THE HOMO-DIMERIC FORM OF ADP-RIBOSYL CYCLASE IN SOLUTION

ADP-ribosyl cyclase is a multi-functional enzyme that catalyzes the fo rmation of two Ca2+ signaling molecules, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). X-ray crystall ography of three different crystal forms shows that it is a non-covale nt dimer. Chemical cross-linking and dynamic light scattering were use d in this study to determine if the cyclase is also a non-covalent dim er in solution. Treatment of the cyclase in dilute solution (0.05 mg/m l ) with dimethylsuberimidate resulted in complete conversion to a spe cies with molecular weight about twice that of the monomeric cyclase. Prolonged cross-linking of the cyclase at four times higher concentrat ion produced also only the covalently linked dimers and no multimer fo rmation was observed. The cross-linked dimer retained full enzymatic a ctivity and readily catalyzed the formation of cADPR from NAD, NAADP f rom NADP, cyclic ADP-ribose phosphate from NADP, and cyclic GDP-ribose from nicotinamide guanine dinucleotide. Analysis of the autocorrelati on functions obtained from dynamic light scattering measurements indic ated the cyclase solution (2 mg/ml) was composed of a single molecular species and its diffusion coefficient was measured to be 7.4x10(-7) c m(2)/s. Computer modeling using the crystallographic dimensions of the non-covalent cyclase dimer, a donut shaped molecule with a central ca vity and overall dimensions of 7x6x3 nm, gave a value for the diffusio n coefficient essentially the same as that measured. These results ind icate the cyclase is a non-covalent dimer in solution. (C) 1998 Elsevi er Science B.V. All rights reserved.