Metalloproteinase activities expressed during development and maturation of the rat prostatic complex and seminal vesicles.

The objective of this study was to characterize proteinase activities expressed during development and maturation of the prostate gland and seminal vesicles of the rat by using gelatin-and casein-containing SDS polyacrylamide gel zymography. The prostatic complexes of 2- and 10-day-old animals and the individual lobes of the prostate (ventral, dorsolateral, and anterior [coagulating gland]) and the seminal vesicles of 15-day-old animals expressed prominent gelatinolytic activities of approximately 64, 71, and 76 kDa. These activities had properties of metalloproteinases; i.e., they were stimulated by Ca2+ and inhibited by EDTA and EGTA. They were greatly diminished by 52 days of age (immediately postpuberty) and were not detected in the dorsal lobe of the adult. Less active gelatinolytic proteinases with molecular masses of approximately 34 and 43 kDa were expressed in the developing prostatic complexes and individual lobes and seminal vesicles, but they were not detected in postpubertal animals. Weak gelatinolytic activities of 82, 85, and 89 kDa were found in the prostatic complexes; these activities were greatly diminished in all prostate lobes with sexual maturation but were expressed in the seminal vesicles at all ages. A large-molecular-mass Ca(2+)-independent proteinase of 130 kDa or greater was first detected in the dorsolateral prostate at 21 days of age. This activity was expressed in both the lateral and dorsal lobes of the adult but was greater in the lateral lobe. Proteinase activities of about 22 and 26 kDa that were not stimulated by Ca2+ were detected in the ventral prostate at 15 days of age by means of both gelatin and casein gels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production and partial characterization of extracellular proteinases from <Emphasis Type="Italic">Streptomyces malaysiensis</Emphasis>, isolated from a Brazilian cerrado soil

Streptomyces malaysiensis AMT-3, isolated from a Brazilian cerrado soil, showed proteolytic activities detected by gelatin–sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum proteinase production was obtained when using 2.5% wheat bran and 0.1% yeast extract in the culture medium, after 5 days incubation at 30°C. The enzymatic complex degraded gelatin optimally at pH 7.0, and under these conditions eight proteolytic bands (four serine-proteinases and four metaloproteinases), ranging from 20 to 212 kDa, were detected on the culture supernatant filtrates. In addition, a 35-kDa proteinase was thermostable at 60°C for 120 min. These results point out to the applicability of gelatin zymograms in the characterization of crude enzymatic complexes. According to our results, this enzymatic complex could be used for biotechnological applications.     

DNA polymerases and DNA topoisomerases solubilized from nuclear matrices of regenerating rat livers

DNA topoisomerase activity together with the activities of DNA polymerase were detected in a form tightly associated with rat liver nuclear matrices. DNA polymerase activities were solubilized from the nuclear matrices of regenerating rat livers by sonic treatment followed by extraction of these activities with detergent and salt. The predominant activity was mainly α-polymerase as judged from the size determined by sucrose density gradient centrifugation. However, only β-polymerase activity was detected in the matrix of normal rat livers. DNA topoisomerase activity, detected in both regenerating and normal liver nuclear matrices, showed a molecular size of about 4 S in sucrose gradient, and was active in the presence of EDTA. These results suggest that this enzyme belongs to type I topoisomerase.     

Gelatinolytic proteinase activities in human seminal plasma

Proteinase activities in human seminal plasma were detected using gelatin-containing sodium dodecyl sulphate-polyacrylamide gel electrophoresis zymography. Three prominent bands of activity of Mr 60,000, 66,000 and 90,000 were observed as well as 9 other bands of less intensity (34,000-158,000). These proteinases were dependent upon calcium for optimal activity, did not hydrolyse casein, and were predominantly in the soluble portion of seminal plasma. Examination of seminal plasma of men with different sperm concentrations, split ejaculates, and prostatic secretions indicated that the prostate gland was a source of most of these activities. Proteinase activities of Mr 34,000, 37,000, 82,000 and 120,000 were expressed more frequently in seminal plasma from normozoospermic men than from seminal plasma of oligo- or azoospermic men, indicating that they may also arise from spermatozoa in the semen sample. The proteinases of Mr 60,000 and 66,000 were found in all seminal plasmas whereas there was variation in the expression of the other molecular forms of enzyme, even in the normozoospermic samples. There are multiple forms of gelatinolytic proteinase activities in human seminal plasma which appear to arise from multiple sources in the reproductive tract including the Cowper's/urethral glands, the prostate gland, seminal vesicle and/or spermatozoa. Their function(s) in semen remains to be established.     

