Activation of transglutaminase during embryonic development

Abstract

Incorporation of [3H]putrescine into proteins was shown to increase markedly in sea urchin eggs upon fertilization. Emetine, an inhibitor of protein synthesis, had no effect on the rate of protein labeling. However, the reaction could be prevented by the addition of 2-[3-(diallylamino)-propionyl]benzothiophene, a noncompetitive inhibitor of transglutaminase, and also by dansylcadaverine, which is a substrate for transglutaminase. The inert N alpha-dimethyl analogue of dansylcadaverine had no influence. Considering the complexity of the incorporation of the [3H]putrescine tracer in this system, it was deemed essential to prove by rigorous analytical methods that the reaction was, indeed, consistent with a transglutaminase mechanism. gamma-Glutamyl[3H]putrescine could be recovered in 80-90% yield from the proteolytic digest of proteins from the 20-min fertilized cell. Another sign of the in vivo activity of transglutaminase was the isolation of substantial amounts of epsilon-(gamma-glutamyl)lysine from proteins of sea urchin embryo, yielding a frequency value for this cross-link as high as 1 mol/400 000 g of protein in the 32-cell-stage material.

Intramolecular crosslinking of monomeric fibrinogen by tissue transglutaminase.

“…To the dried precipitate, 0.5 ml of 0.1 M ammonium bicarbonate and a small crystal of thymol were added. Enzymatic digestion was carried out by the procedure of Cariello et al (11) with the sequential addition of subtilisin (10 jig and 4 pug), pronase (10 Ag and 4 pug), carboxypeptidase Y (5 ,tg), leucine aminopeptidase and prolidase (2 pug each), and, again, leucine aminopeptidase (2 pkg). The samples were dried and dissolved in 0.2 ml of water, and amino acid analysis was performed according to the o-phthalaldehyde derivatization procedure of Griffin et al (12).…”

Cloning and characterization of a cluster of genes encoding polypeptides present in the insoluble fraction of the spore coat of Bacillus subtilis

“…Peroxidase-mediated tyrosine-tyrosine cross-links are involved in the hardening of the sea urchin fertilization membrane (6,13), and a variety of cross-links involving lysine oxidase are present in collagen (10,35).…”

Spermidine is bound to a unique protein in early sea urchin embryos.

“…Comparable studies in sea urchin embryos reported incorporation of 18.2 pmol of putrescine equivalents per g of protein (15 (19).…”

“…The possibility that dansylcadaverine interferes with cell uptake of spermidine might be considered. In this regard, however, it is worth noting that recent studies of sea urchin (Arbacia) embryogenesis proved conclusively that posttranslational modification of protein by the polyamine putrescine is mediated by transglutaminase (15).…”

“…Significant amounts of spermidine are found in rodent seminal plasma proteins (20), small amounts are present in several small proteins in activated human lymphocytes (20) (Mr <20,000), in human amniotic fluid (21) (Mr 10,000-30,000), in conjugated form in human plasma (22,23), in nuclear and nucleolar proteins from calf liver (24), in HTC (15), and in Friend erythroleukemia cells (25), and spermine is evident in human seminal plasma proteins (unpublished studies). However, a specific protein with bound spermidine has not been identified in any of these examples.…”