Collagenase-Released Non-Collagenous Proteins of Cortical Bone Matrix

Two distinct groups of non-collagenous components were isolated from rat cortical bone gelatin which had previously been digested with purified bacterial col-lagenase. One component was disulfide-bonded, strongly acidic, trypsin-labile glycoprotein aggregate with a molecular mass of more than 100, 000 daltons. When reduced with β-mercaptoethanol this protein disaggregated into subunits with a molecular mass of about 60, 000 daltons. The other components consisted of a group of polypeptides with a molecular mass of about 5, 000 daltons. The latter group was present in collagenase digests prepared from normal bone gelatin but was hardly detectable or absent in digests of gelatin prepared from either autolyzed, trypsinized or lathyritic bone, or from the residue of neutral salt extracted rat tail tendon.

A recently discovered group of non-collagenous proteins is tightly bound to the highly crosslinked insoluble structure of collagen fibrils of bone and dentin. Dische et al ¹ predicted the existence of these proteins in bone by analyzing the products of KOH hydrolysis of the EDTA-insoluble residues of cortical bone collagen. Comparable products were separated following digestion of bone matrix with collagenase by Herring², and oxidation of dentin matrix by alkaline sodium metaperiodate by Shuttleworth and Veis.³ Leaver et al ⁴ proposed the name collagenase-released proteins (CRP) for non-collagenous proteins obtained from the EDTA-insoluble residue of bone and dentin. There are at least two limitations in information about CRP in the above-cited reports. The first is that the collagenases were not certified to be protease-free. The second is that EDTA-insoluble residues of cortical bone were only partly digestable by bacterial collagenase. We report here isolation and partial characterization of CRP from a special preparation of bone matrix gelatin which is quantitatively digested by a protease-free bacterial collagenase-gelatinase purified by the method of Peterkofsky and Diegelmann.^{5, 6}     

An antigenic antimorphogenetic bone hydrophobic glycopeptide (AHG).

A hydrophobic glycopeptide, isolated from fresh marrow-free cortical bone of the rat, extinguished the bone morphogenetic response of insoluble bone matrix gelatin which otherwise invariably produces new bone from migrating mesenchymal cells after implantation in muscle of the rat. Incubation of the 10 to 100 mug of the glycopeptide per milligram of bone gelatin in phosphate or Tris-HCl buffer, pH = 7.4, for 24 hours recombines the hydrophobic polypeptide and gelatin. Incubated with doses of 50 to 100 mug, implants of gelatin in Sprague-Dawley recipients are rapidly resorbed by a lymphocyte-plasma cell-macrophage infiltrate without developing any new bone. With 10 to 20 mug, infiltrate appears and bone develops inversely in proportion to the dose. Bone develops at all dose levels only when bone matrix gelatin and glycopeptide were prepared from an inbred strain of Lewis rats and recipients were of the same strain, and thereby suggests that only allogeneic AHG, produces an antigenic antimorphogenetic resonse. The glycopeptide was isolated from bone together with lipids and other hydrophobic proteins or polypeptides by chloroform-methanol (1:1) extraction and further purified by chromatography on ion-exchange columns and molecular sieves. The molecular weight established by Sephadex G-100 filtration and by calibrated dialysis is about 5000 daltons. Analysis of the biologically active fraction shows 75% of proteinaceous material and close to 25% carbohydrate with trace amounts of hexosamines and sialic acid. Amino acid analysis shows high levels of alanine, valine and leucine in comparison to other chloroform-methanol extractable but immunogenically relatively-inactive fractions.     

Chick bone collagenase inhibitor and latency of collagenase

Collagenase and collagenase inhibitor were isolated from the culture fluid of embryonic chick bone. The inhibitor, separated as a high molecular weight aggregate (160,000–200,000 daltons) during gel filtration in 1M NaCl, dissociated in 6M urea to species of approx 25,000 daltons. The inhibition of collagenase activity by the addition of inhibitor was not reversed by the addition of trypsin or p-aminophenylmercuric acetate. However, isolated inhibitor alone was inactivated by treatment with either trypsin or p-aminophenylmercuric acetate. The results suggest that the latent form of chick bone collagenase is a proenzyme which converts into an active form without a detectable change in molecular weight and that this occurs after the inactivation of collagenase inhibitor.     

