The gene for the membrane polypeptide D2 has been mapped on the pea (Pisum sativum) chloroplast genome. The nucleotide sequence of the gene and its flanking regions is presented. The only large open reading frame in the sequence codes for a protein of MW 39.5 kD. A potential ribosome binding site is located 6 nucleotides upstream from the initiation codon and there are two sets of putative promotor sequences in the 5' flanking region. The polypeptide has a high content of hydrophobic amino acids, predominatly grouped in clusters of 20 or more residues. The 3' end of the D2 gene is overlapped by 50 nucleotides of a second open reading frame (UORF I) which is at least 369 nucleotides long. Based on current data we suggest the D2 polypeptide to be a constituent of photosystem II (PSII).
The essential role of cytochrome P450 3A4 (CYP3A4) in human small intestine is well established, and CYP3A5 seems also to be present in most subjects. However, the role of CYP3A7 in the small intestine remains poorly characterized. We have therefore studied the expression of these CYP3A enzymes in the duodenal tissue from 19 patients, using a specific RT‐PCR (reverse transcriptase‐polymerase chain reaction) method. CYP3A4 and CYP3A5 were present at the mRNA level in the duodenum of 18 and 19 of the 19 patients studied, respectively. In contrast, mRNA for CYP3A7 was not found in the duodenum in any of the patients. These findings strongly suggest that, unlike CYP3A4 and CYP3A5, CYP3A7 is not expressed in human duodenum.
The genes encoding the two P700 chlorophyll a-apoproteins of the photosystem I complex were localized on the pea (Pisum sativum) chloroplast genome. The nucleotide sequence of the genes and the flanking regions has been determined. The genes are separated by 25 bp and are probably cotranscribed. The 5' terminal gene (psaA1) codes for a 761-residue protein (MW 84.1 kD) and the 3' terminal gene (psaA2) for a 734-residue protein (MW 82.4 kD). Both proteins are highly hydrophobic and contain eleven putative membrane-spanning domains. The homology to the corresponding polypeptides from maize are 89% and 95% for psaA1 and psaA2, respectively. A putative promoter has been identified for the psaA1 gene, and potential ribosome binding sites are present before both genes.
Chromatofocusing of soybean (Glycine max L.) leaf lipoxygenases revealed three distinct peaks of activity. Based on their isoelectric points (pis), pH optima, and mutant analysis it appears that the leaf isozymes are different from those described from mature soybean seed. At least one leaf lipoxygenase appears to differ from those found in hypocotyls. The pis of the main bands of the three leaf lipoxygenase peaks are 6.67, 5.91, and 5.67. The pH optima curves of three active fractions exhibit peaks at pH 6.2, 5.5, and 8.5, respectively. One of the fractions has two polypeptides with slightly different molecular weights, both of which react to soybean seed lipoxygenase antibodies. The other two fractions contain a polypeptide of unit molecular weight reacting with the lipoxygenase antibodies.LOXs2 (linoleate: oxygen oxidoreductase, EC 1. 13.11.12) are found throughout the plant (8) and animal kingdoms (9). They catalyze, in plants, the hydroperoxidation of cis,cis-1,4-pentadiene structures present in unsaturated fatty acids. The physiological role of LOX in plants remains ambiguous, although roles in growth and development, senescence or wound response, and pest resistance have been suggested ( 1 3, 15, 19).While soybean seed LOX has been extensively studied with three different isozymes described (2,12) sunlight. Leaves were measured at the maximum width, weighed, and ground in one-half volume of water with a mortar and pestle. Samples were centrifuged at 12,000g for 15 min and the supernatants were analyzed for LOX activity (11). Enzyme PurificationThe middle leaflets of soybean (Glycine max L. Merr.) trifoliates were harvested at random from plants growing in a greenhouse, frozen in liquid nitrogen, and ground with a mortar and pestle. Following grinding, 5 mM Na acetate (pH 4.5) was added (80 mL per 20 leaflets) and the resulting slurry was centrifuged for 7.5 min at 10,000g. The supernatant was made 10% in sucrose and loaded onto a Sephadex G-50 (coarse, 55 x 5 cm) (Pharmacia) column and eluted in 5 mM Na acetate, pH 4.5 at about 8 mL min-'. The protein containing fractions were analyzed for LOX activity by measuring the change in absorbance at 234 nm with time (11). Sodium linoleate was used as the LOX substrate. This was adjusted to an absorbance of 0.45 with previously prepared (partially oxidized) substrate. The most active fractions were pooled and centrifuged for 1.5 h at 140,000g to remove microsomal particles. The resulting supernatant was loaded onto a PBE94 chromatofocusing column (Pharmacia) previously equilibrated to pH 7.4 with 25 mM imidazole HCl solution. The proteins were eluted using Polybuffer 74 (Pharmacia), which had been diluted 1 to 8 and adjusted to pH 4.0 with 1 M HCl.Every other fraction collected from the chromatofocusing column was analyzed for LOX activity, and the approximate pls of the LOX proteins were determined by comparison to pI standards (Bio-Rad) on IEF-PAGE gels (see below). The samples with the highest LOX activity in a single peak were pooled and concentrated ...
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