Purification and characterization of bovine interstitial collagenase and tissue inhibitor of metalloproteinases.

In this report we describe the purification of bovine interstitial collagenase and provide information on its substrate specificity, kinetic parameters of catalytic activity, and amino terminal protein sequence. In addition, we present a simplified protocol for the purification of bovine tissue inhibitor of metalloproteinases (TIMP). Collagenase was purified by sequential chromatography through heparin-Sepharose, DEAE-Sepharose, and green-agarose, resulting in a product that was greater than 95% pure as judged by polyacrylamide electrophoresis. Typical of other interstitial collagenases, the isolated bovine protein was activated by protease and organomercurial treatment. It also demonstrated a kinetics and substrate specificity similar to those of human collagenase. TIMP was purified by sequential chromatography through heparin-Sepharose and DEAE-Sepharose followed by reverse-phase HPLC. The purified protein had a size, N-terminal sequence, and inhibitor activity similar to those of other mammalian TIMPs. Partial peptide sequences suggested that bovine collagenase and TIMP have strong sequence homology to their human homologues.     

Mouse bone collagenase inhibitor: purification and partial characterization of the inhibitor from mouse calvaria cultures

The organ culture of neonatal mouse calvaria produced both collagenase and collagenase inhibitor. The inhibitor was purified by a series of column chromatographies: DEAE-cellulose and CM-cellulose ion-exchange chromatography, concanavalin A-Sepharose and heparin-Sepharose affinity chromatography, and finally by Sephacryl S-200 gel filtration. The purified inhibitor migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular mass of 28,000. The inhibitor was purified 140-fold to a specific activity of 163 units/mg with a yield of 18% over the first step of the purification by DEAE-cellulose chromatography. The inhibitor stained positively for carbohydrate with periodic acid-Schiff's reagent indicating, in conjunction with its affinity to concanavalin A, that the inhibitor is a glycoprotein. In addition to mouse bone collagenase, this inhibitor also inhibited chick bone, rat bone, rabbit corneal, and human gingival collagenase, but did not inhibit bacterial collagenase.     

Purification and characterization of bovine dental pulp collagenase inhibitor

Root pulps from bovine unerupted wisdom teeth produce a potent collagenase inhibitor together with latent collagenase when cultured in Eagle's minimal essential medium (Biochem. Int. 5, 763, 1982). The inhibitor was purified more than 700-fold from the explant medium using Con A-Sepharose, Ultrogel AcA 44 and DE-52 cellulose columns. It showed a single band (MW = 36,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but showed multiple bands on basic (pH 8.3) polyacrylamide gel electrophoresis and electrofocusing. The inhibitor is a sialo-glycoprotein containing approx. 20% carbohydrate by weight and its composition suggests that it contains complex-type oligosaccharides. The electrophoretic heterogeneity of the inhibitor was proved to be due to the attachment of different numbers of sialic acid residues. All the SH groups were demonstrated to exist as six disulfide linkages which might be involved in the inhibitory activity. The bovine pulp inhibitor does not combine with collagen. The addition of the inhibitor to activated collagenase resulted in dose-dependent inhibition of the enzyme activity, but the interaction between the inhibitor and activated collagenase is not tight enough for the complex to remain intact during gel filtration column chromatography. A rabbit antiserum was prepared against the inhibitor, and immunoglobulin purified from the antiserum can completely abolish the inhibitory activity of the inhibitor.     

Isolation and characterization of an inhibitor of neovascularization from scapular chondrocytes

An inhibitor of neovascularization from the conditioned media of scapular chondrocytes established and maintained in serum-free culture has been isolated and characterized. To determine whether this chondrocyte-derived inhibitor (ChDI) was capable of inhibiting neovascularization in vivo, this protein was assayed in the chick chorioallantoic membrane assay. ChDI was a potent inhibitor of angiogenesis in vivo (4 micrograms = 87% avascular zones). This inhibitor is also an inhibitor of fibroblast growth factor-stimulated capillary endothelial cell (EC) proliferation and migration, as well as being an inhibitor of mammalian collagenase. ChDI significantly suppressed capillary EC proliferation in a dose-dependent, reversible manner with an IC50 (the inhibitory concentration at which 50% inhibition is achieved) of 2.025 micrograms/ml. Inhibition by ChDI of growth factor-stimulated capillary EC migration was also observed using a modified Boyden chamber assay (IC50 = 255 ng/ml). SDS-PAGE analysis followed by silver staining of ChDI purified to apparent homogeneity revealed a single band having an M(r) of 35,550. Gel elution experiments demonstrated that only protein eluting at this molecular weight was anti-angiogenic. These studies are the first demonstration that chondrocytes in culture can produce a highly enriched, potent inhibitor of neovascularization which also inhibits collagenase.     