Comparison, characterization, and identification of proteases and protease inhibitors in epididymal fluids of domestic mammals. Matrix metalloproteinases are major fluid gelatinases

The testicular and epididymal fluids of ram, boar, and stallion were analyzed by means of one-dimensional and two-dimensional gelatin gel zymography. Five main gelatinolytic bands were revealed in the ram and at least seven were observed in the boar and stallion. These proteolytic bands showed regionalized distribution throughout the organs. The two main proteolytic activities at around 54-66 kDa retrieved in all three species were inhibited by EDTA and phenanthroline, indicating that they were metallo-dependent enzymes. The activity of some of the low-molecular-weight gelatinases was also decreased by EDTA, whereas others were inhibited by serine protease inhibitors. One of the main proteases at 60-62 kDa from the caput fluid of the stallion and the ram was N-terminal sequenced; in both cases, high sequence homology was found with the N-terminal of the matrix-metalloproteinase-2 pro-form (pro-MMP-2). Antibodies against MMP-2, MMP-3, and MMP-9 gelatinases confirmed the regional distribution in the fluids of pre -, pro-, active, or degraded forms of these metalloproteases in all three species. We also observed the presence of acrosin in epididymal fluids, which was probably released by dead spermatozoa, but this enzyme did not explain all the serine protease activity. Moreover, the majority of this enzyme is bound to the protease inhibitor alpha(2)-macroglobulin, which is present in the fluids of all three species. TIMP-2, a potent inhibitor of MMPs, was present in the fluid of the caput regions in the ram and boar, and in the caput and caudal fluids of the stallion. This study demonstrated that similar types of proteases and inhibitors are regionally distributed in the epididymal fluids of three domestic species, suggesting an identical role in the sperm maturation process, the plasticity of this organ, or both.     

Collagenase activation of latent matrix-degrading proteinases from human plasma fibronectin

Fibronectin contains two latent gelatinolytic enzymes, FN-gelatinase and FN-laminase that can be activated in the presence of Ca2+ from the purified cathepsin D-produced 190-kDa fibronectin fragment. The results of this work show that Achromobacter collagenase cleaves fibronectin and generates an active FN-gelatinase. In contrast to the cathepsin D digest, the collagenase digest directly exhibits gelatinolytic activity without additional activation. The gelatinolytic activity of the total collagenase digest can be inhibited by phenylmethanesulfonyl fluoride, a serine proteinase inhibitor and by pepstatin A, an aspartic-acid proteinase inhibitor. FN-laminase activity, when assayed with its synthetic substrate GPAGPR and also with laminin was revealed after separation of the collagenase digest of fibronectin on heparin Ultrogel. FN-gelatinase and FN-laminase activities were found in heparin unretained and heparin strongly retained fractions. These results have demonstrated that in contrast to cathepsin D, Achromobacter collagenase activates two matrix-degrading proteinases from fibronectin, FN-Gelatinase und FN-Laminase.     

Urokinase directly activates matrix metalloproteinases-9: a potential role in glioblastoma invasion

Previous reports showed that urokinase plasminogen activator (uPA) converts plasminogen to plasmin which then activates matrix metalloproteinases (MMPs). Here, we report that uPA directly cleaved pro-MMP-9 in a time-dependent manner at both C- and N-terminus and generated two gelatinolytic bands. uPA-activated-MMP-9 efficiently degraded fibronectin and blocked by uPA inhibitor B428 and recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1). B428 inhibited basal and PMA-induced active MMP-9 in glioblastomas (GBM) U1242 cell media as well as cell invasion in vitro. A combination of MMP-9 and uPA antibodies more significantly inhibited U1242 cell invasion than uPA or MMP-9 antibody alone. Both uPA and MMP-9 were highly expressed in U1242 cell and GBM patient specimens. Furthermore, two active MMP-9 fragments with identical molecular weights to the uPA-activated MMP-9 products were detected in GBM patient specimens. These results suggest that uPA-mediated direct activation of MMP-9 may promote GBM cell invasion.     