A thermostable collagenolytic protease with a very large molecular mass produced by thermophilic Bacillus sp. strain MO-1

A collagenolytic protease was purified to homogeneity from thermophilic Bacillus sp. strain MO-1. The protease from strain MO-1 showed high activity toward type I and IV collagens and gelatin. However, peptide substrates (4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg and 2-furylacryloyl-Leu-Gly-Pro-Ala) for collagenases were inert as substrates. The collagenolytic protease cleaved oxidized insulin B-chain at 11 sites and degraded type I and IV collagens into anonymous small pieces, suggesting that the protease digests collagens at multiple sites. The collagenolytic protease was far more thermostable than a mesophilic Clostridium histolyticum collagenase. The collagenolytic protease possesses two salient features: (1) it has a very large molecular mass, 210 kDa, and consists of two, identical 105-kDa subunits; (2) it belongs to a serine protease group. The high molecular mass is unique among serine proteases but common for collagenases. The features of the enzyme from strain MO-1 suggest that it is a new collagenolytic protease which is distinct from previously reported collagenases and serine proteases.     

Purification and physico-chemical properties of collagenase synthesized by a bacterium of the type Acinetobacter sp.]

The Acinetobacter spec collagenase has been almost completely purified. This enzyme is a true collagenase the activity of which is high on collagen. The enzyme is active on insoluble collagen, gelatin and the synthetic Pz-peptide, but has no proteolytic activity on casein or bovine serum-albumin. The collagenase was obtained on a simple medium with gelatin and yeast extract. The enzyme was purified by (NH4)2SO4 precipitation. DEAE cellulose column chromatography, Sephadex G 200 gel-filtration. The molecular weight of the enzyme was found to be 102 000 daltons, and its isoelectric point was found to be 7,7 +/- 0,2. The optimum pH and temperature for insoluble collagen hydrolysis were 7.6 and 37 degrees C, respectively; so, this collagenase corresponds to true collagenase. Hydrolysis of Pz-peptide is activated by Ca2+ and inhibited by metal ions (Cu2+, Fe3+, Zn2+, Pb2+, Hg2+). EDTA and o-phenanthroline induced a very significant reduction in enzyme activity. Iodoacetate and p-CMB induced a slight reduction in enzyme activity only at high concentrations (10-2M). The collagenase is most stable for temperatures less than or equal to 50 degrees C.     

Purification and properties of a small latent matrix metalloproteinase of the rat uterus

A small metalloproteinase that digests Azocoll was found in the uterus of the rat. Its activity increased to high levels during the postpartum period in parallel with the breakdown of the extracellular matrix exclusive of collagen (Sellers, A., and Woessner, J.F., Jr. (1980) Biochem. J. 189, 521-531). This enzyme has now been purified almost 7,000-fold to homogeneity from 12 g of tissue using molecular sieve chromatography, blue sepharose chromatography, and zinc-chelate chromatography. Gel electrophoresis with sodium dodecyl sulfate and dithiothreitol gives Mr = 28,000 for the latent form of the enzyme and Mr = 19,000 for the active form that arises spontaneously or by treatment with aminophenylmercuric acetate. The enzyme digests components of the extracellular matrix including gelatins of types I, III, IV, and V, fibronectin, and proteoglycan. It digests the alpha 2(I) chain of gelatin in preference to the alpha 1(I) chain and cleaves dinitrophenyl-Pro-Leu-Gly-Ile-Ala-Gly-Pro-D-Arg. It cleaves the B chain of insulin at two points: Ala14-Leu15 and Tyr16-Leu17. It has no action on collagens of types I, III, IV, or V at 26 degrees C and no action on elastin or phenylazo-Pro-Leu-Gly-Pro-D-Arg. The pH optimum is at pH 7 and the pI at 5.9. The enzyme requires zinc and calcium ions for activity; cobalt and strontium can partially replace these metal ions. The enzyme is not inhibited by low levels of phosphoramidon or Zincov. Its properties clearly distinguish it from collagenase, gelatinase (matrix metalloproteinase 2), and stromelysin (matrix metalloproteinase 3); it therefore constitutes a further member of the family of extracellular matrix metalloendopeptidases. The name matrix metalloproteinase 7 is proposed.     