Purification and characterization of a collagenase inhibitor produced by bovine vascular smooth muscle cells

A potent polypeptide inhibitor of mammalian collagenases was purified to homogeneity from medium conditioned by bovine aortic smooth muscle cells maintained in culture. This inhibitor was purified by a series of molecular sieve and heparin-Sepharose chromatographic procedures; it had an apparent Mr of 28,500 and was a major protein secreted by the smooth muscle cells. It was found to be active against several mammalian collagenases including those obtained from rabbit and human fibroblasts and a tumor-specific type IV collagenase. In contrast, it had minimal inhibitory activity for bacterial collagenase and was inactive against the serine proteases plasmin and trypsin. The inhibitor shared many characteristics with tissue inhibitor of metalloproteinases including the ability to irreversibly inhibit susceptible proteinases, heat and acid resistance, and sensitivity to trypsin degradation and reduction-alkylation. A polyclonal rabbit antiserum with blocking activity which recognized the Mr 28,500 protein was obtained. This inhibitor, which is likely produced by bovine vascular smooth muscle cells in vivo to protect the collagen matrix of blood vessels, may play an important role in pathological conditions associated with alteration of collagen metabolism in tissues.     

Metalloenzyme inhibitor from kidney beans: partial purification and characterization

Inhibitory activity directed against metalloenzymes has been highly purified from extracts of red kidney beans (Phaseolus vulgaris). The inhibitor is a substance of small molecular weight and appears to be a chelator of Zn²⁺. One milligram of the preparation inhibited 23 milligrams carboxypeptidase A. The inhibitor also strongly inhibited carboxypeptidase B and alkaline phosphatase and could activate phosphoglucomutase that had previously been inactivated with Zn²⁺. The isoelectric point of the inhibitor is 4.7. The inhibitor activity was abolished by preincubation with Zn²⁺, Ni²⁺, Co²⁺, or Cu²⁺. The mechanism of inhibition of carboxypeptidases and alkaline phosphatase by the bean inhibitor is apparently due to the complexing and complete removal of Zn²⁺ from the enzymes.     

Adenosine deaminase from bovine brain: purification and partial characterization.

Bovine brain adenosine deaminase cytoplasmatic form was purified about 450 fold by salt fractionation, column chromatography on DEAE-cellulose, octyl-sepharose 4B and affinity chromatography on CH-sepharose 4B 9-(p-aminobenzyl)adenine. The purified enzyme was homogeneous on disc gel electrophoresis; the enzyme had a molecular mass of about 65 kDa with an isoelectric point at pH 4.87. The Km values for adenosine and 2'-deoxyadenosine were 4 x 10(-5) and 5.2 x 10(-5) M, respectively. The enzyme showed a great stability to temperature with a half life of 15 hours at 53 degrees C significantly different compared to that known for other mammalian forms of this enzyme. Aza and deaza analogs of adenosine and erythro-9-(2-hydroxy-3-nonyl) adenine were good inhibitors of the bovine brain enzyme with little difference with respect to those reported for the adenosine deaminases purified from other sources. Kinetic constants for the association and dissociation of coformycin and 2'-deoxycoformycin with the bovine brain adenosine deaminase are reported.     

Purification and characterization of bovine dental sac collagenase

1. Active type collagenase was purified as much as 140-fold from the explant medium of bovine dental sacs and showed a single band on disc gel electrophoresis. Purified collagenase cleaved native collagen at only one locus under physiological conditions, but hydrolyzed neither gelatin nor alpha-casein. The optimal pH was about 7.8. 2. The molecular weight of active type enzyme was 35,000 by gel filtration and 34,000 by gel electrophoresis. The activation of latent type of collagenase resulted in the reduction of molecular weight from 45,000 to 38,000 by gel filtration. 3. A small but detectable amount of collagenase was directly extracted from frozen and thawed bovine dental sacs. In explant media of frozen and thawed tissue and fresh tissue with actinomycin D, some activity was detected for the first 2 days, but essentially no collagenase activity was detected in the explant medium after day 3. 4. The latent type collagenase was activated by trypsin, 4-aminophenylmercuric acetate (4-APMA), thiocyanate and deoxycholate (DOC). DOC showed irreversible dissociation of latent type enzyme in similar fashion to that exerted by 4-APMA. 5. The purified collagenase was inhibited by bovine serum, EDTA, o-phenanthroline, cysteine and dithiothreitol.     