Extracellular matrix remodeling and metalloproteinase involvement during intestine regeneration in the sea cucumber Holothuria glaberrima

The sea cucumber, Holothuria glaberrima, has the capacity to regenerate its internal organs. Intestinal regeneration is accomplished by the thickening of the mesenteric border and the invasion of this thickening by mucosal epithelium from the esophagus and the cloaca. Extracellular matrix (ECM) remodeling has been associated with morphogenetic events during embryonic development and regeneration. We have used immunohistochemical techniques against ECM components to show that differential changes occur in the ECM during early regeneration. Labeling of fibrous collagenous components and muscle-related laminin disappear from the regenerating intestine and mesentery, while fibronectin labeling and 4G7 (an echinoderm ECM component) are continuously present. Western blots confirm a decrease in fibrous collagen content during the first 2 weeks of regeneration. We have also identified five 1,10-phenanthroline-sensitive bands in collagen gelatin zymographs. The gelatinolytic activities of these bands are enhanced during early stages of regeneration, suggesting that the metalloprotease activity is associated with ECM remodeling. Inhibition of MMPs in vivo with 1,10-phenanthroline, p-aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamate or N-CBZ-Pro-Leu-Gly hydroxamate produces a reversible inhibition of intestinal regeneration and ECM remodeling. Our results show that significant changes in ECM content occur during intestine regeneration in the sea cucumber and that the onset of these changes is correlated to the proteolytic activities of MMPs.     

The identification, purification and characterisation of an inhibitor of collagenase (20K) produced by neoplastic epithelial cells

A rat carcinoma cell line (T2/H7) constitutively synthesised interstitial collagenase. When these cells were incubated with 12-O-tetradecanoylphorbol 13-acetate (TPA) they secreted an inhibitor of collagenase, which resulted in a net decrease of collagenolytic activity being detected in conditioned medium. Using reverse zymography, the Mr of the inhibitor was found to be 20,000 which suggests that it may be the rat homologue of inhibitor of metalloproteinase 2 (IMP2; TIMP-2), as it inhibited both the gelatinolytic and collagenolytic activities of rat collagenase. The inhibitor was separated from collagenase by filtration through a YM30 membrane. The inhibitor was purified further by sequential chromatography on heparin-Sepharose and Con A-Sepharose. It bound to heparin-Sepharose in 75 mM NaCl and was eluted with 300 mM NaCl. It did not bind to Con A-Sepharose, suggesting that it was a non-glycosylated molecule. The inhibitor was resistant to treatment with either trypsin, APMA or heat.     

Genetically Controlled Variation of "Acid" β-Galactosidase Detected in Rattus norvegicus by Isoelectric Focusing

Two genetically variant forms of rat "acid" beta-galactosidase were found to differ in isoelectric point and pH dependence, but not in thermostability or sensitivity to inhibition by p-mercuribenzoate (PMB). The results of two backcrosses and an intercross indicated that the isoelectric focusing phenotypes are controlled by two codominant alleles at a single autosomal locus, for which we propose the name Glb-1. No significant linkage between Glb-1 and albino (LG I), brown (LG II), or hooded (LG VI) was observed. Strain-specific differences in total levels of kidney beta-galactosidase were detected, but it is not yet known whether the variation is controlled by genes linked to Glb-1. Experiments in which organ homogenates were incubated with neuraminidase indicated that the genetically variant forms do not result from differences in sialylation, though sialylation does appear to be largely responsible for the presence of multiple bands within each phenotype and for differences in the banding patterns of beta-galactosidases derived from different organs. The beta-galactosidase present in the bands used for Glb-1 typing resembles human GM1 gangliosidase (GLB1) with respect to pH optimum, substrate specificity, and susceptibility to inhibition by PMB. It also appears that Glb-1 is homologous with the Bgl-e locus of the mouse. In rats as in mice the genetically variant bands of beta-galactosidase are active at acid pH and have relatively high isoelectric points. In both species these bands are readily detectable in kidney homogenates, and can be revealed in homogenates of liver or spleen following treatment with neuraminidase. The presence of the same beta-galactosidase bands in homogenates of rat kidney and small intestine as well as in neuraminidase-treated homogenates of liver and spleen suggests that the Glb-1 variants differ by one or more point mutations in the structural gene for "acid" beta-galactosidase.     

