Search PubMed⌕ Search

Biomedical subjects

H Chuan

Publications and source records attributed to H Chuan.

7 recordsLinked to original sources
PubMed1989

Determination of the roles of active sites in F1-ATPase by controlled affinity labeling.

The affinity reagents 3'-O-(5-fluoro-2,4-dinitrophenyl) [alpha-32P]ATP (FDNP-[alpha-32P]ATP) and 3'-O-(5-fluoro-2,4-dinitrophenyl) [8-14C]ATP (FDNP-[14C]ATP) were synthesized and used to characterize the structure and function of the three active sites in F1-ATPase. FDNP-[alpha-32P]ATP was found to bind covalently to F1 up to two DNP-[alpha-32P]ATP labels per F1 in the absence of Mg2+ without decreasing the ATPase activity. However, when MgCl2 was subsequently added to the reaction mixture, the enzyme could be further labeled with concomitant decrease in ATPase activity that is consistent with the complete inactivation of one enzyme molecule by an affinity label at the third ATP-binding site. Partial hydrolysis of the FDNP-[14C]ATP-labeled enzyme and sequencing of the isolated peptide indicated that the affinity label was attached to Lys-beta 301 at all three active sites. Samples of F1 with covalent affinity label on Lys-beta 301 were also used to reconstitute F1-deficient submitochondrial particles. The reconstituted particles were assayed for ATPase and oxidative phosphorylation activities. These results show that the catalytic hydrolysis of ATP either by F1 in solution or by F0F1 complex attached to inner mitochondrial membrane takes place essentially at only one active site, but is promoted by the binding of ATP at the other two active sites, and that ATP synthesis during oxidative phosphorylation takes place at all three active sites [corrected].

Adenosine Triphosphatases↗
PubMed1989

8-Azido-2'-O-dansyl-ATP. A fluorescent photoaffinity reagent for ATP-binding proteins and its application to adenylate kinase.

The photoaffinity reagent 8-azido-2'-O-[14C]dansyl-ATP (AD-ATP) has been synthesized for labeling and monitoring the active sites of ATPases and kinases. In its first application, the reagent is used to explore the active site of adenylate kinase from rabbit muscle. In the dark, AD-ATP inhibits adenylate kinase reversibly and competitively with KI = 0.25 +/- 0.01 microM. Under weak UV illumination, AD-ATP labels adenylate kinase irreversibly. The photoinactivation data also show KI = 0.25 +/- 0.02 microM. The ratio (r) of the specific activity of AD-ATP-labeled adenylate kinase to that of the unlabeled enzyme has been determined as a function of the number (n) of label/enzyme. The linear plot of r versus n with slope equal to -1 shows that the labeling is very specific, i.e. each label completely inactivates an enzyme molecule. After the labeled enzyme was partially hydrolyzed and the radioactive peptides analyzed and sequenced, it was found that Leu-115, Cys-25, and probably His-36 were labeled, in agreement with previous conclusions on the structure of the active site of this enzyme based on amino acid sequence, x-ray diffraction, and NMR studies. The environment-sensitive fluorescent dansyl group of AD-ATP can function as an in situ probe for monitoring ligand or conformation changes at the active site. The fluorescence of AD-ATP-labeled enzyme with n = 0.9 is not affected by ATP but increases with the concentration of AMP in solution. This observation is also in agreement with the previous conclusion that ATP does not bind to the AMP site of adenylate kinase. The observed enhancement of fluorescence indicates that binding of AMP by this enzyme causes environmental change at its ATP site. The possible usefulness of AD-ATP as an effective biological inhibitor or as a molecular probe for studying the structure and regulation of ATP-binding proteins is discussed.

Adenosine Triphosphate↗
PubMed1989

A potent fluorescent ATP-like inhibitor of cAMP-dependent protein kinase.

