggest that the acidic cysteine proteinase of T. vaginalis may play a dual role for parasite survival in conferring escape from host humoral defense by degradation of immunoglobulins, and in supplying nutrients to parasites by degradation of hemoglobin.     

Purification of prophenoloxidase from crayfish blood cells,and its activation by an endogenous serine proteinase

A prophenoloxidase was purified from blood cells of the crayfish Pacifastacus leniusculus. The purified proenzyme was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and had a molecular mass of 76 kDa under both non-reducing and reducing conditions. The crayfish prophenoloxidase was a glycoprotein, with an isoelectric point of about 5.4.A 36 kDa serine proteinase, isolated and purified from crayfish blood cells (Aspán et al., 1990b, Insect Biochem.20, 709–718), could convert the 76 kDa prophenoloxidase to phenoloxidase by an apparent proteolytic cleavage, since the molecular masses of two active enzymes, phenoloxidases, were 60 and 62 kDa. A commercial serine proteinase, trypsin, activated prophenoloxidase to phenoloxidase, and as a result a 60 kDa protein was produced.In the blood cells of crayfish four serine proteinases or ³H-DFP binding proteins are present, with masses of 36, 38, 50 and 67 kDa. However, ³H-DFP labelling of proteins in blood cells lysate, prepared in its inactive form, only yielded labelled bands of 50 and 67 kDa, whereas addition of an elicitor to prophenoloxidase system activation, a β-1,3-glucan, resulted in the appearance of four ³H-DFP labelled proteins, with molecular masses of 67, 50, 38 and 36 kDa, respectively. Thus, the 36 kDa endogenous serine proteinase, the prophenoloxidase activating enzyme, ppA, may be present as an inactive precursor in crayfish blood cells. The 38 and 36 kDa proteinases could both cleave the chromogenic peptide S-2337 [Bz-Ile-Glu-(γ-O-Piperidyl)-Gly-Arg-p-nitroaniline], and specifically bind prophenoloxidase.These results show that crayfish prophenoloxidase, the terminal enzyme of the prophenoloxidase activating cascade, a proposed defence pathway in arthropod blood, can be converted to active enzyme by an apparent proteolytic cleavage, not only by a commercial proteinase, but also by an endogenous serine type proteinase.     

Digestive physiology and characterization of digestive cathepsin L-like proteinase from the sugarcane weevil Sphenophorus levis

Sugarcane is an important crop that has recently become subject to attacks from the weevil Sphenophorus levis, which is not efficiently controlled with chemical insecticides. This demands the development of new control devices for which digestive physiology data are needed. In the present study, ion-exchange chromatography of S. levis whole midgut homogenates, together with enzyme assays with natural and synthetic substrates and specific inhibitors, demonstrated that a cysteine proteinase is a major proteinase, trypsin is a minor one and chymotrypsin is probably negligible. Amylase, maltase and the cysteine proteinase occur in the gut contents and decrease throughout the midgut; trypsin is constant in the entire midgut, whereas a membrane-bound aminopeptidase predominates in the posterior midgut. The cysteine proteinase was purified to homogeneity through ion-exchange chromatography. The purified enzyme had a mass of 37 kDa and was able to hydrolyze Z-Phe-Arg-MCA and Z-Leu-Arg-MCA with k_cat/K_m values of 20.0 ± 1.1 μM⁻¹ s⁻¹ and 30.0 ± 0.5 μM⁻¹ s⁻¹, respectively, but not Z-Arg-Arg-MCA. The combined results suggest that protein digestion starts in the anterior midgut under the action of a cathepsin L-like proteinase and ends on the surface of posterior midgut cells. All starch digestion takes place in anterior midgut. These data will be instrumental to developing S. levis-resistant sugarcane.     

Purification and some physico-chemical and enzymic properties of a calcium ion-activated neutral proteinase from rabbit skeletal muscle

Ca²⁺-activated neutral proteinase was purified from rabbit skeletal muscle by a method involving DEAE-Sephacel chromatography, affinity chromatography on organomercurial–Sepharose and gel filtration on Sephacryl S-200 and Sephadex G-150. The SDS (sodium dodecyl sulphate)/polyacrylamide-gel-electrophoresis data show that the purified enzyme contains only one polypeptide chain of mol.wt. 73000. The purification procedure used allowed us to eliminate a contaminant containing two components of mol.wt. about 30000 each. Whole casein or α₁-casein were hydrolysed with a maximum rate at 30°C, pH7.5, and with 5mm-CaCl₂, but myofibrils were found to be a very susceptible substrate for this proteinase. This activity is associated with the destruction of the Z-discs, which is caused by the solubilization of the Z-line proteins. The activity of the proteinase in vitro is not limited to the removal of Z-line. SDS/polyacrylamide-gel electrophoresis on larger plates showed the ability of the proteinase to degrade myofibrils more extensively than previously supposed. This proteolysis resulted in the production of a 30000-dalton component as well as in various other higher- and lower-molecular-weight peptide fragments. Troponin T, troponin I, α-tropomyosin, some high-molecular-weight proteins (M protein, heavy chain of myosin) and three unidentified proteins are degraded. Thus the number of proteinase-sensitive regions in the myofibrils is greater than as previously reported by Dayton, Goll, Zeece, Robson & Reville [(1976) Biochemistry 15, 2150–2158]. The Ca²⁺-activated neutral proteinase is not a chymotrypsin- or trypsin-like enzyme, but it reacted with all the classic thiol-proteinase inhibitors for cathepsin B, papain, bromelain and ficin. Thus the proteinase was proved to have an essential thiol group. Antipain and leupeptin are also inhibitors of the Ca²⁺-activated neutral proteinase.     

