ABSTRACT
In the genital tract of the male rat two different forms of the enzyme transglutaminase (TGase) could be identified and characterized. The coagulating gland and the dorsal prostate secrete a glycosylated and acylated TGase with a molecular weight of 65,000 dalton and pI value of 8.7. This secretory form was purified to homogeneity using preparative isoelectric focusing and gel filtration on a Superdex 200 column. Running fast protein liquid chromatographic gel filtration on a Superose 12 column in the presence of calcium ions, high-molecular-weight aggregates were physically formed which could only be eluted using drastic conditions (0.1 M sodium hydroxide). In the presence of 10 mM EDTA this tendency to aggregate was greatly diminished. Utilizing a Superdex 200 column for gel filtration, the secretory TGase was even eluted as a monomeric protein. Testicular TGase was isolated by ion-exchange fast protein liquid chromatography on a Mono Q and by gel filtration on a Superdex 200 column. This enzyme represents a tissue-type TGase with a molecular weight of 82,000 dalton and pI value of 5.25. Hydrophobic interaction chromatography on a phenyl-Superose column showed no further enrichment of the GTP-binding form of transglutaminase.
Subject(s)
Prostate/enzymology , Testis/enzymology , Transglutaminases/isolation & purification , Animals , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Isoelectric Focusing , Male , Molecular Weight , Rats , Rats, Inbred Strains , Transglutaminases/metabolismABSTRACT
A transglutaminase (TGase, EC 2.3.2.13) was isolated from the secretion of rat coagulating gland (CGS-TGase). The protein consists of a single polypeptide chain and has a molecular mass of 65 kDa. During purification the net charge changes from pI 7.6 in the crude extract to pI 8.5-8.7 for the purified protein. Nearly equal numbers of glutamyl- and lysyl-residues were detected by amino acid analysis. The protein therefore represents an appropriate substrate of autocatalytic crosslinking. The total number of cysteine residues is 18-19, six of which being present in free form. One of the thiol groups is essential for the enzymic activity. The protein core is glycosylated with mannosyl residues and in addition substituted with saturated acyl residues and phosphoinositol. The phosphoinositol anchor was demonstrated by use of a specific antibody. Removal of the acyl- and glycosyl-residues or of the total anchor group results in autoaggregation and decrease of enzymic activity. In contrast to tissue-type TGases, Ca2+ dependent enzymic activity of CGS-TGase is not inhibited by GTP. The secretory TGase shows no immunological cross-reactivity to tissue-type enzyme or blood factor XIII.
Subject(s)
Genitalia, Male/enzymology , Transglutaminases/metabolism , Amino Acids/analysis , Animals , Blotting, Western , Calcium/metabolism , Electrophoresis, Polyacrylamide Gel , Guanosine Triphosphate/metabolism , Guinea Pigs , Male , Protein Processing, Post-Translational , Rats , Rats, Inbred Strains , Solubility , Transglutaminases/chemistry , Transglutaminases/isolation & purificationABSTRACT
The distribution of transglutaminase immunoreactivity in the male genital system of the rat has been studied at the light and electron microscopic levels using a highly specific polyclonal antibody. The antibody is obviously secreted in an apocrine fashion in the dorsal prostate as well as the coagulating gland. This moiety is responsible for semen clotting. In addition to the secretory moiety, epididymal sperm contain endogenous immunoreactive material in the cytoplasmic droplet. Whether this is added to the sperm within the epididymis--as is the immunoreactive material on the sperm head--or already present in developing sperm has to be elucidated by further immunoelectron microscopic studies.