The fluorescent ATP analogue 8-azido-2'-O-[14C]dansyl-ATP ([ 14C]AD-ATP) was used to probe the ATP-binding site in the catalytic (C) subunit of cAMP-dependent protein kinase. AD-ATP was found to inhibit the phosphotransferase activity of C subunit with extremely high specificity. Complete inhibition was observed when each mol of C subunit was covalently labeled with 1 mol of this fluorescent ATP analogue. The labeling can be accelerated by the presence of Mg2+ or Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly), whereas high concentrations of ATP can almost completely protect the enzyme from AD-ATP. Detailed studies indicated that AD-ATP competes with ATP for binding to C subunit. Analysis of the kinetic data gave dissociation constants of 2.9 and 13 microM for AD-ATP and ATP bound to C subunit, respectively. AD-ATP has a fluorescence emission peak at 510 nm in pH 7.0 aqueous buffer containing 25% glycerol. After covalent binding to C subunit this emission peak shifts to 455 nm, which suggests that the label at ATP site is in an endogenous hydrophobic environment. Upon the binding of Mg2+ or Kemptide, the fluorescence of AD-ATP-labeled C subunit can be enhanced by 50 and 45%, respectively. This enhancement suggests that the binding of either the peptide substrate or Mg2+ induces conformational change at the active site of C subunit. Analysis of the fluorescence data shows that the values of Kd for Mg2+ and Kemptide bound to AD-ATP-labeled C subunit are 0.2 mM and 2.1 microM, respectively. The normal procedure for the preparation of the C subunit from the bovine heart muscle has been simplified to require only one-fifth of the usual working time to obtain the homogeneous enzyme with 70% yield from the crude extract.

Adenosine Triphosphate↗
PubMed1988

3'-O-(5-fluoro-2,4-dinitrophenyl)ADP ether and ATP ether. Affinity reagents for labeling ATPases.

The affinity reagents 3'-O-(5-fluoro-2,4-dinitrophenyl)ADP ether (FDNP-ADP) and 3'-O-(5-fluoro-2,4-dinitrophenyl)ATP ether (FDNP-ATP) were synthesized and characterized. FDNP[14C]ADP was found to label the active site of mitochondrial F1-ATPase slowly at room temperature but with high specificity. F1 was effectively protected from the labeling reagent by ATP or ADP. An average number of 1.3 covalent label per F1 is sufficient for 100% inhibition of the ATPase. About 73% of the radioactive label was found covalently attached to beta subunits, 9% on alpha, practically none on gamma, delta, and epsilon. Cleavage of the labeled enzyme by pepsin and sequencing of the major radioactive peptide showed that the labeled amino acid residue in beta subunit was Lys beta 162. These results show that Lys beta 162 is indeed at the active site of F1 as assumed in the recently proposed models (Fry, D. C., Kuby, S. A., and Mildvan, A. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 907-911; Duncan, I. M., Parsonage, D., and Senior, A. E. (1986) FEBS Lett. 208, 1-6).

Adenosine Diphosphate↗
PubMed1987

P1-(5'-adenosyl)-P2-N-(2-mercaptoethyl)diphosphoramidate. An affinity reagent for demonstrating the presence of Tyr-beta 311 at the hydrolytic site of F1-ATPase.

The compound P1-(5'-adenosyl)-P2-N-(2-mercaptoethyl)diphosphoramidate (AMEDA) was synthesized as an ATP analogue for in situ reaction with the 4-nitro-2,1,3-[14C]benzoxadiazolyl group (NBD) in the labeled F1-ATPase (F1). AMEDA was found to reactivate O-[14C]NBD-F1 via a dual-path mechanism. The principal path involves the binding of AMEDA at a site in F1 with Kd = 14.5 microM and subsequent reaction with the [14C]NBD label. The second slower path involves the direct biomolecular reaction of AMEDA with the radioactive label on F1. The rate of reactivation of O-[14C]NBD-F1 by AMEDA was decreased by ADP or ATP which competes with the ATP analogue for binding to the labeled enzyme. The reaction product was found to contain one adenine group, two phosphate groups, and one [14C]NBD label per molecule as expected from the structure of the compound AMEDA-[14C]NBD. Purified AMEDA-[14C]NBD was found to bind to unlabeled F1 with Kd = 2 microM. These observations demonstrate the in situ reaction of bound AMEDA with the nearby [14C]NBD label attached to Tyr-beta 311 and support the assumed presence of Tyr-beta 311 near the phosphate groups of ATP bound at the hydrolytic site of F1-ATPase. The possible locations of Tyr-beta 364, His-beta 427, and Tyr-beta 345 relative to Tyr-beta 311 in F1 are discussed, and the validity of the previously proposed model for F1-ATPase with one hydrolytic site assisted by two auxiliary sites is examined and compared with that of the widely accepted alternating sites model.

Adenosine Diphosphate↗