Stimulation of prostaglandin synthesis in cultured rat synovial cells by a factor derived from polymorphonuclear leukocytes

Stimulation of synovial cell prostaglandin production by a factor obtained from casein-induced peritoneal polymorphonuclear (PMN) cells has been investigated. Both the extract and short time cultured medium of rat peritoneal PMN cells stimulate prostaglandin (PG)E2 production as well as collagenase production in the culture of rat synovial cells. PGE2 production by the cells in the presence of the PMN factor is much faster (5 to 24 hr) than collagenase production (24 hr or later, Biomedical Res. 3, 506-516, 1982). This stimulating factor is confirmed to be derived from PMN cells, based on the purification of the cells from peritoneal exudate cells by the Ficoll-Urographin method. Elution profile of the factor on gel filtration has indicated that both PGE2 and collagenase productions by synovial cells are stimulated by the same effluent fractions corresponding to molecular weights of 15,000 - 20,000 daltons and 30,000 - 40,000 daltons. These results suggest that PMN cells are involved in PG production as well as collagenase production in the inflamed tissue by stimulating connective tissue cells such as synovial cells.     

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.     

Comparison of bone inductive proteins of rat and porcine bone matrix

Subcutaneous implantation of demineralized bone matrix in allogenic rats induces a sequence of events resulting in de novo formation of cartilage, bone and bone marrow. In the present study endochondral bone formation by demineralized porcine matrix was studied and compared with the rat bone matrix. Endochondral bone formation was induced by 4M guanidine hydrochloride fraction IV (less than 50,000 daltons) of Sepharose CL-6B gel filtration but not by whole extract or by demineralized porcine bone matrix. Sephacryl S-200 gel filtration of the osteoinductive proteins of fraction IV showed the Porcine osteoinductive factor to be associated with protein fraction III (less than 20,000 daltons) whereas the rat with fraction II (between 20,000 and 30,000 daltons) of the chromatographic profile indicating an apparent difference in molecular weight of the osteoinductive factors between these two species.     

Collagen synthesis by murine bone marrow cell culture

Collagen types synthesized by murine bone marrow cells were studied and the effect of lithium chloride on collagen biosynthesis in vitro was investigated. In the liquid culture system used, an adherent, mixed cell population supports hemopoiesis. Radioactive labeling of cell cultures and subsequent fractionation with ammonium sulfate, enzyme digestion, immune precipitation, and gel electrophoresis indicated that the bone marrow cells synthesized precursors to collagen types I, III, and IV, and fibronectin. A previously undescribed molecule or fragment with an apparent molecular weight of 17,000 daltons that was susceptible to bacterial collagenase and containing no interchain disulfide bonds was also identified in the culture media of both control and lithium-treated cells. Lithium treatment did not affect the types of collagen synthesized, although the relative proportions of collagen types may differ from controls. However, lithium does have an effect on the appearance of some, as yet unidentified, non-collagenous components in the cell culture media.     

Detection and characterization of endothelin-1-like immunoreactivity in rat plasma

Using two radioimmunoassays (RIAs) for endothelin-1 (ET-1) with and without a substantial cross-reactivity with ET-3, we have measured the plasma ET-1-like immunoreactivity (-LI) level in rat plasma. ET-1-LI was detected in plasma from male Wistar rats. ET-1-LI in rat plasma consisted of three components with molecular weights of 6K, 4K and 2.5K daltons by gel permeation chromatography. Two of the components were eluted at positions of big ET (4K) and synthetic ET-1 (2.5K). The remaining component was eluted at the preceding fraction (6K). No difference was observed in ET-1-LI of the small molecular form of ET (2.5K) between the two RIAs. Thus, there is little or no ET-3 in rat plasma, which has the sequence found originally in the rat genome. The concentration of the small molecular form of ET, presumably ET-1, in rat plasma was about 4 pg/ml.     

Controlled release of all-trans-retinoic acid from PEGylated gelatin nanopaticles by enzymatic degradation

A new targeting drug carrier for anticancer drug, all-trans-retinoic acid (atRA), was proposed by using angiogenesis which is one of the specific physiological properties of cancer cells. The proposed drug carrier was prepared as PEGylated gelatin nanoparticle (176 nm size). The gelatin molecules were aggregated by coupled deoxycholic acid and the surface of the nanoparticles was covered by polyethylene glycol to reduce reticuloendothelial system (RES) uptake. To prove the feasibility of the nanoparticles as a targeting drug carrier, the degradation of the nanoparicles by collagenase IV and the release pattern of atRA from the nanoparticles by enzymatic degradation were evaluated. The PEGylated gelatin nanoparticles were significantly degraded by collagenase IV within 10 seconds, with most of them degraded within 1 min. When atRA loaded in the PEGylated gelatin nanoparticles was released in phosphate buffered saline (PBS), only twelve percent of atRA were released for one hour. However, when the nanoparticles were put into PBS with collagenase IV of 0.1 μM, a burst effect of atRA was about 40% for the initial 10 min, followed by a continuous release of atRA upto 75% for 5 hr. Therefore, the PEGylated gelatin nanoparticles released anticancer drug very sensitively by collagenase IV, which is one of major matrix metalloproteases involved in angiogenesis. These results showed a feasibility that PEGylated gelatin nanoparticles could be used as a new targeting anticancer drug carrier using angiogenesis as a specific physiological property of cancer cells.     

