The effects of lipid composition on the rate and extent of heme binding to membranes

The effects of membrane composition on heme binding to large unilamellar vesicles were examined using 30 separate phospholipid mixtures. Although there was some variation, most lecithins with Tm values less than or equal to 20 degrees C showed overall equilibrium partition constants equal to approximately 5 x 10(5) and association and dissociation partition rate constants equal to approximately 3 x 10(6) s-1 and 7 s-1, respectively, for CO-heme binding at 30 degrees C. A sharp decrease in the association rate for CO-heme uptake was observed as the lipid vesicles changed from liquid-crystalline to the gel phase. The addition of dicetyl phosphate or dimyristoylphosphatidylglycerol, which are negatively charged at neutral pH, decreased the affinity of the vesicles for CO-heme. The association rate and equilibrium partition constants for CO-heme uptake in unsaturated lecithins were unaffected by cholesterol content at levels up to 40%/mol. The affinity of saturated dimyristoylphosphatidylcholine (DMPC) vesicles for CO-heme decreased with increasing cholesterol content at 30 degrees C. This effect appears to be related to the influence of cholesterol on the DMPC phase transition temperature (Tm) since at low temperatures (less than or equal to 20 degrees C) little CO-heme binds to vesicles composed of DMPC even in the absence of cholesterol.     

Amino acid sequence of rubber elongation factor protein associated with rubber particles in Hevea latex

The amino acid sequence of rubber elongation factor, a recently discovered protein tightly bound to rubber particles isolated from the commercial rubber tree Hevea brasiliensis, is presented. The role of this protein in rubber elongation and its interaction with prenyltransferase and rubber particles have been discussed in the preceding paper in this series (Dennis, M. S., and Light, D. R. (1989) J. Biol. Chem. 264, 18608-18617). Trypsin, Staphylococcus protease, chymotrypsin, acetic acid, and hydroxylamine cleavage were used to generate peptide fragments that were isolated by reverse phase high pressure liquid chromatography and analyzed by amino acid composition and automated Edman degradation. Each digest contained one blocked peptide identified as the amino terminus. The blocked amino-terminal peptide from the tryptic digest was analyzed by amino acid composition, fast atom bombardment mass spectrometry (molecular ion 1659.9), subdigested with Staphylococcus protease for partial sequence analysis, and finally deblocked with bovine liver acyl-peptide hydrolase removing an acetylalanine to allow analysis by Edman degradation. Rubber elongation factor is 137 amino acids long, has a molecular mass of 14,600 daltons, and lacks four amino acids: cysteine, methionine, histidine, and tryptophan. The NH2 terminus is highly charged and contains only acidic residues (5 of the first 12 amino acids). The first four amino acids are highly represented in other known NH2-terminally acetylated proteins. Comparison of the sequence of rubber elongation factor with other known sequences does not reveal significant sequence similarities that would suggest an evolutionary relationship.     

Purification of a prenyltransferase that elongates cis-polyisoprene rubber from the latex of Hevea brasiliensis

We have purified "rubber transferase" from latex of the commercial rubber tree Hevea brasiliensis and find that it is a dimer with a monomeric molecular mass of 38,000 Da, requires Mg2+, and is stabilized by thiols in agreement with studies of a partially purified preparation previously described (Archer, B. L., and Cockbain, E. G. (1969) Methods Enzymol. 15, 476-480). Greater than 90% of the [1-14C]isopentenyl pyrophosphate which is incorporated into deproteinated rubber particles by the purified prenyltransferase is added to high molecular mass polyisoprene (greater than 20,000 Da). Purified prenyltransferase and deproteinated rubber particles reconstitute 40-60% of the biosynthetic activity of whole latex in samples matched for rubber content. Incorporation is linear with added rubber particles up to at least 10 mg/ml rubber or 20 microM rubber molecules (based on a number average molecular mass of 500,000 Da). Prenyltransferase concentrations estimated in whole latex (0.37% or 160 nM) are sufficient to saturate all elongation sites in whole latex, and addition of purified prenyltransferase does not increase [1-14C]isopentenyl pyrophosphate incorporation. Deproteinated rubber particles can be titrated with the pure enzyme (Kd = 9 nM) demonstrating that the fraction of rubber molecules available for addition is low (approximately 0.01%). An estimated 7,000 isoprene units are added per complex at a rate of 1/s in a typical assay. Hevea prenyltransferase catalyzes the formation of cis-isoprene in the presence of rubber particles. However, in the absence of rubber particles and in the presence of dimethylallyl pyrophosphate, the purified prenyltransferase catalyzes the formation of geranyl pyrophosphate and all trans-farnesyl pyrophosphate as demonstrated by thin layer chromatography, gas chromatography, and molecular exclusion chromatography.     

Expression cloning of cDNA by phage display selection.

Expression cloning of a mouse kappa chain fragment has been achieved from a cDNA library by display of expressed proteins on filamentous phage and affinity selection for binding to anti-mouse Fab antibodies. Expressed proteins were anchored to the phage coat by a synthetic, anti-parallel leucine zipper, which had been selected from a semi-randomized zipper library for the ability to connect a test protein to phage. From a library of 4 x 10(6) transformants, two separate clones displaying different size cDNA inserts were recovered after four selection rounds. These results further demonstrate the utility of phage display for cDNA expression cloning.     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

Regulation of hydroxydocosanoic acid sophoroside production in Candida bogoriensis by the levels of glucose and yeast extract in the growth medium.

Cells of Candida bogoriensis produce as a major extracellular lipid 13-[(2'-O-beta-D-glucopyranosyl-beta-D-glucopyranosyl)oxy]docosanoic acid 6',6'-diacetate (Ac2Glc2HDA), the diacetylated sophoroside of 13-hydroxydocosanoic acid (HDA), along with mono- and unacetylated derivatives. The HDA glycolipid production is greater than 2 g/liter when cells are grown on a "standard" medium of 3% glucose and 0.15% yeast extract. Either lowering the glucose concentration (0.5 to 2.0% glucose, at 0.2% yeast extract) or raising the yeast extract concentration (2 to 4% yeast extract at 3% glucose) greatly decreased the yield of this glycolipid, as well as its rate of synthesis measured by [14C]acetate incorporation. Total HDA production was also depressed on the low glucose medium, as was the activity of UDP-glucose:HDA glucosyltransferase, the first enzyme involved in the synthesis of Ac2Glc2HDA from HDA. Levels of acetyl-CoA:Glc2HDA acetyltransferase were not decreased by growth on a low glucose medium, however, even under conditions in which glycolipid production was less than 4% of that found in the standard medium. Low levels of the HDA glycolipids were monitored by high pressure liquid chromatography of their p-bromophenacyl esters, formed by the action of alpha,beta-dibromoacetophenone on the sodium salt of the lipid in the presence of a crown reagent catalyst. This regulation of extracellular Ac2Glc2HDA production by the nutrient composition of the growth medium may represent an important property in the adaptation of C. bogoriensis to its natural environment, the phyllosphere.