The homo-dimeric form of ADP-ribosyl cyclase in solution.
ADP-ribosyl cyclase is a multi-functional enzyme that catalyzes the formation of two Ca2+ signaling molecules, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). X-ray crystallography of three different crystal forms shows that it is a non-covalent dimer. Chemical cross-linking and dynamic light scattering were used in this study to determine if the cyclase is also a non-covalent dimer in solution. Treatment of the cyclase in dilute solution (0.05 mg/ml) with dimethylsuberimidate resulted in complete conversion to a species with molecular weight about twice that of the monomeric cyclase. Prolonged cross-linking of the cyclase at four times higher concentration produced also only the covalently linked dimers and no multimer formation was observed. The cross-linked dimer retained full enzymatic activity and readily catalyzed the formation of cADPR from NAD, NAADP from NADP, cyclic ADP-ribose phosphate from NADP, and cyclic GDP-ribose from nicotinamide guanine dinucleotide. Analysis of the autocorrelation functions obtained from dynamic light scattering measurements indicated the cyclase solution (2 mg/ml) was composed of a single molecular species and its diffusion coefficient was measured to be 7. 4x10-7 cm2/s. Computer modeling using the crystallographic dimensions of the non-covalent cyclase dimer, a donut shaped molecule with a central cavity and overall dimensions of 7x6x3 nm, gave a value for the diffusion coefficient essentially the same as that measured. These results indicate the cyclase is a non-covalent dimer in solution.
Munshi
C
C
6-182 Lyon Laboratory, Department of Physiology, University of Minnesota, Minneapolis, MN 55455, USA.
Baumann
C
C
Levitt
D
D
Bloomfield
V A
VA
Lee
H C
HC
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000737:chemistry*; D001048:Aplysia / Q000201:enzymology*; D003432:Cross-Linking Reagents / Q000378:metabolism; D019281:Dimerization; D004120:Dimethyl Suberimidate / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006150:Guanine Nucleotides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D009244:NAD+ Nucleosidase / Q000737:chemistry*; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation; D012542:Scattering, Radiation
D015703:Antigens, CD; D000943:Antigens, Differentiation; D003432:Cross-Linking Reagents; D006150:Guanine Nucleotides; D009243:NAD; D004120:Dimethyl Suberimidate; D009249:NADP; C102728:nicotinamide guanine dinucleotide; D036541:ADP-ribosyl Cyclase; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1