Isozymes of S-adenosylmethionine synthetase from rat liver: isolation and characterization

S-Adenosylmethionine synthetase exists in at least two distinct forms, alpha- and beta-forms, in adult liver. The beta-form was purified to homogeneity from the soluble fraction of rat liver with a yield of about 10%. An antiserum directed against the purified beta-form from rat liver was prepared by injecting the purified enzyme into a rabbit. Ouchterlony double diffusion analysis and immunochemical titrations revealed that the isozymes, alpha- and beta-forms, are identical. Thus, the alpha-form was isolated from rat liver as a single protein using immunoaffinity chromatography against the beta-form. The molecular weights of the beta- and alpha-forms were determined to be 48,000 each by sodium dodecyl sulfate disc gel electrophoresis, and about 100,000, and 200,000, respectively, by Sephacryl S-200 gel filtration. These results indicate that the beta-form consisted of two subunits of 48,000 daltons and the alpha-form of four subunits of 48,000 daltons. The sedimentation coefficient was calculated to be 5.5S for the beta-form and 8.0S for the alpha-form.     

CRYSTALLINE TRYPSIN : II. GENERAL PROPERTIES

A method is described for isolating a crystalline protein of high tryptic activity from beef pancreas. The protein has constant proteolytic activity and optical activity under various conditions and no indication of further fractionation could be obtained. The loss in activity corresponds to the decrease in native protein when the protein is denatured by heat, digested by pepsin, or hydrolyzed in dilute alkali. The enzyme digests casein, gelatin, edestin, and denatured hemoglobin, but not native hemoglobin. It accelerates the coagulation of blood but has little effect on the clotting of milk. It digests peptone prepared by the action of pepsin on casein, edestin or gelatin. The extent of the digestion of gelatin caused by this enzyme is the same as that caused by crystalline pepsin and is approximately equivalent to tripling the number of carboxyl groups present in the solution. The activity of the preparation is not increased by enterokinase. The molecular weight by osmotic pressure measure is about 34,000. The diffusion coefficient in ½ saturated magnesium sulfate at 6°C. is 0.020 ±0.001 cm.² per day, corresponding to a molecular radius of 2.6 x 10^–7 cm. The isoelectric point is probably between pH 7.0 and pH 8.0. The optimum pH for the digestion of casein is from 8.0–9.0. The optimum stability is at pH 1.8.     

Synthesis and physical characterization of poly(ethylene glycol)-gelatin conjugates

Poly(ethylene glycol) (PEG) with the terminal group of active ester was coupled to the amino group of gelatin to prepare PEG-grafted gelatin (PEG-gelatin). The affinity chromatographic study revealed that the PEG-gelatin with high degrees of PEGylation did not adsorb onto the gelatin affinity column, in remarked contrast to gelatin alone and the PEG-gelatin with low PEGylation degrees. The former PEG-gelatin showed a critical micelle concentration while it had the apparent molecular size of about 100 nm and a surface charge of almost zero. These findings indicate that the PEG-gelatin formed a micelle structure of which the surface is covered with PEG molecules grafted. When the body distribution of 125I-labeled gelatin and PEG-gelatin after intravenous injection was evaluated, the radioactivity of micellar PEG-gelatin was retained in the blood circulation compared with that of gelatin and the PEG-gelatin of no micelle formation. At the same PEGylation degree, the blood concentration was significantly higher for the PEG-gelatin prepared from PEG with a molecular weight of 12 000 than that of molecular weights of 2000 and 5000. It is concluded that the PEG-gelatin is a drug carrier with a micelle structure which retains in the blood circulation.     

A comparison of bone matrix and tendon with particular reference to glycoprotein content.

