Osteoblast collagenase: collagenase synthesis by clonally derived mouse osteogenic (MC3T3-E1) cells

Latent collagenase was isolated by heparin-Sepharose affinity chromatography from the culture medium of clonally derived mouse osteogenic (MC3T3-E1) cells. Collagenase synthesis by MC3T3-E1 cells was significantly stimulated by the addition of parathyroid hormone to the serum-containing culture medium. The cellular origin of the isolated collagenase was confirmed by demonstrating the characteristic 3/4 and 1/4 fragments of collagen alpha-chain, as well as inhibition of the enzyme by anti-mouse bone collagenase antibody.     

Collagenase synthesis in clonal monkey kidney epithelial cells (JTC-12 cells)

Cells of clone JTC-12 derived from monkey kidney epithelial cells were found to produce latent collagenase in serum-containing cultures. Collagenase production by JTC-12 cells was stimulated more than ten-fold with the addition of 12-0-tetradecanoyl-phorbol-13-acetate. The JTC-12 cell collagenase was purified 2,370-fold over the conditioned medium to a specific activity of 5,940 units/mg with a yield of 79% by heparin-Sepharose, ion-exchange and immunoadsorption chromatography. Thus the JTC-12 cell culture appears to be a useful model for studying the initial degradation of surrounding connective tissues by epithelial tissue in carcinogenesis.     

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 from a human hepatoma cell line of a basic fibroblast growth factor-like molecule that stimulates capillary endothelial cell plasminogen activator production, DNA synthesis, and migration.

A 17,500-dalton protein which stimulates plasminogen activator production in cultured bovine capillary endothelial cells has been purified from a SK-Hep-1 human hepatoma cell lysate by using heparin affinity chromatography and fast protein-liquid ion exchange chromatography. The purified molecule stimulated plasminogen activator production in a dose-dependent manner between 0.01 and 1 ng/ml. It also stimulated collagenase synthesis, DNA synthesis, and motility in capillary endothelial cells in the same concentration range. This molecule was identified as a basic fibroblast growth factor-like molecule on the basis of its biological activity, its affinity for heparin-Sepharose, and its cross-reactivity with a polyclonal antibody raised against the human placental basic fibroblast growth factor.     

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.     

Purification and characterization of an endothelial cell growth factor from serum-free culture medium of human diploid fibroblast cells

Serum-free culture supernatants of human embryo fibroblast cells contain endothelial cell growth factor (f-ECGF) which supports the serial propagation of human umbilical vein endothelial cells in the serum-free culture medium (medium A). This growth-stimulating activity has been partially purified from serum-free culture-conditioned medium. The stability of the activity to acid (pH 4.0-4.5) was utilized for the first step in purification. f-ECGF had a high affinity to heparin-Sepharose CL-6B, and was isolated by the methods of heparin affinity, of ion-exchange and gel filtration chromatography from the serum-free culture-conditioned medium preparation. The purified f-ECGF had an isoelectric point in the pH range 4.5-6, and a molecular weight of approx. 30 kDa determined by either gel filtration or SDS-polyacrylamide gradient gel electrophoresis. The f-ECGF has high affinity for concanavalin A column, and the activity was partially eluted from the column with ethylene glycol and alpha-methylmannose. The results indicate that f-ECGF is an acidic-glyco-protein with heterogeneous sugar chain(s).     

Collagenase synthesis by cloned rabbit pulp cells--molecular and immunological identity of pulp cell and fibroblast enzyme.

Cultures of cloned rabbit pulp (RP) cells without stimulation produced collagenase of a concentration as high as reference rabbit skin fibroblast cultures which were stimulated with phorbol myristate acetate (PMA, 100 ng/ml). The RP cell collagenase was compared with reference fibroblast collagenase in Western blot analysis using monoclonal antibodies prepared against RP cell collagenase and a polyclonal antibody prepared against rabbit fibroblast collagenase. Both enzyme preparations revealed, with either antibody, identical bands of approximate molecular masses 57,000, 52,500, and 45,000. These antibody preparations variously inhibited RP cell collagenase activity. Intracellular collagenase in RP cells in culture was demonstrated by the indirect immunofluorescence antibody technique using polyclonal anti-fibroblast collagenase antibody. RNA samples from RP cells hybridized with rabbit fibroblast collagenase cDNA (clone H9) and showed a distinct band at 2.7 kb. Both control and PMA-stimulated RP cells and PMA-stimulated reference skin fibroblasts demonstrated strong cytoplasmic hybridization between H9 and collagenase mRNA. The results indicate that RP cell collagenase is identical to rabbit fibroblast collagenase, and that the RP cell line provides a useful in vitro reference system for the study of collagenolysis in the rabbit model.     