Partial purification and characterization of latent human leukocyte collagenase

Latent and active collagenase were extracted from human polymorphonuclear leukocytes. Separation of the two forms of the enzyme was performed by gel filtration on Sepharose 6 B. The latent form of the enzyme was detected from chromatographic fractions after a brief treatment with trypsin or exposure of the fractions to the sulfhydryl reagent phenylmercuric chloride. Latent enzyme eluted before active enzyme from the column, indicating a higher apparent molecular weight. Partially purified latent enzyme exhibited an apparent molecular size of 70-75 kDa as estimated by gel filtration. A value of 50-55 kDa was obtained for active enzyme. Without activation the latent enzyme did not degrade soluble collagen substrate. This was demonstrated by a quantitative viscometric assay and also by sodium dodecyl sulfate polyacrylamide gel electrophoresis, when no typical cleavage products of collagen could be seen. Latent enzyme could not be obtained unless serine protease inhibitors were present during the extraction and purification procedures. The effects of the activators trypsin, phenylmercuric chloride, phenylmethyl sulfonyltrypsin, and N-ethylmaleimide on the latent human polymorphonuclear leukocyte collagenase were studied. Contrary to the suggestion that inactive proteases activate latent human polymorphonuclear leukocyte collagenase, the inactive phenylmethyl sulfonyl-trypsin could not activate latent collagenase.     

Partial purification and characterization of sialate O-acetylesterase from bovine brain

From bovine brain an esterase was purified 2,600-fold in an overall yield of 5.6%. For the isolation ion-exchange chromatographies, gel filtration, and preparative isoelectric focusing were used. The molecular mass is 56 kDa after gel chromatography on Sephacryl S-200 and 51 kDa after HPLC, the pH-optimum at 7.4, and the isoelectric point in the range of pH 5.8-6.1, as estimated from preparative isoelectric focusing. The substrate specificity of this enzyme was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. Besides aromatic acetyl esters such as e.g. alpha-naphthyl acetate, the highest preference was for N-acetyl-9-O-acetylneuraminic acid, followed by N-acetyl-4-O-acetylneuraminic acid. Other primary acetyl esters such as 6-O-acetylated D-glucose and 2-acetamido-2-deoxy-D-mannose were not hydrolyzed. The 9-O-acetyl derivative of the naturally occurring unsaturated sialic acid 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, however, is a substrate for this esterase. Whereas N-acetyl-9-O-acetylneuraminic acid as a component of sialyllactose is nearly as well hydrolyzed as the corresponding free sialic acid, O-acetylated sialoglycoconjugates with high molecular weights (mucins, serum glycoproteins, gangliosides) are not hydrolyzed by this esterase. N-Acetylated sialic acids are better substrates than the analogous N-glycoloyl derivatives. Esterification of the carboxyl function of sialic acids prevents the action of the esterase on the O-acetyl groups. The enzyme has no carboxyl esterase or amidase activity, and does not act on acetylcholine. It hydrolyzes almost exclusively acetyl esters. Inhibition studies suggest that it has a catalytically active serine residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of high-buoyant-density proteoglycans from bovine femoral-head cartilage.

Proteoglycans were extracted from bovine (15-18 months old) femoral-head cartilage. The heterogeneity of the A1D1 proteoglycan fraction was examined by gel chromatography, sedimentation velocity, sucrose rate-zonal centrifugation and CS2SO4 isopycnic centrifugation. In all cases polydisperse but unimodal distributions were obtained. Chemical analysis of the preparation yielded a galactosamine/glucosamine molar ratio of 7:1, and 13C n.m.r. spectroscopy showed that the chondroitin sulphate comprised equal proportions of the 4- and 6-sulphate isomers. Gel chromatography of a papain and Pronase digest of the proteoglycan indicated that the chondroitin sulphate chains had a Mn of approx. 10500. The mean buoyant density of the proteoglycan in pure CS2SO4 was 1.46 g/ml. Physical characterization of the proteoglycan preparation in 4M-guanidine hydrochloride, pH 7.4, by using conventional light-scattering gave a radius of gyration of 42 nm and a Mw of 0.96 X 10(6). Quasi-elastic light-scattering in the same solvent yielded a translational diffusion coefficient, D020, of 5.41 X 10(-8) cm2 X S-1, and ultracentrifugation gave a sedimentation coefficient, S020, of 12.0S. Thus from sedimentation-diffusion studies a Mw of 1.36 X 10(6) was calculated. The possible origins for the differences in the two molecular-weight estimates are discussed. It is concluded that the high-buoyant-density proteoglycans from bovine articular cartilage are significantly smaller than those from bovine nasal septum, and that this is largely due to the smaller size of their chondroitin sulphate chains.     

