Abstract
Evidence is presented that transglutaminase is composed of a single polypeptide chain of molecular weight 80,000 to 90,000. (a) Polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and mercaptoethanol gave a single band with a mobility corresponding to a molecular weight of approximately 85,000. (b) Gel filtration in guanidine HCl of the 14C-carbamidomethyl carboxymethylated enzyme protein showed a single peak of absorbance and radioactivity from which a molecular weight of approximately 85,000 was estimated. (c) Amino-terminal analysis by conventional methods showed no evidence of free α-amino groups. A peptide, believed to contain the amino-terminal residue, was obtained by Pronase digestion and was isolated at levels of 0.75 and 0.8 mole/90,000 g of enzyme. The sequence of this peptide was determined as pyroglutamylalanylaspartylleucine. (d) Digestion by carboxypeptidase A of the carboxymethylated enzyme protein in denaturing solvents released glycine and serine at equal rates to the level of 1 mole/90,000 g of protein. Hydrazinolysis gave approximately 1 mole of glycine. These findings, together with earlier evidence that the molecular weight of the native enzyme is 80,000 to 90,000 and that the enzyme protein contains 17 or 18 —SH groups, but no disulfide bonds, form the basis for the view of an unbridged monomeric structure of transglutaminase. Indication that transglutaminase performs its catalytic functions in the monomeric form was obtained from a comparison of the gel filtration patterns for the enzyme protein in the presence and absence of calcium ion. The identical nature of these patterns is in accord with the suggestion that this metal, which is essential for activation of transglutaminase, does not affect a change in enzyme molecular weight. A revised enzyme purification procedure is presented. Rabbit antiserum against transglutaminase has been prepared and used to characterize the enzyme purified by this procedure as immunologically homogeneous.
Highlights
90,000. (a) Polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and mercaptoethanol gave a single band with a mobility corresponding to a molecular weight of approximately 85,000. (b) Gel filtration in guanidine HCl of the 14C-carbamidomethyl carboxymethylated enzyme protein showed a single peak of absorbance and radioactivity from which a molecular weight of approximately 85,000 was estimated. (c) Amino-terminal analysis by conventional methods showed no evidence of free a-amino groups
Indication that transglutaminase performs its catalytic functions in the monomeric form was obtained from a comparison of the gel filtration patterns for the enzyme protein in the presence and absence of calcium ion
In order to determine whether this essential metal ion alters the molecular weight, i.e. causes polymerization, of the enzyme protein, we examined the gel filtration characteristics of the native enzyme in the presence and absence of CaCL The finding of the identical gel filtration pattern in each case (Table V) is evidence that Ca++
Summary
90,000. (a) Polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and mercaptoethanol gave a single band with a mobility corresponding to a molecular weight of approximately 85,000. (b) Gel filtration in guanidine HCl of the 14C-carbamidomethyl carboxymethylated enzyme protein showed a single peak of absorbance and radioactivity from which a molecular weight of approximately 85,000 was estimated. (c) Amino-terminal analysis by conventional methods showed no evidence of free a-amino groups. (a) Polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and mercaptoethanol gave a single band with a mobility corresponding to a molecular weight of approximately 85,000. (b) Gel filtration in guanidine HCl of the 14C-carbamidomethyl carboxymethylated enzyme protein showed a single peak of absorbance and radioactivity from which a molecular weight of approximately 85,000 was estimated. (d) Digestion by carboxypeptidase A of the carboxymethylated enzyme protein in denaturing solvents released glycine and serine at equal rates to the level of 1 mole/. Hydrazinolysis gave approximately 1 mole of glycine These findings, together with earlier evidence that the molecular weight of the native enzyme is. Indication that transglutaminase performs its catalytic functions in the monomeric form was obtained from a comparison of the gel filtration patterns for the enzyme protein in the presence and absence of calcium ion. The identical nature of these patterns is in accord with the suggestion that this metal, which is essential for activation of transglutaminase, does not affect a change in enzyme molecular weight
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