Mineralization induced by beta-glycerophosphate in cultures leads to a marked increase in collagenase synthesis by mouse osteogenic MC3T3-E1 cells under subsequent stimulation with heparin

The clonally derived mouse osteoblast-like cell line MC3T3-E1 was shown to produce latent collagenase (approximately 0.2 units/ml) under stimulation with either heparin or parathyroid hormone in confluent cultures. However, it was found that MC3T3 E1 cultures which were first induced to undergo mineralization by the addition of beta-glycerophosphate and were subsequently stimulated with heparin showed an approximately ten-fold increase in collagenase synthesis. MC3T3-E1 cell collagenase from a small sample of serum-free culture medium was purified 49-fold to a specific activity of 200 units/mg protein with a yield of 14% by heparin-sepharose affinity chromatography and ion-exchange high performance liquid chromatography. This new mineralization-primed cell culture system may be a valuable model for the study of osteoblast collagenase.     

Secretion of DNA synthesis factor (DSF) by A431 cells that can grow in protein-free medium

A431 cells grew in protein-free Coon's modified Ham's F12 medium at a similar rate to that in medium supplemented with calf serum and secreted a growth factor capable of stimulating DNA synthesis in BALB/c3T3 cells. This factor had strong affinity for heparin and was partially purified from the conditioned medium by heparin-Sepharose affinity chromatography and molecular sieving on Bio-Gel P-60. The apparent molecular weight of the factor was 20-30K. Its activity was inhibited by heparin at concentrations of above 0.03 microgram/ml.     

Purification of a metalloproteinase inhibitor from human rheumatoid synovial fluid.

A metalloproteinase inhibitor present in human rheumatoid synovial fluid was purified by a combination of heparin-Sepharose chromatography, concanavalin A-Sepharose chromatography, ion-exchange chromatography and gel filtration. The Mr of the purified inhibitor was 28000 by SDS/polyacrylamide-gel electrophoresis and 30000 by gel filtration. The inhibitor blocked the activity of the metalloproteinases collagenase, gelatinase and proteoglycanase, but not thermolysin or bacterial collagenase. The serine proteinase trypsin was not inhibited. The inhibitory activity was lost after treatment with trypsin (0.5 micrograms/ml) at 37 degrees C for 30 min, 4-aminophenylmercuric acetate (1 mM) at 37 degrees C for 3 h, after incubation for 30 min at 90 degrees C and by reduction and alkylation. These properties suggest that the inhibitor closely resembles the tissue inhibitor of metalloproteinases ('TIMP') recently purified from connective-tissue culture medium.     

Tyrosine hydroxylase purification from rat PC12 cells.

Tyrosine hydroxylase was purified in high yield from rat PC12 cells. This three-day procedure consisted of differential ammonium sulfate precipitation, anion-exchange chromatography, and heparin-Sepharose affinity chromatography. It yielded an average of 15 mg of purified protein from 100 flasks of PC12 cells, with greater than 40% recovery of tyrosine hydroxylase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single protein band with a molecular weight of approximately 60,000. The protein had a specific activity of 670 nmol/min/mg and had a Km for its reducing cofactor tetrahydrobiopterin of 1.8 mM. The purified protein can be phosphorylated and activated by cyclic AMP-dependent protein kinase.     

Purification of pig synovial collagenase to high specific activity.

1. Pig synovium in tissue culture secretes a specific collagenase in a latent form. 2. The latent enzyme was concentrated by (NH4)2SO4 precipitation and activated with 4-aminophenylmercuric acetate, and the active enzyme was purified by chromatography on Ultrogel AcA44, DEAE-cellulose, heparin-Sepharose and a zinc-chelate medium to a specific activity of 53 400 units/mg. of protein. 3. The enzyme was shown to be essentially homogeneous by polyacrylamide-gel electrophoresis. 4. The purified collagenase digested collagen to give the characteristic three-quarter and one-quarter pieces.     

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.     

Rapid and high-yield purification of porcine heart tissue-type plasminogen activator by heparin-sepharose choromatography

A rapid and high-yield procedure for the purification of single polypeptide tissue-type plasminogen activator (t-PA) from porcine heart tissue has been developed. Delipidated heart tissue was extracted with 0.45 M potassium acetate. The extract was fractionated with ammonium sulfate and purified by a combination of affinity chromatography on heparin-Sepharose CL-6B and gel filtration on Toyopearl HW-55S. The final product had a specific activity of 220,000 IU/mg protein and gave a single protein band (apparent molecular weight; 67,000) in SDS-polyacrylamide gels in the presence or absence of a reducing agent. The increase in specific activity was 3,200-fold, most of which was achieved in the step of heparin-Sepharose chromatography. The yield calculated from the active ammonium sulfate precipitate was about 90% and 500 micrograms or more of the purified enzyme was obtained from 1 kg wet tissue. This procedure may also be useful for the large-scale production of highly purified t-PA from other tissues or tissue culture cells.     

