No abstract

In recent years, acyl dihydroxyacetone phosphate [acyl-DHAPt) has been shown to be a precursor of glycerolipids and glycerol-ether lipids.'.' Acyl-DHAP was discovered as a rapidly labeled lipid that was formed in crude mitochondrial fraction from 3'Pi or y-32P[ATP].3 This rapid labeling was due to the enzymatic dephosphorylation and rephosphorylation of endogenous acyl-DHAP present in the crude mitochondrial fraction (equation 1): Acyl-DHAP 2-/-Acyl-DHA (1) ADP ATP Acyl-DHAP was later shown to be biosynthesized by a direct acylation of DHAP with acyl-CoA. catalyzed by DHAP acyltransferase (EC 2.3.1.42) (equation 2):4

The mechanism of nucleotide binding and hydrolysis by dnaB protein and dnaB X dnaC protein complex has been studied by using fluorescent nucleotide analogues. Binding of trinitrophenyladenosine triphosphate (TNP-ATP) or the corresponding diphosphate (TNP-ADP) results in a blue shift of the emission maximum and a severalfold amplification of the fluorescence emission of the nucleotide analogues. Scatchard analysis of TNP-ATP binding indicates that TNP-ATP binds with a high affinity (Kd = 0.87 microM) and a 8.5-fold enhancement of fluorescence emission of the nucleotide. Only three molecules of TNP-ATP or TNP-ADP bind per hexamer of dnaB protein in contrast to six molecules of ATP or ADP binding to a dnaB hexamer. TNP-ATP and TNP-ADP are both competitive inhibitors of single-stranded (SS) DNA-dependent ATPase activity of dnaB protein. TNP-AMP neither binds to dnaB protein nor inhibits the ATPase activity. Formation of dnaB X dnaC complex by dnaC protein results in diminution of the TNP-ATP fluorescence enhancement and a concomitant decrease in the SS DNA-dependent ATPase activity. Kinetic analysis of the ATPase activity of dnaB X dnaC complex indicates that the decrease in the ATPase activity on complex formation is due to a reduction of the maximal velocity (Vmax). The dnaB protein hydrolyzes both TNP-ATP and dATP, however, with an extremely slow rate in the presence of single-stranded M13 DNA. The 2'-OH group of the nucleotide most likely plays an important role in the hydrolysis reaction but not in the nucleotide binding.

A simple and reliable method, based on that described by W. Seubert (1960, Biochem Prep. 7, 80-83), has been developed for the chemical synthesis of radioactive acyl coenzyme A's. I-14C-labeled fatty acids (palmitic, oleic, and linoleic) are converted to their acyl chlorides with oxalyl chloride. The [l-14C]acyl chlorides are then condensed with a two-to threefold molar excess of coenzyme A in a bicarbonate-buffered tetrahydrofuran solution to form the corresponding [l-14C]acyl coenzyme A's. The overall yields are near 75%, and the purities are greater than 90% based on spectral, chromatographic, and enzymatic properties.

Previous energy transfer studies [Squier, T. C., Bigelow, D. J., de Ancos, J. G., & Inesi, G. (1987) J. Biol. Chem. 262, 4748-4754] have utilized fluorescent iodoacetamide derivatives covalently bound to the Ca2+-ATPase of sarcoplasmic reticulum (SR), using labeling conditions that completely modify the most reactive of the protein's surface sulfhydryls to a final level of 9 nmol/mg of SR protein. Unambiguous interpretation of these results requires localization of these labeling sites with respect to the primary structure of the Ca2+-ATPase. In the present study, we have used the probe 6-(iodoacetamido)fluorescein (IAF) as a marker for these sites. The IAF-labeled Ca2+-ATPase was completely proteolyzed with trypsin, followed by centrifugation to remove (unlabeled) membrane-associated portions. The soluble IAF-labeled tryptic peptides were purified by size-exclusion and reverse-phase high-performance liquid chromatography. Two IAF-peptides resulted. The major (4.1 nmol of IAF/mg of starting protein) and minor (1.9 nmol/mg) IAF-peptides were sequenced and were identified, respectively, as Ala673-IAF-Cys674-Cys675-Phe676-Ala677+ ++-Arg678 and as Glu668-Ala669-IAF-Cys670-Arg671. A model is proposed to explain the selectivity of IAF for Cys670 and Cys674 of the approximately 14 surface sulfhydryls of the Ca2+-ATPase. The labeling region, Arg667 through Arg678, has been predicted to be alpha-helical; Cys670 and Cys674 would be adjacent in the helix and imbedded in an Arg cluster. The Arg residues would both attract the anionic IAF and enhance sulfhydryl reactivities by lowering their pK values.