Masako Sakamoto

Collagenase and bone resorption: isolation of collagenase from culture medium containing serum after stimulation of bone resorption by addition of parathyroid hormone extract.

Summary A new method is described for the isolation of tissue collagenase from culture medium containing serum. The method was used to isolate mouse bone collagenase from an in vitro tissue culture system utilizing mouse bone calvaria in which rapid bone resorption was stimulated by the addition of parathyroid hormone extract. The amount of collagenase activity in the tissue culture medium was found to correlate well with the extent of bone resorption observed morphologically. The results provide further and more direct evidence that the synthesis and release of bone collagenase is involved in and related to the removal of bone collagen during bone resorption.

The effect of substrate composition and condition on resorption by isolated osteoclasts

Osteoclasts isolated from 10-day-old rabbits were studied in a cell culture system to obtain further insight into the possible mechanisms involved in the process of bone resorption. Osteoclasts were cultured on the surfaces of devitalized mammalian bones, a composite of polycrystalline synthetic hydroxyapatite and geological single crystal hydroxyapatite. The structural interaction of osteoclasts with these various substrates was morphologically characterized by using a scanning electron microscope. Viable osteoclasts were observed to adhere and extend pseudopodia and filopodia for all cases of calcium-phosphate surfaces. However, actual resorbing process was only observed in devitalized bone surfaces. Resorption pits of various sizes were found in multiple series and showed a preference for mineral-exposed area over collagen-exposed area. The difference observed between resorption of those areas of bone surface and the failure of osteoclasts to resorb non-tissue substrates are discussed in relation to current theories of the mechanisms of mineral dissolution and resorption in mammalian calcified tissues.

Mouse bone collagenase: Purification of the enzyme by heparin-substituted sepharose 4B affinity chromatography and preparation of specific antibody to the enzyme

Heparin-substituted Sepharose 4B gel, affinity chromatography used to purify mouse bone collagenase increased its specific activity 1340-fold (2850 units/mg) with an overall yield of 9% from the original pooled tissue culture medium. The purified enzyme moved as a single band and had a molecular weight of 47,000 as determined by analytical disc gel electrophoresis in the presence of 1% sodium dodecyl sulfate. Antiserum to the purified enzyme was produced in rabbits, and its monospecificity was established by immunoelectrophoresis. The γ-globulin fraction of the antiserum, freed of α 2 -macroglobulin by gel filtration, inhibited collagenase activity. The antibody was purified approximately 70-fold by immunoadsorption using enzyme-substituted Sepharose 4B gel. The extent of the immunoinhibition of various tissue collagenases by mouse bone collagenase antibody varied depending on the tissue and species from which the enzyme was obtained.

Studies on the interaction between heparin and mouse bone collagenase

Mouse bone collagenase was found to be tightly bound to a heparin-substituted gel at low ionic strength. The bond was reversible, however, and the collagenase could be elutted at high ionic strength. In addition to providing a method for purifying the enzyme with high yield, the results suggest that the strong ionic bond between heparin and collagenase may partially explain the mechanism wherein heparin enhances the activity of mouse bone collagenase.

Mineralization induced by β-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.

Latent collagenase from the culture medium of embryonic chick bones.

Enzymatically inactive collagenases have been isolated from several tissues which may be activated by tissue culture medium or trypsin [l-5] , suggesting synthesis by the cells of a precursor zymogen which is then enzymatically activated by scission of a portion of the larger precursor molecule. However, 3 M NaSCN was found [6,7] , which dissociates a number of antigen-antibody complexes, was as effective as trypsin in converting the inactive form of the collagenase to an active form, suggesting that the inactive form is a non-covalently bound complex of active enzyme and inhibitor. Additional evidence for this latter proposal is provided [g-lo]. We have recently been able to isolate a latent collagenase from the tissue culture medium of embryonic chick bones and have studied the nature of the latent enzyme.

Isolation of tissue collagenase from homogenates of embryonic chick bones

Abstract An enzyme capable of digesting undenatured collagen in solution and in the solid state as reconstituted collagen fibrils at neutral pH was extracted from demineralized embryonic chick bone homogenates in 1.0M NaCl at neutral pH. The enzyme could be dissociated from the small amount of collagen which was also solubilized in 1.0M NaCl by the serial use of Diaflo XM-300 and PM-10 membranes, which procedures also concentrated the enzyme. The enzymatic activity was inhibited by EDTA, cysteine and horse serum, and was enhanced by the addition of heparin.

Chick bone collagenase inhibitor and latency of collagenase

Abstract 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.

Collagenase activity and morphological and chemical bone resorption induced by prostaglandin E2 in tissue culture.

Summary The isolation of collagenase present in serum-containing medium harvested from bone cultures was effected by heparin-Sepharose affinity chromatography. Almost all of the bone tissue collagenase released into the medium was of the latent, inactive type, which could be activated in vitro with trypsin. Over the course of the experiment a 20-fold greater amount of collagenase was released into the medium by the resorbing cultures stimulated with prostaglandin E2 as compared to the control cultures. A significant increase in calcium released into the medium correlated in time with rapid bone resorption as measured microscopically. The increase in collagenase activity did not demonstrate the same precise time course.

Biosynthesis of O‐phosphoserine‐containing phosphoproteins by isolated bone cells of mouse calvaria

Five groups of isolated bone cells from mouse calvaria were incubated with [3H]serine and the presence and amount of O[3H]phosphoserine used as an indication of phosphoprotein synthesis. Cells in the osteoblastic fraction were the most active in synthesizing phosphoproteins, and unlike the other cell groups, released most of the phosphoproteins into the tissue culture medium. When subjected to molecular sieving and ion‐exchange chromatography, the phosphoproteins synthesized by the bone cells of the osteoblastic group behaved like the phosphoproteins extracted from mouse calvaria by EDTA.