Mono-ADP-ribosylation in the developing rat heart

Abstract

Mono-ADP-ribosylation is a posttranslational covalent modification which transfers the ADP-ribose moiety from NAD* to cellular acceptor proteins. The objective of the dissertation research was to study mono-ADP-ribosylation in the developing rat heart. In the adult rat heart, a 52 kDa mono-ADP-ribosylation product was present in the plasma membrane. However, this product was absent in the 1-day-old rat heart. Chemical hydrolysis demonstrated that the ADP-ribose group was attached to an arginine residue. The 52 kDa product displayed isoelectric point heterogeneity on two-dimensional electrophoresis with a pl between 5.5 and 5.8. The addition of low concentrations of NADP* (0.1 mM or less) to the reaction mixture prevented NAD* breakdown and enhanced ADPribosylation. Higher concentrations of NADP* decreased the ADP-ribosylation.

It was investigated whether the 52 kDa substrate was the stimulatory guanine nucleotide binding protein Gsa. However, two antibodies directed against two different regions of Gsa, did not recognize the 52 kDa protein. The arginine-specific ADP-ribosyltransferase was characterized in the rat heart. The enzyme had an apparent Km for NAD* of 330 /LIM and a Vmax of 1.1 nmole/mg/min. The pH optimum was 7.0.

The age-dependent difference in ADP-ribosylation was further investigated. The 52 kDa ADP-ribosylation product was present in low to non-detectable levels between days 1 and 15. Thereafter, it was gradually increased. Arginine-specific ADP-ribosyltransferase activity was also very low between days 1 and 15 and increased thereafter. In both adult and 1-day-old rat hearts, the majority of the activity was detected in the plasma membrane fraction.

These results demonstrate that ADP-ribosylation occurs in the rat heart plasma membrane and that an arginine-specific ADP-ribosyltransferase is present. The changes in ADP-ribosylation with developmental age suggest a possible role for ADP-ribosylation during cardiac development or an effect of cardiac development on ADP-ribosylation.