Evidence of endogenous mono-ADP-ribosylation of cardiac proteins via anti-ADP-ribosylarginine immunoreactivity.

Arginine-specific mono-ADP-ribosylation of proteins and arginine-specific mono-ADP-ribosyltransferase occur in heart. We developed a polyclonal antiserum, R-28, against ADP-ribosylpolyarginine that recognized mono-ADP-ribosylated proteins and identified the major mono-ADP-ribosylation products of quail heart. Treatment of Immobilon-bound ADP-ribosylated Gs protein with hydroxylamine under conditions that remove ADP-ribose from its arginines eliminated R-28 immunoreactivity to Gs. Also, R-28 immunoreactivity to quail heart proteins was removed by NaOH and phosphodiesterase I treatments. Similar treatment with mercuric chloride did not remove the immunoreactivity but did remove exogenously (via in vitro pertussis toxin treatment) added ADP-ribose from cysteine of cardiac Gi/Go proteins. The antiserum did not appear to react with ADP-ribosylasparagine of Rho (formed by C3 toxin), ADP-ribosyldiphthamide of elongation factor 2 (formed by diphtheria toxin) in quail heart preparations, or polyADP-ribosylated proteins of a neonate rat cardiac nuclear preparation. Thus, the R-28 antiserum appears to contain predominantly antibodies directed against ADP-ribosylarginine. To test the usefulness of R-28, immunoblotting of subcellular fractions of quail heart was performed. R-28 showed the greatest immunoreactivity in the sarcolemma with significant immunoreactivity in denser membrane fractions. The cytosol also contained an immunoreactive band distinct from those found in the membranes. Hydroxylamine treatment eliminated immunoreactivity in the sarcolemma and denser membrane fractions but not the cytosol, suggesting the membranous immunoreactive bands contain ADP-ribosylarginine. In conclusion, a polyclonal antiserum that recognizes ADP-ribosylarginine proteins has been raised. The usefulness of the antiserum is demonstrated by the characterization of endogenous arginine mono-ADP-ribosylation products in quail heart. The quail heart has several sarcolemmal and denser membrane fraction proteins that appear to be mono-ADP-ribosylated on arginines.

In vitro reconstitution of microtubule plus end-directed, GTPgammaS-sensitive motility of Golgi membranes.

Purified Golgi membranes were mixed with cytosol and microtubules (MTs) and observed by video enhanced light microscopy. Initially, the membranes appeared as vesicles that moved along MTs. As time progressed, vesicles formed aggregates from which membrane tubules emerged, traveled along MTs, and eventually generated extensive reticular networks. Membrane motility required ATP, occurred mainly toward MT plus ends, and was inhibited almost completely by the H1 monoclonal antibody to kinesin heavy chain, 5'-adenylylimidodiphosphate, and 100 microM but not 20 microM vanadate. Motility was also blocked by GTPgammaS or A1F4- but was insensitive to A1C13, NaF, staurosporin, or okadaic acid. The targets for GTPgammaS and A1F4- were evidently of cytosolic origin, did not include kinesin or MTs, and were insensitive to several probes for trimeric G proteins. Transport of Golgi membranes along MTs mediated by a kinesin has thus been reconstituted in vitro. The motility is regulated by one or more cytosolic GTPases but not by protein kinases or phosphatases that are inhibited by staurosporin or okadaic acid, respectively. The pertinent GTPases are likely to be small G proteins or possibly dynamin. The in vitro motility may correspond to Golgi-to-ER or Golgi-to-cell surface transport in vivo.

IQGAP1, a Rac- and Cdc42-binding protein, directly binds and cross-links microfilaments.

Activated forms of the GTPases, Rac and Cdc42, are known to stimulate formation of microfilament-rich lamellipodia and filopodia, respectively, but the underlying mechanisms have remained obscure. We now report the purification and characterization of a protein, IQGAP1, which is likely to mediate effects of these GTPases on microfilaments. Native IQGAP1 purified from bovine adrenal comprises two approximately 190-kD subunits per molecule plus substoichiometric calmodulin. Purified IQGAP1 bound directly to F-actin and cross-linked the actin filaments into irregular, interconnected bundles that exhibited gel-like properties. Exogenous calmodulin partially inhibited binding of IQGAP1 to F-actin, and was more effective in the absence, than in the presence of calcium. Immunofluorescence microscopy demonstrated cytochalasin D-sensitive colocalization of IQGAP1 with cortical microfilaments. These results, in conjunction with prior evidence that IQGAP1 binds directly to activated Rac and Cdc42, suggest that IQGAP1 serves as a direct molecular link between these GTPases and the actin cytoskeleton, and that the actin-binding activity of IQGAP1 is regulated by calmodulin.

Developmental and biochemical characteristics of the cardiac membrane-bound arginine-specific mono-ADP-ribosyltransferase.

ADP-ribosylation of protein in heart membrane preparations has been shown to be present in adult tissue but absent from early neonate tissue [Piron and McMahon (1990) Biochem. J. 270, 591-597]. To further this observation, the cardiac membrane-bound form of arginine-specific mono-ADP-ribosyltransferase (EC 2.4.2.31) has been characterized. Apparent Km values of 330 and 470 microM were found in heart membrane preparations from rat and quail respectively. The Vmax. value depended greatly on the species of animal studied, and was 1.1 and 48 nmol/min per mg in rat and quail preparations respectively. The specific activity of the enzyme was lowest in pig, intermediate in rat, dog and rabbit, and highest in mouse and quail cardiac membranes. In the rat, the ADP-ribosylation of protein and enzyme activity were very low in heart preparations from 1-15-day-old animals. Thereafter the ADP-ribosylation and enzyme activity increased gradually to adulthood. Bacillus cereus phosphatidylinositol-specific phospholipase C, known to hydrolyse glycosylphosphatidylinositol anchors of proteins, released the mono-ADP-ribosyltransferase from membrane preparations of both rat and quail in a dose-dependent, Zn(2+)-inhibited manner. Thus it appears that a membrane-bound form of arginine-specific mono-ADP-ribosyltransferase is present in heart membranes from a variety of species and is not species-specific. The activity of this ADP-ribosyltransferase appears to be developmentally regulated and to be bound to the cardiac membranes by a glycosylphosphatidylinositol anchor.