An immunoradiometric assay for 1,25-dihydroxyvitamin D3 receptor

A ligand-independent, quantitative assay has been developed for the measurement of 1,25-dihydroxyvitamin D receptor utilizing purified receptor from pig intestine as a standard and two high affinity monoclonal antibodies directed to two different epitopes on the receptor. In this assay a fixed amount of 125I-labeled antibody is incubated with a fixed amount of a second antireceptor antibody linked to biotin and increasing amounts of purified receptor protein or sample. Antibody-receptor complexes can then be immunoprecipitated with avidin-Sepharose beads and counted. This method is highly reproducible and can detect 150 pg of 1,25-dihydroxyvitamin D3 receptor in crude extracts with intra- and interassay coefficients of variation of 8.6 and 18.2%. The monoclonal antibodies used recognize both native and denatured receptors from several different species, including human. This immunoradiometric assay should prove useful for studies of receptor regulation, occupancy, distribution, and turnover.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

Resolution and reconstitution of soluble components of rat liver microsomal vitamin D3-25-hydroxylase

Solubilized components of the vitamin D₃-25-hydroxylase, isolated from intact rat liver microsomes known to catalyze the C-25 oxidation of vitamin D₃in vitro, have been separated into two submicrosomal fractions enriched in detergent-solubilized NADPH-cytochrome c reductase and cytochrome P-450 or P-448. The P-450 hemoprotein-containing fraction was obtained by solubilization with cholic acid followed by treatment with the nonionic detergent, Emulgen 911, yielding a final preparation with a specific content of 7.25 nmol/mg microsomal protein. The reduced triphosphopyridine nucleotide-dependent cytochrome P-450 reductase activity, as detected by its ability to reduce the artificial electron acceptor, cytochrome c, was isolated free of cytochromes b₅ or P-450 by solubilization with deoxycholate and chromatography on DEAE-cellulose. The reductase component was found to exhibit kinetic properties with Michaelis constants: K_m(NADPH) = 3.14 μM, K_m(NADH) = 31.25 μM, and K_{m(cyt c)} = 12.34 μM. The NADPH-cytochrome c reductase activity was sensitive to NADPH-reversible inhibition by NADP, but not rotenone or cyanide. When the isolated components were incubated in the presence of an NADPH-generating system and carbon monoxide under anaerobic conditions, enzymatic reduction of the P-450 hemoprotein was measured by the appearance of characteristic absorbances at 420 and 450 nm of the reduced carbon monoxide vs. reduced difference spectrum. Furthermore, when the soluble submicrosomal components were reconstituted with excess reduced triphosphopyridine nucleotide, ³H-labeled vitamin D₃, and soluble cytosolic supernatant, full vitamin D₃-25-hydroxylase activity was restored at rates of up to 7.68 pmol/h/mg protein, with an apparent turnover number of cytochrome P-450 of 1.16 to 1.20 under conditions where the concentrations of the hemoprotein were rate limiting for net product formation. These results strongly support the hypothesis that the rat liver microsomal mixed-function oxidase, vitamin D₃-25-hydroxylase, consists of at least two membrane-bound protein components, NADPH-cytochrome c reductase and a cytochrome P-450 terminal oxidase, for the catalytic conversion of vitamin D₃ to 25-hydroxyvitamin D₃.     

Post-translational processing of 7S and 11S components of soybean storage proteins