Matrix metalloproteinase activities of turkey (Meleagris gallopavo) bile

The bile from turkey (Meleagris gallopavo) gall bladders was found to contain substantial matrix metalloproteinase (MMP) activities using gelatin, collagen, and casein substrate zymography, [³H]labeled collagen degradation assays, and gelatin–agarose affinity purification. Five major bands corresponding to approximate M_w of 64, 60, 46, 40 and 36 kDa showed gelatinolytic activities. On incubation with p-aminophenylmercuric acetate or thimerosal, the densities of both the 64- and 46-kDa bands decreased with increasing intensities of the 60- and 40-kDa bands. Both the 64- and 60-kDa bands showed collagenolytic activities whereas the caseinolytic activities appeared as diffuse bands corresponding to M_w of approximately 60, 40 and 36 kDa. Using [³H]collagen as substrate, the bile enzymes showed both a time and concentration-dependent degradation, which could be inhibited by the MMP inhibitors such as EDTA, phenanthroline, and N-[(2R)-2-(hydroxyamido carbonylmethyl)-4-methylpentanonyl]-l-tryptophan methylamide, but not by serine and cysteine protease inhibitors like trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane, phenylmethylsulfonyl fluoride or leupeptin. Both 60- and the 40-kDa gelatinolytic bands showed affinity adsorption to a gelatin–agarose matrix. The physiological roles of bile MMPs are not clear, but their involvement in the digestive functions of birds are likely.     

Enzyme activity in partly dissociated fragments of rat intestinal maltase/glucoamylase.

Pure rat intestinal maltase/glucoamylase was partially inactivated in 1% sodium dodecyl sulphage by heating at 40--70 degree C for 5 min at pH 7.5, or by lowering the pH to 5.4--6.6 at 24 degree C. When partially active preparations were electrophoresed in the presence of sodium dodecyl sulphate, a complicated protein band pattern of incompletely dissociated fragments of the enzyme was observed. Complete dissociation of the enzyme in sodium dodecyl sulphate, induced by boiling or by pH values below 5.4, was accompanied by total loss of enzyme activity and simplification of the protein pattern to five major species. Although the original enzyme band was absent from some partially dissociated preparations, enzyme activity was present and was associated with several transient protein bands on the gels. Maltase and alpha-glucosidase activities were detected in these bands, but glucoamylase activity was absent.     

Diazaborine treatment of Baker's yeast results in stabilization of aberrant mRNAs

Upon Northern blotting, Saccharomyces cerevisiae that was treated with diazaborine showed aberrant mRNAs that were extended at the 3'-end and terminated at secondary processing sites. These bands were also detected in untreated Deltaupf1, Deltaxrn1, and rat7-1 mutants. This finding demonstrates that the aberrant mRNAs also occur in untreated strains in small quantities and can reach the cytoplasm, where they are normally degraded by Xrn1p. Diazaborine treatment stabilizes these mRNAs. The detection of the aberrant bands in the untreated rat7-1 strain indicates that Rat7 is involved in quality control of RNA. The aberrant mRNAs were not detected after diazaborine treatment of a DRG1-1 mutant. Drg1p, a member of the family of AAA (ATPases associated with a variety of cellular activities) proteins, which are thought to represent specific chaperones, may be involved in the process of unfolding the mRNA-ribonucleoprotein complex or in the recognition of aberrant mRNA molecules in the cytoplasm.     