Bone matrix and tendon are compared in terms of their carbohydrate and non-collagenous protein composition. The collagen content of both tissues was similar (90-91%), but bone matrix had at least three times as much sialic acid (0.28%) as tendon (0.08%). Smaller differences were found in the analysis of hexoses and hexosamines. After digestion with bacterial collagenase, about 9% of the total protein from both tissues was non-diffusible on dialysis, and this contained only 0.15% (bone) and 0.7% (tendon) of the original hydroxyproline; recovery of sialic acid was 86-87%. The collagenase-resistant soluble material amounted to about 9% (bone matrix) and 5% (tendon); the insoluble residues were 1 and 4% respectively. There were clear differences in the carbohydrate contents of the digests, but the amino acid compositions were similar. When the soluble digests were chromatographed on DEAE-cellulose, the elution profiles indicated the presence in each tissue of a variety of glycoproteins and a proteoglycan fraction, and showed clearly that an acidic glycoprotein corresponding to bone sialoprotein was not present in tendon.     

Fibronectin and fibrinogen degradation products stimulate PMN-leukocyte and mast cell degranulation.

The ability of various peptides cleaved by plasmin from human fibrinogen and fibronectin or fibrinogen- and fibronectin- related synthetic peptides to induce histamine release from mast cells and collagenase and elastase from PMN-leukocytes was examined. Low molecular weight fibrinogen degradation products showed dose dependent secretion of collagenase. These peptides (mol. wt. 1.4 kD) at the concentration of 10(-5) M released about 47% of collagenase and 13% of elastase. Synthetic fibrinopeptides A and B had a similar strong collagenase releasing potency and also released histamine from mast cells. Peptides from plasmin digestion of fibronectin containing cell attachment site with sequence Arg-Gly-Asp-Ser and also synthetic peptide reproducing this amino-acid sequence at the concentration of 1000 micrograms/ml released about 50% of collagenase and 55% of elastase from PMN-leukocytes. Moreover peptides containing cell attachment and gelatin binding site induced histamine release from mast cells. The association of fibrinogen and fibronectin degradation with activation of mast cells may motivate the treatment with antihistaminic drugs of all pathological conditions where the intensive protein degradation takes place.     

The molecular weight of the cell-free translation product of αl(I) procollagen mRNA

Chick embryo calvaria RNA has been translated in a nucleasetreated reticulocyte lysate system. The products have been characterized by their susceptibility to collagenase digestion and precipitability by Type I procollagen chain antibodies. The largest product, pre procollagen αl(I), migrates more slowly than proto procollagen αl(I) isolated from calvaria, contains initiator methionine and is cleaved to proto procollagen αl(I) by dog pancreas microsomes. The molecular weight of pre procollagen αl(I) was estimated to be 165,000 and by a similar analysis, pre procollagen α2 was identified and its weight determined to be approximately 165,000 daltons. Bacterial collagenase digestion of pre procollagen αl(I) generated two new low molecular weight hydrophobic components.     

Molecular forms of immunoactive atrial natriuretic peptide in the rat hypothalamus and atrium

Acid extracts of rat hypothalamus and atrium were prepared by a procedure previously shown to minimize proteolytic degradation of peptides. The majority of the immunoactive material in the atrial extracts had a molecular weight of approximately 9,000 to 15,000 daltons, while that in the hypothalamic extracts had a molecular weight of about 1,500 to 1,800 daltons. The major molecular weight forms of atrial natriuretic peptide from each extract were further distinguishable when analyzed by RP-HPLC. These results suggest that small peptides such as atriopeptins I, II, and III, may not be authentic post-translational processing products in the atrium, and that the hypothalamus and atrium may differentially cleave pro-atrial natriuretic peptide to form tissue-specific products.     

The interaction of purified rabbit bone collagenase with purified rabbit bone metalloproteinase inhibitor.

1. Pure rabbit bone metalloproteinase inhibitor (TIMP) bound tightly to pure rabbit bone collagenase with an apparent Kd of 1.4 X 10(-10) M. 2. The molecular weight of the enzyme-inhibitor complex was found to be 54 000, but no enzyme activity could be recovered from the complex after treatment with either mercurials or proteinases. The complex thus differed from latent collagenase in terms of size, susceptibility to mercurials and behaviour on concanavalin A-Sepharose. 3. The interaction of the purified components was compared with that of crude collagenase and crude inhibitor in culture medium. Mercurial treatment partially reversed the inhibition in the crude system, but not when the purified components were used. 4. The significance of the results is discussed in relation to the extracellular control of the activity of collagenase.