Systematic separation and purification of elastase, gelatinase (matrix metalloproteinase 9), and collagenase (matrix metalloproteinase 8) from polymorphonuclear leukocytes in dialyzers previously used by patients with renal failure

We developed a simple and effective method for the systematic separation and purification of human polymorphonuclear leukocyte (PMN) proteinases, elastase, gelatinase (matrix metalloproteinase 9, type IV collagenase), and collagenase (matrix metalloproteinase 8), derived from the extracts of hollow fiber dialyzers that had been utilized in the treatment of patients with renal failure. The fraction containing elastase was grossly separated from that containing gelatinase and collagenase by heparin-Sepharose chromatography and purified in an aprotinin column. The remaining two enzymes were then separated using the gelatin-Sepharose column after gel chromatography following ammonium sulfate precipitation. Gelatinase and collagenase were further purified by gelatin-Sepharose chromatography as a latent form and by collagen-Sepharose chromatography as an activated form. This novel method offers procedural advantages over existing methods that separate PMNs from the whole blood of volunteers for experimental research purposes.     

Characterization of the metalloproteinase inhibitor produced by bovine articular chondrocyte cultures

Primary cultures of bovine articular chondrocytes release a latent metalloproteinase which is activated by incubation with organomercurials to degrade proteoglycans. All the enzyme present in the culture medium is latent and binds to columns of heparin-Sepharose. The yield of activity from the heparin-Sepharose columns (measured after organomercurial treatment) is approximately 300–1000% depending on the chondrocyte culture batch. Recombination of column fractions shows that the increase in activity is due to the separation of an inhibitor of the metalloproteinase by the chromatographic step. The metalloproteinase inhibitor has a molecular weight of approximately 35 000 (determined by Bio-Gel P-60 chromatography) and binds reversibly to columns of concavalin A-Sepharose. It is relatively heat stable (30 min at 60°C) and resistant to inactivation by trypsin (2 h, 37°C, 10 μg/ml trypsin). The inhibitor is active against rat uterine collagenase and gelatinase but does not affect bacterial metalloproteinases such as thermolysin and Clostridium histolyticum collagenase.     

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.     

Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity.

The purpose of this study was to identify the herpes simplex virus glycoprotein(s) that mediates the adsorption of virions to cells. Because heparan sulfate moieties of cell surface proteoglycans serve as the receptors for herpes simplex virus adsorption, we tested whether any of the viral glycoproteins could bind to heparin-Sepharose in affinity chromatography experiments. Two glycoproteins, gB and gC, bound to heparin-Sepharose and could be eluted with soluble heparin. In order to determine whether virions devoid of gC or gB were impaired for adsorption, we quantitated the binding of wild-type and mutant virions to cells. We found that at equivalent input concentrations of purified virions, significantly fewer gC-negative virions bound to cells than did wild-type or gB-negative virions. In addition, the gC-negative virions that bound to cells showed a significant delay in penetration compared with wild-type virus. The impairments in adsorption and penetration of the gC-negative virions can account for their reduced PFU/particle ratios, which were found to be about 5 to 10% that of wild-type virions, depending on the host cell. Although gC is dispensable for replication of herpes simplex virus in cell culture, it clearly facilitates virion adsorption and enhances infectivity by about a factor of 10.     

Purification of basic fibroblast growth factor from rat brain: identification of a Mr 22,000 immunoreactive form

A 18,000-dalton protein which stimulates plasminogen activator (PA) activity in endothelial GM 7373 cells has been purified from rat brain by using heparin affinity chromatography and ion-exchange chromatography. The purified molecule stimulates PA activity in a dose-dependent manner between 1 and 30 ng/ml. It also stimulates proliferation of GM 7373 cells and DNA synthesis in NIH 3T3 cells in a similar concentration range. The molecule has been identified as a bFGF-like molecule on the basis of its biological activity, its affinity for heparin-Sepharose, and its cross-reactivity with anti-human bFGF antibodies. In the final preparation of the rat brain bFGF, trace amounts (less than 5%) of a contaminant were detectable. This contaminant has a molecular weight of 22,000 and cross reacts with several anti-human placental bFGF antibodies. On the basis of its affinity for heparin-Sepharose and its immunological characteristics, this protein appears to be an high molecular weight form of bFGF.     

Synthesis of procollagen by odontogenic cells of rabbit tooth germ

Studies were performed to determine whether cultured odontogenic cells from rabbit tooth germ (RP cell) could synthesize dentine-like collagen. When cells were cultured with [¹⁴C]proline, 33% of the total incorporated proteins present were collagenous. Cultured RP cells were labelled with [¹⁴C]proline in the presence of β-aminopropionitrile. The resulting fractions, on analysis by CM-cellulose chromatography, contained three radioactive protein peaks, α1(I), [α1(III)]₃, α2. From the radioactive measurements, RP cells synthesized a significant amount of type III collagen, comparable to type I collagen.DEAE-cellulose chromatography was used to separate collagen molecules from collagen precursors. The results showed that 60% of total collagen precursor was type III precursor and the remainder was type I precursor.CM-cellulose chromatography of CNBr peptides of collagen from culture medium and cell extract revealed the presence of type I and type III collagen. Thus, the RP cell, which is a diploid cell, is unique in the predominance of type III collagen in culture, differing thereby from the character of collagen in vivo.