Subcellular localization of cyclic ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities in porcine airway smooth muscle.
Recent studies have provided evidence for a role of cyclic ADP-ribose (cADPR) in the regulation of intracellular calcium in smooth muscles of the intestine, blood vessels and airways. We investigated the presence and subcellular localization of ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of beta-NAD(+) to cADPR, and cADPR hydrolase, the enzyme that degrades cADPR to ADPR, in tracheal smooth muscle (TSM). Sucrose density fractionation of TSM crude membranes provided evidence that ADP-ribosyl cyclase and cADPR hydrolase activities were associated with a fraction enriched in 5'-nucleotidase activity, a plasma membrane marker enzyme, but not in a fraction enriched in either sarcoplasmic endoplasmic reticulum calcium ATPase or ryanodine receptor channels, both sarcoplasmic reticulum markers. The ADP-ribosyl cyclase and cADPR hydrolase activities comigrated at a molecular weight of approximately 40 kDa on SDS-PAGE. This comigration was confirmed by gel filtration chromatography. Investigation of kinetics yielded K(m) values of 30.4+/-1.5 and 695. 3+/-171.2 microM and V(max) values of 330.4+/-90 and 102.8+/-17.1 nmol/mg/h for ADP-ribosyl cyclase and cADPR hydrolase, respectively. These results suggest a possible role for cADPR as an endogenous modulator of [Ca(2+)](i) in porcine TSM cells.
White
T A
TA
Department of Veterinary PathoBiology, College of Veterinary Medicine, University of Minnesota, St Paul, 55108, USA.
Johnson
S
S
Walseth
T F
TF
Lee
H C
HC
Graeff
R M
RM
Munshi
C B
CB
Prakash
Y S
YS
Sieck
G C
GC
Kannan
M S
MS
D036541:ADP-ribosyl Cyclase; D000818:Animals; D015153:Blotting, Western; D019757:Carbon-Oxygen Lyases / Q000378:metabolism*; D002458:Cell Fractionation; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D009130:Muscle, Smooth / Q000201:enzymology* / Q000648:ultrastructure; D010761:Phosphorus Radioisotopes; D019761:Phosphorus-Oxygen Lyases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine; D014132:Trachea / Q000201:enzymology* / Q000648:ultrastructure
D010761:Phosphorus Radioisotopes; D036541:ADP-ribosyl Cyclase; D019757:Carbon-Oxygen Lyases; C019621:ADP-ribose-histone hydrolase; D019761:Phosphorus-Oxygen Lyases