Identification of a nucleotide pyrophosphohydrolase from articular tissues in human serum.
OBJECTIVE
To characterize the nucleotide pyrophosphohydrolase (NTPPHase) in human serum.


METHODS
NTPPHase activity and kinetic analysis were performed using thymidine monophosphate paranitrophenyl ester (TMPNP) or 32Pgamma-labeled ATP as substrate. Sera were chromatographed (dye column), and peak fractions were analyzed kinetically and by immunoblot using antibodies to 127-kd articular cartilage vesicle (ACV) NTPPHase as well as to PC-1 and to 58 kd, two plasma membrane ecto-NTPPHases. Enzyme activity was measured before and after sample ultracentrifugation.


RESULTS
NTPPHase activity was found in all sera tested (2 normal subjects, 9 arthritis patients). Specific activity was increased 9-32-fold after chromatography; 60-80% of total activity was recovered in a single peak containing an approximately 100-kd soluble peptide related to the 127-kd ACV enzyme. The apparent Km of this peptide (TMPNP) was virtually identical to that of the porcine ACV 127-kd enzyme. No immunoreactivity against PC-1 or 58-kd NTPPHase was found.


CONCLUSIONS
Human serum NTPPHase is derived from 127-kd ACV-related enzyme.
Cardenal
A
A
Department of Medicine, Medical College of Wisconsin, Milwaukee 53226, USA.
Masuda
I
I
Haas
A L
AL
Ono
W
W
McCarty
D J
DJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002358:Cartilage, Articular / Q000201:enzymology*; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008297:Male; D008875:Middle Aged; D011755:Pyrophosphatases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013938:Thymidine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D014461:Ultracentrifugation
C043805:4-nitrophenyl 3'-thymidylate; D013938:Thymidine Monophosphate; D000255:Adenosine Triphosphate; D011755:Pyrophosphatases; C021895:nucleoside triphosphate pyrophosphatase