In addition to its cGMP-selective catalytic site, cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains two allosteric cGMP-binding sites and at least one phosphorylation site (Ser92) on each subunit [Thomas, M.K., Francis, S.H. & Corbin, J.D. (1990) J. Biol. Chem. 265, 14971-14978]. In the present study, prior incubation of recombinant bovine PDE5 with a phosphorylation reaction mixture...

The interaction of lin-benzoadenosine di- and triphosphates with the catalytic subunit and type II holoenzymes of adenosine cyclic 3',5'-monophosphate (cAMP) dependent protein kinase has been investigated by steady-state kinetics and fluorescence spectroscopy. lin-Benzo-ADP is a competitive inhibitor of the catalytic subunit with respect to ATP with a Ki (8.0 microM) similar to the Ki for ADP (...

The asymmetric forms (17 S and 13 S ) of acetylcholinesterase isolated from Torpedo californica electric organs have been subjected to collagenolytic and tryptic digestions and the hydrodynamic properties and component peptides of the products analyzed. The asymmetric forms contain the catalytic subunits and a trypsin-sensitive structural subunit attached to a filamentous collagen-like tail uni...

We describe here the genetic and biochemical analyses of two classes of mutations in the soluble phosphofructokinase (PFK I) of Saccharomyces cerevisiae: those leading to the loss of activity and those giving rise to a kinetically altered enzyme. Complementation and allele-testing between these two classes of mutants show that loss of enzyme activity in vitro can come about not only by mutation...

The crystal structure of a novel aluminium fluoride inhibited form of bovine mitochondrial F(1)-ATPase has been determined at 2 A resolution. In contrast to all previously determined structures of the bovine enzyme, all three catalytic sites are occupied by nucleotide. The subunit that did not bind nucleotide in previous structures binds ADP and sulfate (mimicking phosphate), and adopts a "half...

An X-ray structure of the F1 portion of the mitochondrial ATP synthase shows asymmetry and differences in nucleotide binding of the catalytic beta subunits that support the binding change mechanism with an internal rotation of the gamma subunit. Other structural and mutational probes of the F1 and F0 portions of the ATP synthase are reviewed, together with kinetic and other evaluations of catal...

The type V cyclic GMP phosphodiesterase was partially purified from the high-speed supernatant of guinea-pig lung. The isoenzyme displayed linear kinetics for cyclic GMP hydrolysis, with Km = 2.2 ± 0.2 ,tM and Vmax = 1.2 ± 0.08 nmol/ min per mg. The selective type V phosphodiesterase inhibitor Zaprinast inhibited cyclic GMP hydrolysis with IC50 (concn. giving 50% inhibition) = 0.45 + 0.08 /,M. ...

For the second time this year random cDNA sequencing, in combination with data from unicellular eukaryotes, has made a significant contribution to the analysis of human telomerase. Two groups have reported mammalian homologues of the Tetrahymena p80 telomerase-associated protein, in both cases the key breakthrough being mammalian cDNA clones with database matches to Tetrahymena p80. This has no...

It was recently observed' that uridine diphosphate galactose4-epimerase from Saccharomyces fragilis exhibits greatly enhanced activity in the presence of various cations. This activation appears to be primarily due to an increase in the affinity of the enzyme for its substrate, UDP-galactose.1' 2 Many different types of cation are effective in this regard, including univalent and divalent metal...

Telomerase is an RNA-directed DNA polymerase, composed of RNA and protein subunits, that replicates the telomere ends of linear eukaryotic chromosomes. Using a genetic strategy described here, we identify the product of the EST2 gene, Est2p, as a subunit of telomerase in the yeast Saccharomyces cerevisiae. Est2p is required for enzyme catalysis, as mutations in EST2 were found to result in the ...