Site-directed mutageneses of rat liver cytochrome P-450d: catalytic activities toward benzphetamine and 7-ethoxycoumarin

Catalytic activities toward benzphetamine and 7-ethoxycoumarin of 11 distal mutants, 9 proximal mutants, and 3 aromatic mutants of rat liver cytochrome P-450d were studied. A distal mutant Thr319Ala was not catalytically active toward benzphetamine, while this mutant retained activity toward 7-ethoxycoumarin. Distal mutants Gly316Glu, Thr319Ala, and Thr322Ala displayed higher activities (kcat/Km) toward 7-ethoxycoumarin that were 2.4-4.7-fold higher than that of the wild-type enzyme. Although kcat/Km values of four multiple distal mutants toward benzphetamine were less than half that of the wild type, activities of these mutants toward 7-ethoxycoumarin were almost the same as or higher than the wild-type activity toward this substrate. The distal double mutant Glu318Asp, Phe325Tyr showed 6-fold higher activity than the wild-type P-450d toward 7-ethoxycoumarin. Activities of the proximal mutants Lys453Glu and Arg455Gly toward both substrates were much lower (less than one-seventh) than the corresponding wild-type activities. Catalytic activities of three aromatic mutants, Phe425Leu, Pro427Leu, and Phe430Leu, toward benzphetamine were less than 7% of that of the wild type, while the activities of these aromatic mutants toward 7-ethoxycoumarin were more than 2.5 times higher than the wild-type activity toward this substrate. From these findings, in conjunction with a molecular model for P-450d, we suggest that (1) the relative importance to catalysis of various distal helix amino acids differs depending on the substrate and that these differences are associated with the size, shape, and flexibility of the substrate and (2) the proximal residue Lys453 appears to play a critical role in the catalytic activity of P-450d, perhaps by participating in forming an intermolecular electron-transfer complex.     

Ammo-Terminal Amino Acid Sequences of Pea Phytochrome II Fragments Obtained by Limited Proteolysis

Apical shoot of pea seedling contains two immunochemically distinctspecies of phytochrome, type I (PI) and type II (PII) [Abe etal. (1985) Plant Cell Physiol. 26: 1387-1399]. A PII samplewas prepared from crude extracts from apical shoot of 7-day-old,light-grown pea (Pisum sativum L. cv. Alaska) seedlings, usingbrushite, DEAE-agarose and an agarose linked monoclonal anti-peaPII antibody (mAPll) column chromatography. More than 75% ofthe PII sample consisted of one major 115 kDa chromopeptide,and less than 25% of PII was composed of two minor non-chromophoric49 and 48 kDa fragments, as judged by SDS-polyacrylamide gelelectrophoresis and zinc-induced chromophore fluorescence analysis.Limited proteolysis of PII was performed with either trypsinor Staphylococcus aureus V8 protease, and the resulting fragmentswere separated by SDS-polyacrylamide gel electrophoresis andblotted electrophoretically onto a polyvinylidene difluoridemembrane. Amino-terminal amino acid sequences of the four fragmentsof PII cut from the membrane were determined by a gas-phasesequencer. The sequence of the determined regions (76 aminoacid residues in total), reveals that PII was 63% homologouswith PI. Hence the primary structure of PII is distinct fromthat of PI. (Received July 21, 1989; Accepted September 1, 1989)     

Isolation and Identification of Diacylglyceryl-O-4'-(N,N,N-trimethyl)-homoserine from the Fern Adiantum capillus-veneris L.

Diacylglyceryl-O-4'-(N,N,N-trimethyl)-homoserine was isolatedfrom fronds of the fern Adiantum capillus-veneris L. and identifiedby chemical and spectral analyses. Positional analysis of thefatty acids by lipase treatment showed that palmitic acid wasesterified at position 1, but linoleic, linolenic and arachidonicacids at position 2 of the glycerol moiety of the lipid. Althoughthe trimethylhomoserine lipid has been found in some algal species,this is the first report that it exists in a vascular plant. (Received April 9, 1983; Accepted June 22, 1983)     

Conversion of 5' xanthylic acid to guanine and guanine nucleotides by a mutant of Brevibacterium ammoniagenes

A decoyinine resistant, KY 13501, isolated after nitrosoguanidine treatment from Brevibacterium ammoniagenes ATCC 6872 converted 5′XMP added in fermentation media to guanine derivatives and accumulated them in the media. The converted substances were identified as guanine, 5′GMP, 5′GDP, and 5′GTP. The conditions for the conversion were examined and the following points were clarified. (1) Very low concentration of manganese ion (Mn²⁺) showed profound effects on the conversion and the excessive amounts of the ion severely repressed the conversion. (2) Under limitation of Mn²⁺, 5′XMP was converted most efficiently when added at inoculation time. (3) The inhibition of the conversion by excessive amount of Mn²⁺ was completely released by addition of a surface activating agent, polyoxyethylene stearylamine. (4) For the conversion, it was essential to maintain pH of the media at 7.5 to 8.0 and supply ammonium ion.     

Triglucosylation on the biotransformation of (+)-menthol by cultured cells of Eucalyptus perriniana.

Cultured cells of Eucalyptus perriniana biotransformed (+)-menthol to its gentiobioside and triglucoside [2,6-di-O-(beta-D-glucopyranosyl)-beta-D-glucopyranoside]. The structures of these compounds were determined by means of NMR techniques.     

Isolation and partial characterization of platelet α-granule membranes

Porcine alpha-granules, prepared by a modification of pre-existing methods, were found to be essentially homogeneous by transmission electron microscopy. Freeze-fractured samples of isolated granules revealed numerous intramembranous particles on the EF (exoplasmic fracture) surface and to a lesser extent on the PF (protoplasmic fracture) surface whereas the PS (protoplasmic) surface was relatively smooth. The granules appear to be sealed, as evidenced by: a) the retention of their electron dense core material; b) the inability of impermeant labels to react with the granule contents, and c) the finding that the intragranular proteins are refractory to mild hydrolysis by externally added proteases. Membranes were isolated by alkali extraction of the granules and used for biochemical characterization. Approximately 87% of the protein, but only insignificant amounts of phospholipid were removed by this procedure, which yielded membrane vesicles devoid of the dense core. The membranes contain one major and several minor polypeptides of molecular weights ranging from 28,000 to 230,000, as determined by polyacrylamide gel electrophoresis. The major polypeptide contains carbohydrate residues. The exposure of specific proteins on the cytoplasmic surface of the granule membrane was determined by a combination of surface-specific labeling and proteolysis of intact granules, followed by membrane isolation and analysis. In sealed granules, only a limited number of bands are modified by the reagents whereas most of them are affected following granule lysis, indicating asymmetry in their transmembrane disposition. The fraction eluted by alkali extraction was also analyzed and found to contain nine major polypeptides of molecular weights ranging from 230,000 to 43,000. These are compared to the weights of the macromolecules believed to be secreted from alpha-granules, as determined by radioimmunological techniques.