Active site labeling of erythrocyte transglutaminase by o-phthalaldehyde.

Tissue-type transglutaminase is inactivated in a time-dependent way during incubation with submillimolar concentrations of o-phthalaldehyde, with affinity labeling kinetics. The rate of inactivation by the reagent is greatly enhanced in the presence of the essential enzyme cofactor calcium and is decreased by GTP, an allosteric inhibitor. A fluorescent isoindole derivative is formed during the modification apparently through crosslinkage of active site Cys 277 to a lysine residue. These data and the quenching of fluorescence by addition of calcium ions suggest that the enzyme active site is directly involved in the inactivation process.

Properties of particulate transglutaminase from Yoshida tumor cells.

Homogenates of Yoshida hepatoma cells, cultured as ascite suspension in vivo, display significant transglutaminase activity in both the cytosolic and the particulate fraction. The enzyme, however, is predominantly membrane-bound. Transglutaminase was solubilized from the membranes either by extraction with detergents or treatment with neutralized hydroxylamine or proteinases. We observed similar molecular weight under denaturing conditions, catalytic and immunologic properties for purified cytosolic and solubilized transglutaminase, and identity of the limited proteolytic maps. These results suggest that transglutaminase isoforms actually consist of the same protein undergoing translocation by unknown mechanisms.

Utilization of phytohaemagglutinin for in vivo karyological studies in teleost fish.

A simple technique is described for increasing the number of mitoses in circulating blood and in the kidney of teleost fish. This method employed phytohaemagglutinin-P solution, a T-like cell mitogen, which was injected (1 mg/100 g body wt) intraperitoneally, and 53 h later many blasts suitable for karyological studies, blocked in metaphase, were present both in the circulating blood and in the kidney.

On the interaction between xanthine oxidase and actin.

Xanthine oxidase increases the rate of actin polymerization. This occurs at oxidase concentrations as low as 40 nM provided the concentration of the polymerizing agent is low (0.5 mM MgCl2). In the presence of 0.1 M KCl plus 1 mM MgCl2 as the polymerizing agents, xanthine oxidase does not affect the rate of the polymerization but increases significantly the rate of the conversion of F(ATP)actin into F(ADP.Pi)actin and probably also the rate of the orthophosphate release.

On an Mg2+-dependent interaction of actin with glyceraldehyde-phosphate dehydrogenase. The fundamental role of KCl in the organization of F-actin.

In the presence of Mg2+, the formation of actin filaments is hindered by glyceraldehyde-3-phosphate dehydrogenase. This effect, which increases with the square of Mg2+ concentration, is counteracted by 0.15 M KCl. Thus KCl, at concentrations found in the intracellular compartment, appears to be strictly required for the correct formation of actin filaments in all tissues in which the glyceraldehyde-phosphate dehydrogenase concentration is high.

The experimental basis of some recent hypotheses on the mechanism of the polymerization of actin: a reappraisal.

We have studied the effect of sonication on the fluorescence of N-(1-pyrenyl)iodoacetamide-labeled F-actin as well as of native actin-pyrenyl-actin mixed oligomers in which the subunits were covalently attached to each other by phenylenebismaleimide. In both cases the fluorescence of the solution was largely decreased by sonication. We have found that this effect is due (a) to a 20-30% decrease of the specific fluorescence of the polymers. These results question the validity of the novel mechanism for the polymerization of actin recently proposed (D. Pantaloni et al. (1984) J. Biol. Chem. 259, 6274-6283). In these studies, in fact, the implicit assumption was made that the quenching of the fluorescence of the solution under sonication was due exclusively to the conversion of F-actin into G-actin.

An ADPase from sarcoplasmic reticulum of rabbit muscle cleaves ADP bound to F-actin.

We have found that sarcoplasmic reticulum from rabbit muscle contains an ADPase which cleaves ADP bound to F-actin. The interaction is not of the simple Michaelis-Menten type, the order of the reaction being larger than the first. A possible explanation of this behaviour could be that ADPase binds to two adjacent actin monomers with a preferred orientation thus cleaving preferentially the nucleotide of one of the two monomers.

Fructose-1,6-bisphosphate aldolase from rabbit muscle. Kinetic resolution of the enamine phosphate from the enamine-aldehyde intermediate at low temperature.

At or below -12 degrees C and in the presence of 40% ethylene glycol, only two out of the four dihydroxyacetone phosphate binding sites of aldolase are catalytically active. At these same temperatures and at pH* 8.3, the equilibrium between the pre-enamine and the enamine plus the post-enamine intermediates is largely shifted in favor of the latter. The enamine phosphate and the enamine-aldehyde phosphate intermediates have been resolved by studying the rate of their formation at -13 degrees C and pH* 5.28 and the trapping by DL-glyceraldehyde 3-phosphate at -24 degrees C and pH* 5.24.