The synthesis and processing of the major storage proteins in soybean cotyledons was studied both in vivo and in vitro. The and subunits of 7S as well as the 11S proteins are synthesized as higher molecular weight-precursors on membrane-bound polysomes. The initial translation products of the 7S are proteolytically cleaved during translation suggesting the removal of a signal peptide as evidenced by the presence of 2 and 2 peptides immunoreactive with 7S antibody in the in vitro chain completion products of the membrane-bound polysomes. This is followed or accompanied by cotranslational glycosylation, which increases their size equivalent to that of initially-synthesized precursors. In vivo pulse-labelled 7S and products are of slightly higher molecular weights than the immunoprecipitable chain-completion products, indicating further post-translational modifications. A slow post-translational processing during a period of 1.5 to 16 h yields the final 7S and glycoproteins.Acidic and basic subunits of the 11S protein appear to be synthesized from common large molecular weight (60K-59K) precursors. Antibodies to the 11S acidic component recognize both acidic and basic domains in the precursor while those raised against basic subunits appear to be specific for that region only. The processing of the 11S precursor is also very slow and occurs post-translationally. This slow rate of processing, coupled with a temporal difference in the synthesis of 7S and 11S components, suggests a highly coordinated mechanism for synthesis and packaging of these proteins into protein bodies during seed development.     

Epoxysuccinyl peptide-derived cathepsin B inhibitors: modulating membrane permeability by conjugation with the C-terminal heptapeptide segment of penetratin

Besides its physiological role in lysosomal protein breakdown, extralysosomal cathepsin B has recently been implicated in apoptotic cell death. Highly specific irreversible cathepsin B inhibitors that are readily cell-permeant should be useful tools to elucidate the effects of cathepsin B in the cytosol. We have covalently functionalised the poorly cell-permeant epoxysuccinyl-based cathepsin B inhibitor [R-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH; R=OMe] with the C-terminal heptapeptide segment of penetratin (R=epsilonAhx-Arg-Arg-Nle-Lys-Trp-Lys-Lys-NH2). The high inhibitory potency and selectivity for cathepsin B versus cathepsin L of the parent compound was not affected by the conjugation with the penetratin heptapeptide. The conjugate was shown to efficiently penetrate into MCF-7 cells as an active inhibitor, thereby circumventing an intracellular activation step that is required by other inhibitors, such as the prodrug-like epoxysuccinyl peptides E64d and CA074Me.     

Characterization of SPACR, a sialoprotein associated with cones and rods present in the interphotoreceptor matrix of the human retina: immunological and lectin binding analysis

Rod and cone photoreceptors project from the outer retinal surface into a carbohydrate-rich interphotoreceptor matrix (IPM). Unique IPM glycoconjugates are distributed around rods and cones. Wheat germ agglutinin (WGA) strongly decorates the rod matrix domains and weakly decorates the cone matrix domains. This study characterizes the major WGA-binding glycoprotein in the human IPM, which we refer to as SPACR (sialoprotein associated with cones and rods). SPACR, which has a molecular weight of 147 kDa, was isolated and purified from the IPM by lectin affinity chromatography. A polyclonal antibody to SPACR was prepared that colocalizes in tissue preparations with WGA-binding domains in the IPM. Sequential digestion of SPACR with N- and O- glycosidases results in a systematic increase in electrophorectic mobility, indicating the presence of both N- and O-linked glycoconjugates. Complete deglycosylation results in a reduction in the relative molecular mass of SPACR by about 30%. Analysis of lectin binding allowed us to identify some of the structural characteristics of SPACR glycoconjugates. Treatment with neuraminidase exposes Galbeta1- 3GalNAc disaccharide as indicated by positive peanut agglutinin (PNA) staining, accompanied by the loss of WGA staining. Maackia amurensis agglutinins (MAA-1 and MAA-2), specific for sialic acid in alpha2-3 linkage to Gal, bind SPACR, while Sambucus nigra agglutinin (SNA), specific for alpha2-6 linked sialic acid, does not, indicating that the dominant glycoconjugate determinant on SPACR is the O-linked carbohydrate, NeuAcalpha2-3Galbeta1-3GalNAc. The abundance of sialic acid in SPACR suggests that this glycoprotein may contribute substantially to the polyanionic nature of the IPM. The carbohydrate chains present on SPACR could also provide sites for extensive crosslinking and participate in the formation of the ordered IPM lattice that surrounds the elongate photoreceptors projecting from the outer retinal surface.