Changes in protein composition and hydrolytic enzyme activity of Escherichia coli and Hafnia alvei grown in human fluids

Growing of Escherichia coli and Hafnia alvei cells in several cell-free human fluids, such as normal serum, serum from diabetic patients, pleural, ascitic and spinal fluid, revealed that various biochemical changes occurred. Protein profile on SDS-PAGE as well as acid and alkaline phosphohydrolytic enzymes on native gels of cell extracts were affected after culturing of bacteria in the above fluids. Gelatinolytic and hyaluronolytic activity was of interest because both of them are histolytic enzymes. Although there was a potential appearance of gelatinolytic bands on gelatin-SDS-PAGE in cells starved in seawater, none of these activities were expressed in cells grown in human fluids. A hyaluronolytic activity of approximately 45 KDa was present in cells cultured in Mueller Hinton broth. This enzyme was decreased either in cells starved in seawater or in cells grown in human fluids to an almost invisible band on hyaluronan-SDS-PAGE.     

Purification and characterization of a trypsin-like serine proteinase from rat brain slices that degrades laminin and type IV collagen and stimulates protease-activated receptor-2

A trypsin-like serine proteinase was purified from the incubation medium of rat brain slices by gelatin zymography. The purification consisted of ammonium sulfate precipitation, benzamidine-Sepharose 6B affinity chromatography, and carboxymethyl-cellulose and gel filtration chromatographies. The gelatinolytic activity, identified at 22 kDa (P22) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, was eluted as one active peak throughout the purification, and the final preparation gave a single protein peak on reverse-phase HPLC. Diisopropyl fluorophosphate, benzamidine, p-toluenesulfonyl-L-lysine chloromethyl ketone, and aprotinin completely inhibited the activity of P22, whereas phenanthroline, p-toluene-sulfonyl-L-phenylalanine chloromethyl ketone, and elastinal did not. P22 efficiently digested the extracellular matrix proteins laminin and type IV collagen. P22 produced an increase in intracellular Ca2+ concentration in A172 glioblastoma, which was desensitized through prior stimulation with protease-activated receptor-2 agonist peptide SLIGKV, indicating that P22 can stimulate protease-activated receptor-2. Rat brain penetration injury induced gelatinolytic activity in the lesioned area whose molecular size was consistent with that of P22. These results indicated that on incubation of rat brain slices, a trypsin-like serine proteinase was secreted into the medium that was capable of digesting extracellular matrix and stimulating protease-activated receptor-2. It is suggested that the gelatinolytic activity induced by brain injury might be that of P22.     

Trichomonas vaginalis, Tritrichomonas foetus, and Trichomitus batrachorum: comparative proteolytic activity

At least four proteolytic activities were detected in the lysates of each of Trichomonas vaginalis, Tritrichomonas foetus, and Trichomitus batrachorum. These were HPAase, a dithiothreitol-dependent activity on hide powder azure; AZCase, a dithiothreitol-dependent activity on azocasein; and two distinct activities towards peptide nitroanilide derivatives--one was optimally active at pH 7 and stimulated by dithiothreitol; the other had no dithiothreitol requirement and was highly active at pH 5. HPAase and AZCase were active over a broad pH range. Overall, with respect to these four activities, T. batrachorum and T. vaginalis were quite similar. In contrast, T. vaginalis and T. foetus differed from one another in several respects, notably the level of HPAase activity and the properties of the dithiothreitol-independent activity. Multiple bands of proteinase activity were demonstrated with each species after electrophoresis of parasite extracts on polyacrylamide gels containing denatured haemoglobin. They appeared optimally at acid pH and in the presence of dithiothreitol. The proteinase band patterns of T. foetus were similarly complex (at least six bands), whereas T. batrachorum gave a much simpler pattern (three bands). The sensitivities to proteinase inhibitors suggested that all the activities were due to cysteine proteinases. The results show that there are some similarities in the proteolytic activities of all three trichomonad species, and that the two parasites of the urinogenital tracts of mammals possess additional features in common.     