A Kunitz proteinase inhibitor from Archidendron ellipticum seeds: purification, characterization, and kinetic properties

Leguminous plants in the tropical rainforests are a rich source of proteinase inhibitors and this work illustrates isolation of a serine proteinase inhibitor from the seeds of Archidendron ellipticum (AeTI), inhabiting Great Nicobar Island, India. AeTI was purified to homogeneity by acetone and ammonium sulfate fractionation, and ion exchange, size exclusion and reverse phase chromatography (HPLC). SDS-PAGE of AeTI revealed that it is constituted by two polypeptide chains (alpha-chain, M(r) 15,000 and beta-chain, M(r) 5000), the molecular weight being approximately 20 kDa. N-terminal sequence showed high homology with other serine proteinase inhibitors belonging to the Mimosoideae subfamily. Both Native-PAGE as well as isoelectric focussing showed four isoinhibitors (pI values of 4.1, 4.55, 5.27 and 5.65). Inhibitory activity of AeTI remained unchanged over a wide range of temperatures (0-60 degrees C) and pH (1-10). The protein inhibited trypsin in the stoichiometric ratio of 1:1, but lacked similar stoichiometry against chymotrypsin. Also, AeTI-trypsin complex was stable to SDS unlike the SDS unstable AeTI-chymotrypsin complex. AeTI, which possessed inhibition constants (K(i)) of 2.46 x 10(-10) and 0.5 x 10(-10)M against trypsin and chymotrypsin activity, respectively, retained over 70% of inhibitory activity after being stored at -20 degrees C for more than a year. Initial studies on the insecticidal properties of AeTI indicate it to be a very potent insect anti-feedant.     

Partial purification and characterization of dynein adenosine triphosphatase from bovine sperm

Outer dynein arm polypeptides that possess Mg+2-adenosine triphosphatase (ATPase) activity have been extracted from the flagellar axonemes of demembranated bovine sperm. Electron microscopy of intact and salt-extracted sperm demonstrates a relatively selective removal of the outer dynein arms. The salt extract contains a specific ATPase activity of 55 nmoles inorganic phosphate (Pi)/min/mg protein. Sucrose density gradient centrifugation of this extract results in a 6-fold increase in specific activity of ATPase (333 nmole/Pi/min/mg protein), which sediments as a single 13S peak. Concomitant with the increase in specific activity, there is enrichment of three high molecular weight polypeptides (Mr greater than 300,000) characteristic of dynein heavy chains. ATPase activities in the initial extract and in the 13S peak are inhibited by concentrations of vanadate and erythro-9-[3-2-(hydroxynonyl)]adenine similar to those that inhibit ATPase activity in sea urchin sperm dynein. These findings indicate that outer arm dynein ATPase can be extracted and partially purified from bovine sperm.     

Isolation, purification and characterization of collagenase from hepatopancreas of the land snail Achatina fulica

Collagenase (matrix metalloproteinase-1, EC:3.4.24.7) was isolated from the hepatopancreas of Achatina fulica and characterized for its enzymatic activity and immunological properties. Procollagenase was isolated using ammonium sulphate precipitation and gel filtration, followed by purification by reverse-phase high performance liquid chromatography in the presence of trifluoroacetic acid and by dialysis in neutral buffer. In the presence of SDS and beta-mercaptoethanol, the procollagenase resolved into two subunits with molecular masses of 63 and 28 kDa, respectively. The 63 kDa fragment retained its ability to bind and degrade gelatin, but the 28 kDa was inactive. Analysis by 2D gel electrophoresis revealed that the 63 kDa fragment was basic (pIs 7.6, 7.8 and 8.15), while the 28 kDa fragment was acidic (pI 4.7 and 5.1). Western blot analysis confirmed the identity of collagenase, as only matrix metalloproteinase-1 rabbit antibodies against human matrix metalloproteinase-1 (N-terminal region) recognized both the isolated procollagenase and the 63 kDa fragment.     

Partial purification and characterization of cysteine proteinase inhibitor from chicken plasma

A high-molecular-weight cysteine proteinase inhibitor (CPI) was purified from chicken (Gallus gallus) plasma using polyethylene glycol (PEG) fractionation and affinity chromatography on carboxymethyl–papain–Sepharose-4B. The CPI was purified 96.8-fold with a yield of 28.9%. Based on inhibitory activity staining for papain, CPI was shown to have an apparent molecular mass of 122 kDa. No inhibitory activity was obtained under reducing condition, indicating that CPI from chicken plasma was stabilized by disulfide bonds. CPI was stable in temperature ranges from 40 to 70 °C for 10 min; however, more than 50% of the inhibitory activity towards papain was lost within 30 min of heating at 90 °C. CPI was stable in the presence of salt up to 3%. The purified CPI exhibited the inhibitory activity toward autolysis of arrowtooth flounder (Atheresthes stomias) and Pacific whiting (Merluccius productus) natural actomyosin (NAM) in a concentration-dependent manner.