Gerhard E. Gerber

Amino acid analysis by dinitrophenylation and reverse-phase high-pressure liquid chromatography

The determination of amino acids has been achieved by reverse-phase high-pressure liquid chromatography of their dinitrophenyl derivatives. The methods developed permit the quantitation of all amino acids commonly encountered in a protein hydrolysate and the effect of various parameters on this separation was systematically evaluated. The procedure eliminates the need for specialized postcolumn equipment as employed in conventional amino acid analysis and can be obtained by a simple gradient high-pressure chromatograph. The sensitivity obtained is comparable to that available by methods in common usage, being able to determine amino acids quantitatively in the low picomole range.

Determination of the native form of FadD, the Escherichia coli fatty acyl-CoA synthetase, and characterization of limited proteolysis by outer membrane protease OmpT

Several studies have described FadD, the Escherichia coli fatty acyl-CoA synthetase [also known as fatty acid:CoA ligase (AMP-forming); EC 6.2.1.3], as a 42-50 kDa enzyme. Based on sequencing and expression data from the fadD gene, other reports have suggested that FadD is a 62 kDa protein and represents the sole fatty acyl-CoA synthetase in E. coli. We report that the 62 kDa FadD enzyme is a substrate for the outer membrane protease OmpT in vitro, producing a 43 kDa C-terminal fragment and a 19 kDa N-terminal fragment. Immunoblotting with a FadD antibody revealed that only the 62 kDa form of the enzyme is present in vivo, but we utilized the proteolytic sensitivity of FadD to investigate its structure. Photoaffinity labelling experiments revealed that both intact FadD and the 43 kDa fragment bound a long-chain fatty acid. Intact and cleaved FadD were also purified to determine the effect of cleavage on function. When using oleate as a substrate, cleaved FadD displayed 2-fold higher K(m) and V(max) values compared with intact FadD, but the catalytic efficiencies (k(cat)/K(m)) of the two forms were similar. This indicated that cleavage did not adversely affect enzyme activity. Proteolysis of FadD by OmpT was altered by the presence of oleate or ATP, both of which are ligands for the fatty acyl-CoA synthetase. This suggested that FadD undergoes ligand-induced conformational changes and implies that the region surrounding the cleavage site is mobile, a common characteristic of linker domains.

Phospholipid synthesis: effects of solvents and catalysts on acylation

Abstract The peptide dinitrophenylprolylthreoninamide (DNP-Pro-Thr-NH2) was used as a model system to develop better acylation conditions for the synthesis of phospholipids using catalyst-activated anhydrides. The acylation rate was found to be inversely related to the polarity of the solvent, chloroform alone resulting in much better rates of reaction than did pyridine, dimethylformamide (DMF) or mixtures of these solvents. Anhydride activated by 4-pyrrolidinopyridine (PPY) was twice as reactive as that activated by 4-dimethylaminopyridine (DMAP). It was shown that the phosphate group of phosphatidylcholine (PC) interferes with the acylation by a process which could be reserved by means of the addition of a 200-fold excess of PPY. This reversal is not due to base catalysis by the PPY; the results suggest that a mixed anhydride may be formed with the phosphate and that this can be reversed by high catalyst concentrations to produce the reactive acylating agent. The acylation rates for lysophosphatidylcholine (lyso PC) using our optimum conditions were found to be approximately 50 times faster than the best rates reported in the literature, the reaction being complete within 5 min even using only a slight excess of anhydride. acyl group migration was assessed during these reactions and no increase in migration of the acyl groups could be detected due to these reaction conditions. The procedures described provide significant improvements over previous methods described for large scale, as well as highly radioactive microscale phospholipid synthesis.

Specific labeling of Candida tropicalis peroxisomal proteins with photoreactive fatty-acid derivatives

The labeling of Candida tropicalis peroxisomal proteins with photoreactive fatty-acid derivatives was investigated. Proteins having molecular masses of 70 kDa, 48 kDa and 15 kDa were labeled with 11-m-diazirinophenoxy-[11-3H]undecanoate while 11-m-diazirinophenoxy-[11-3H]undecanoyl-CoA labeled proteins of 70 kDa and 55 kDa. The 70 kDa protein labeled with both photoreactive probes was resolved into two bands by electrophoresis on a gradient polyacrylamide gel; immunoprecipitation with anti-fatty acyl-CoA oxidase showed that these proteins are fatty-acyl-CoA oxidases. In purified peroxisomal membranes, two proteins of 36 kDa and 25 kDa were labeled with the photoreactive fatty-acid probe, whereas very little labeling of the above proteins or other proteins was observed with the fatty-acyl-CoA probe. The photoaffinity labeling method described is, thus, clearly capable of identifying and distinguishing between proteins having an affinity for fatty acid or fatty-acyl-CoA. The labeling also identified a fatty-acid-binding site on the 16 kDa peroxisomal matrix protein as well as on two peroxisomal acyl-CoA oxidases. This approach thus provides a general means for the identification of fatty-acid metabolizing enzymes, as well as for the identification of fatty-acid-binding sites on known enzymes.