Purification and properties of a proteinase from Vipera lebetina (snake) venom

A proteinase from the venom of Vipera lebetima was purified by chromatography on Sephadex G-100 and CM-cellulose. The purified proteinase was homogeneous on SDS-polyacrylamide gel electrophoresis and consisted of a single chain with molecular weight of 37 000±1500. The isoelectric point of the proteinase was over 10. The enzyme was active on casein but not on esters and amides of arginine. It split the oxidized insulin B-chain at the peptide bonds of Tyr¹⁶-Leu¹⁷, Phe²⁴-Phe²⁵ and Phe²⁵-Tyr²⁶, and glucagon at the bonds Tyr¹⁰-Ser¹¹, Leu¹⁴-Asp¹⁵ and Leu²⁶-Met²⁷. The enzyme was inhibited by DFP and PMSF, and partially by soybean trypsin inhibitor, but not with EDTA.     

PROTEIN DEGRADATION IN SQUID GIANT AXONS

Axoplasm extruded from giant axons of the Chilean squid, Dosidicus gigas, contained a low level of neutral proteinase-like activity, equivalent to 4 × 10^?6 mg of chymotrypsin per mg of axoplasmic protein. The enzyme was active at physiological pH and ionic strength. Activity was completely inhibited by 1 mM-para-hydroxymercuribenzoate and was enhanced by divalent metal cations, especially Ca²⁺. Axoplasm also exhibited proteinase activity at pH 4.8. Both neutral and acid proteinase like activities were also present in the axonal sheath containing Schwann cells, but their specific activities relative to those in the axoplasm were different. A physiological role, related to the axoplasmic flow of protein, is discussed for the axoplasmic neutral proteinase-like activity.     

Transformation of rat liver cell line by Rous sarcoma virus causes loss of cell surface fibronectin, accompanied with secretion of metallo-proteinase that preferentially digests the fibronectin

An epithelial cell line derived from the liver of a normal Buffalo rat (BRL) was transformed by Rous sarcoma virus (RSV). The RSV-transformed cells were separated into five clones (RSV-BRL1 through 5), which were morphologically different. RSV-BRL cells exhibited the following characteristics distinct from those of BRL cells: tumorigenicity, irregular cell arrangement, loose intercellular junction, growth in soft agar (anchorage-independent growth) except for RSV-BRL3 and 5, and loss of cell surface fibronectin. When BRL cells were cultured in the standard medium supplemented with the serum-free conditioned medium of RSV-BRL cells, the amount of the cell surface fibronectin decreased significantly. It was found that RSV-BRL cells secreted a proteinase capable of hydrolyzing the fibronectin, whereas BRL cells secreted hardly any of this proteinase. The fibronectin-hydrolyzing proteinase (FNase) could also hydrolyze plasma fibronectin added as an exogenous substrate. The hydrolysis of plasma fibronectin was inhibited by ethylenediamine tetraacetate, but stimulated by rho-chloromercuribenzoate and calcium ion. This indicates that FNase is a metallo-enzyme, but not a serine or thiol enzyme. In addition to the proteinase, RSV-BRL cells secreted plasminogen activator and a proteinase inhibitor which inhibited the activity of plasmin but not FNase.     

Extracellular proteinase fromLactobacillus murinus

Lactobacillus murinus CNRZ 313 produced an extracellular proteinase irrespective of the Ca²⁺ content in the culture medium. Proteinase activity was optimal at 37 °C and pH 7.5 in phosphate buffer (0.2 mol/L). It was stimulated by Mg²⁺ and Mn²⁺ and was inhibited by Zn²⁺. Ca²⁺ did not affect the enzymic activity but the proteinase liberated in the presence of this ion is more stable. The enzyme was purified to homogeneity from cell-free culture medium.     

Biosynthesis of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain

The effect of certain nutrients on the growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. Glucose was found to inhibit the synthesis of proteinase in the early (28 h of growth) but not in the late stationary phase (48 h of growth). The inhibitory effect of the other mono-and disaccharides studied was less pronounced. Casamino acids added to the medium at concentrations of 0.1–1% as an additional carbon and nitrogen source stimulated enzyme biosynthesis. Individual amino acids (cysteine, asparagine, glutamine, tryptophan, histidine, and glutamate) also stimulated enzyme biosynthesis in the early stationary phase by 25–30%, whereas other amino acids (valine, leucine, alanine, and aspartate) were ineffective or even slightly inhibitory to enzyme production. The stimulatory effect of the first group of amino acids on the synthesis of proteinase in the late stationary phase was negligible. In contrast, the bivalent ions Ca²⁺, Mg²⁺, and Mn²⁺ stimulated biosynthesis of proteinase in the late stationary phase (by 20–60%) and not in the early stationary phase. The data indicate that there are differences in the biosyntheses of proteinase by the recombinant B. subtilis strain during the early and late periods of the stationary phases.     

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.     

Inhibitory Effect of Glycine on the Production of Amylase and Proteinase by Bacillus subtilis

It was found that the production of amylase and proteinase by washed cells of Bacillus subtilis var. amyloliquefaciens was inhibited by glycine and its peptides but not by glycine derivatives, in which the free amino group was protected with various groups. Incorporation experiments of glycine-C¹⁴ revealed that about 60 per cent of the radioactivity which had been incorporated into the cells was found in the free amino acid fraction of the bacteria. The inhibitory effect of glycine was easily reversed by the addition of amino acid such as alanine, methionine and glutamic acid. Spermine also caused the reversal of inhibition of the enzyme production by glycine.