Insoluble bone gelatin with inclusions of insoluble noncollagenous protein produces new bone when implanted in muscle in allogeneic rats. The implanted residue provides the milieu for expression of bone morphogenetic potential of migratory mesencliymal cells. Neutral buffer solutions activate endogenous enzymes that degrade components essential for cell interactions and differentiation of bone. Chloroform-methanol either denatures or extracts constituents responsible for degradation. Insoluble bone gelatin produces new bone after extraction at 20 with neutral salts, 0.5 M EDTA, 0.1 M Tris HCI, 4 M urea, 0.5 M hydroxylamine, and 10 M KCNS, as well as after limited digestion with pepsin or collagenase, but not after extraction with 5 M guanidine, 7 M urea, water saturated with phenol, or after alkali hydrolysis with 0.1 N NaOH. The specific activity of cell populations interacting with insoluble bone gelatin suggests that a chemical bond between collagen and a noncollagenous protein or part of a protein, cleaved by a neutral proteinase, controls the bone morphogenetic reaction.

Pyruvate kinase (EC 2.7.1.40) was isolated and purified from chicken and turkey breast muscle with a purification procedure very similar to that used for the bovine skeletal muscle isozyme (Cardenas, J., Dyson, R., and strandholm, J. (1973), J. Biol. Chem. 248,6931). A study of the chemical and physical properties of the chicken enzyme revealed that it is a tetramer of four apparently identical subunits, closely resembling in this and most other respects the mamalian type 7 isozyme. The properties of these two enzymes are similar enough to permit subunits of chicken type M pyruvate kinase to combine with subunits of mammalian type L (one of the three mammalian isozymes) to form interspecies tetrameric hybrid isozymes in relative quantities that do not differ makedly from those formed when both the M and L isozymes are of mammalian origin. The similarity between the mammalian and avian type M pyruvates kinases suggests a close evolutionary relationship. Further comparisons among the three mammalian and two avian isozymes of pyruvate kinase are consistent with a common evolutionary origin, perhaps from an ancestral form of the type K isozyme, which is the only pyruvate kinase identified in mammalian and avian embryos.

Data on physicochemical conditions leading to loss of the bone morphogenetic property of bone matrix in neutral buffer solutions support the concept of an enzymic control mechanism better than a chemical blocking reaction or denaturation. The loss is associated with release of 35S-labeled constituents and not prevented by ε-amino caproic acid, an inhibitor of cathepsins. The loss is also associated with release of 35S-cysteine-labeled protein; about 60% of the yield is sustained by the addition of only 3 mmoles/liter of iodoacetic acid. A latent period of about 12 hr, decreased by extraction of bone matrix with CaCl2, is characterized by release of protein polysaccharide and other noncollagenous proteins. Release of sialic acid from the bone matrix by neuraminidase at pH 7.4 has no effect upon bone yield. At 2°C, Tris-HCl buffer or ethylenediaminetetraacetic acid extracts noncollagenous proteins without loss of bone yield; at 37°C, pH 7.4, these solutions also activate endogenous enzymes and reduce bone yield. The component of bone matrix responsible for reduction in bone yield is separable from bone matrix by extraction with phosphate buffer, by catheptic digestion of bone matrix in acidic buffer solutions, by sequential chemical extraction of noncollagenous proteins with cold slightly acidic salt solutions or by extraction-denaturation with chloroform-methanol. Detergents neither extinguish nor denature the morphogenetic property but some solubilize or extract degradative enzymes; hexodecyl trimethyl ammonium bromide, at pH 5.0, is positively charged and extracts hydrophobic proteins, including part of the bone morphogenetic property. A special selection of sulfhydryl chemical inhibitors remarkably different from the selection inhibiting known enzymes preserves the bone morphogenetic property of bone matrix; p-chloromercuribenzoate preservation is reversible by chemical reactions with cysteine. Reduction in bone yield in phosphate buffer is not attributable to a chemical block because chloroform-methanol extraction of the agent does not restore bone yield and is not attributable to denaturation because bone yield sustained by p-chloromercuribenzoate is lost by chemical reactions with cysteine. An hypothetical insoluble bone morphogenetic protein (BMP) firmly bound to collagen is degraded by a soluble neutral proteinase (BMPase). Digestion of the hypothetical BMP occurs without loss of the 640-A electron micrographic image of bone collagen, resembles tryptic digestion and is more selective as well as physiologic in action.