PMID- 1703296
OWN - NLM
STAT- MEDLINE
DCOM- 19910228
LR  - 20190501
IS  - 0027-8424 (Print)
IS  - 1091-6490 (Electronic)
IS  - 0027-8424 (Linking)
VI  - 88
IP  - 2
DP  - 1991 Jan 15
TI  - Purification of a soluble isoform of guanylyl cyclase-activating-factor synthase.
PG  - 365-9
AB  - The soluble form of guanylyl cyclase-activating-factor (GAF) synthase from rat 
      cerebellum was purified to homogeneity by sequential affinity chromatographic 
      steps on adenosine 2',5'-bisphosphate (2',5'-ADP)-Sepharose and 
      calmodulin-agarose. Enzyme activity during purification was bioassayed by the 
      L-arginine-, NADPH-, and Ca2+/calmodulin-dependent formation of a plasma 
      membrane-permeable nitric oxide-like factor that stimulated soluble guanylyl 
      cyclase in RFL-6 cells. With calmodulin and NADPH as cofactors, purified soluble 
      GAF synthase induced an increase of 1.05 mumol of cGMP per 10(6) RFL-6 cells per 
      3 min per mg of protein. The coproduct of this signal-transduction pathway 
      appeared to be L-citrulline. GAF synthase catalyzed the conversion of 107 nmol of 
      L-arginine into L-citrulline per min per mg of protein. Based on these assays, 
      this represents a purification of GAF synthase of approximately 10,076- and 
      8925-fold with recoveries of 16% and 19%, respectively. Rechromatography of the 
      purified enzyme on Mono P (isoelectric point = 6.1 +/- 0.3), Mono Q, and Superose 
      12 or 6 resulted in no further purification or increase in specific activity. A 
      Stokes radius of 7.9 +/- 0.3 nm and a sedimentation coefficient s20,w of 7.8 +/- 
      0.2 S were used to calculate a molecular mass of about 279 +/- 25 kDa for the 
      native enzyme. SDS/PAGE revealed a single protein band with a molecular mass of 
      about 155 +/- 3 kDa. These data suggest that soluble GAF synthase purified from 
      rat cerebellum is a homodimer of 155-kDa subunits and that enzyme activity is 
      dependent upon the presence of calmodulin.
FAU - Schmidt, H H
AU  - Schmidt HH
AD  - Abbott Laboratories, Abbott Park, IL 60064-3500.
FAU - Pollock, J S
AU  - Pollock JS
FAU - Nakane, M
AU  - Nakane M
FAU - Gorsky, L D
AU  - Gorsky LD
FAU - Forstermann, U
AU  - Forstermann U
FAU - Murad, F
AU  - Murad F
LA  - eng
GR  - AR 08080/AR/NIAMS NIH HHS/United States
GR  - DK30787/DK/NIDDK NIH HHS/United States
GR  - HL 28474/HL/NHLBI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Proc Natl Acad Sci U S A
JT  - Proceedings of the National Academy of Sciences of the United States of America
JID - 7505876
RN  - 0 (Isoenzymes)
RN  - 29VT07BGDA (Citrulline)
RN  - EC 1.14.13.39 (Nitric Oxide Synthase)
RN  - EC 1.4.- (Amino Acid Oxidoreductases)
SB  - IM
MH  - Amino Acid Oxidoreductases/*isolation & purification/metabolism
MH  - Animals
MH  - Cerebellum/*enzymology
MH  - Chromatography, Affinity
MH  - Chromatography, Gel
MH  - Citrulline/metabolism
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Isoenzymes/*isolation & purification
MH  - Kinetics
MH  - Molecular Weight
MH  - Nitric Oxide Synthase
MH  - Rats
PMC - PMC50811
EDAT- 1991/01/15 00:00
MHDA- 1991/01/15 00:01
PMCR- 1991/07/15
CRDT- 1991/01/15 00:00
PHST- 1991/01/15 00:00 [pubmed]
PHST- 1991/01/15 00:01 [medline]
PHST- 1991/01/15 00:00 [entrez]
PHST- 1991/07/15 00:00 [pmc-release]
AID - 10.1073/pnas.88.2.365 [doi]
PST - ppublish
SO  - Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):365-9. doi: 10.1073/pnas.88.2.365.