Purification and characterization of a lysosomal form and a variant form of beta-glucuronidase from the rat basophil leukaemia tumour.

Two isoenzyme of beta-glucuronidase from a rat basophil leukaemia tumour were co-purified 4067-fold by (NH4)2SO4 precipitation and sequential chromatography on concanavalin A--Sepharose, Sephadex G-200, DEAE-cellulose, CM-cellulose and phosphocellulose. The purity of the mixture was established by the coincidence of the peaks of enzyme activity and protein at a molecular weight of 300 000 on Bio-Gel P-300, the presence of only two protein bands, both of them enzymically active, in polyacrylamide gels after electrophoresis under non-denaturing conditions, and the presence of a single subunit species, of mol.wt. 75 000, after electrophoresis in polyacrylamide gels under a denaturing conditioning. The major isoenzyme co-migrated with the L form from rat liver during electrophoresis in alkaline polyacrylamide gels, whereas the minor isoenzyme migrated more rapidly than either the lysosomal form or the rat liver microsomal form and was designated the tumour (T) isoenzyme. A mixture of the purified isoenzymes from two preparations had an average specific activity of 1389 units/mg for phenolphthalein beta-D-glycopyranosiduronic acid. The L and T isoenzymes, which had pI5.9 and 5.7 respectively, could be obtained free of cross-contamination by isoelectric focusing and had similar specific activities. Although the T isoenzyme could be a catabolic product of the M or the L form, it could also be a unique tumour product, because it was not detected in extracts of normal rat tissues.     

Antigens in chromatin associated with proliferating and nonproliferating cells

Xenoantisera were raised to total chromatin from the leukemia cell line K562, or materials released through limited deoxyribonuclease I digestion of nuclei or during the control incubation of nuclei without enzyme. The peroxidase-antiperoxidase method of antibody-antigen detection was employed to visualize individual antigens resolved on one-dimensional polyacrylamide gels following transfer to sheets of nitrocellulose (immunotransfers). Each antiserum contained multiple antigen specificities as evidenced by the diverse patterns of reactive bands displayed on the immunotransfers. The most striking difference in antigens recognized between the antisera was observed in the molecular weight region below 50,000, where two highly reactive bands were seen mainly with antiserum to nuclear materials released by deoxyribonuclease I digestion. The antigens detected with all of the antisera were present in chromatins prepared from proliferating cells, while the levels of antigens present in chromatin from non-proliferating peripheral blood lymphocytes were greatly reduced or not detected. Antigens in chromatin from proliferating cells that migrated with apparent molecular weights of 37,000 and 100,000 were not lost once the activities to antigens in lymphocyte chromatin were absorbed out. These two activities were absorbed from antisera with the same amount of chromatins from proliferating cells. Two antigens migrating at molecular weight 52,000 and 76,000 appeared more active in the chromatin from unstimulated lymphocytes than in chromatin from proliferating cells.     

Electrophoretic Forms of Chitinolytic and Lysozyme Activities in Ruminal Protozoa

Homogenates from a mixed ruminal protozoal population and a ruminal protozoon Entodinium caudatum were analyzed for chitinolytic and lysozyme activities by sodium dodecyl sulfate polyacrylamide gel electrophoresis. For chitinase activity, up to eight bands in mixed protozoa and seven bands in E. caudatum were detected. Estimated molecular mass ranged from 70 to 110 kDa. These enzymes did not display lysozyme activity. N-Acetyl-β-glucosaminidase activity was also detected in both samples with an estimated molecular mass of 37 kDa. Lysozyme activity in mixed protozoa was present in two major and three minor bands, where one major band displayed the same motility as chicken egg white (CEW) lysozyme, and the other had an approximate molecular mass of 17.5 kDa. The latter remained active even when denatured in the presence of dithiothreitol and renatured under anaerobic conditions. Entodinium caudatum presented one major band coincident with that of CEW lysozyme and a minor band at the 17.5-kDa point. This study showed that protozoal chitinase and lysozyme activities are originated from several enzymes and that none of these enzymes exhibited both activities.