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Biomedical subjects

J A Putkey

Publications and source records attributed to J A Putkey.

45 records · Page 3Linked to original sources
PubMed1985

The structural organization of the chicken calmodulin gene.

The structural organization of the entire chicken calmodulin (CaM) gene was determined by analysis of overlapping genomic clones obtained from Charon 4A and cosmid DNA libraries. These clones together span 39 kilobases of chicken genomic DNA. The CaM gene is 12 kilobases long and contains 8 exons interrupted by introns of highly variable size. The first intron (A) is only 19 base pairs (bp) long and divides the 5' untranslated region. Intron B separates the ATG from the first nucleotide of the triplet which encodes the NH2-terminal amino acid of CaM (Ala) whereas intron C separates the triplets encoding amino acids 10-11. Introns D, F, and G interrupt the Ca2+ binding subdomains II, III, and IV of CaM whereas intron E is localized in the linker region between the highly homologous NH2- and COOH-terminal halves of the protein. Primer extension studies using chicken brain poly(A+) mRNA and a fragment from the 5' untranslated region of a CaM cDNA identified the presumptive transcription initiation site (cap site) of the mRNA to be 103 bp 5' from the initiation condon ATG. A consensus sequence TATTTAA was localized 29 bp 5' from this cap site while a CCAAT sequence was located further 5' at position -58 bp. The structure of the CaM gene is strikingly similar to genes that encode other Ca2+ binding proteins from sea urchin, chicken, rat, and quail. These data suggest a conservation of genome organization related to the calcium binding and regulatory domains of Ca2+ binding proteins.

Animals↗
PubMed1983

Chicken calmodulin genes. A species comparison of cDNA sequences and isolation of a genomic clone.

A cDNA library, prepared from poly(A+) mRNA isolated from chicken brain, was screened for calmodulin sequences using the cloned full length structural gene from Electrophorus electricus as probe (Lagacé, L., Chandra, T., Woo, S.L.C., and Means, A.R. (1983) J. Biol. Chem. 258, 1684-1688). Fifteen positive signals were detected among 4500 recombinant clones from which two overlapping clones (pCB12 and pCB15) were selected for subsequent DNA sequencing. The combined unique sequences of the two cDNA clones yielded 1395 base pairs and contained the entire coding region for calmodulin, 94 base pairs of the 5'-nontranslated region, and the entire 3'-nontranslated region of 857 base pairs. The derived amino acid sequence of chicken calmodulin is identical with that of the bovine or human protein. Compared to the eel, there is a single conservative amino acid substitution at position 74 which is occupied by Arg in the chicken and Lys in the eel. The overall nucleotide homology between the amino acid coding regions of chicken and eel calmodulin mRNA is 79%. However, the 5'- and 3'-nontranslated regions of the chicken and eel mRNA for calmodulin are highly diverged with sequence homologies of 21 and 29%, respectively. The cDNA clones were used as probes to determine the size and distribution of calmodulin mRNA in a variety of chicken tissues. In all tissues examined, two species of mRNA for calmodulin were detected at 1600 and 1900 nucleotides. Both mRNAs occurred in the cytoplasm with an abundance ratio of 4:1 for the 1600 and 1900 species, respectively. The two mRNAs appear to result from differential processing of transcripts from a single calmodulin gene. Screening of a chicken genomic phage library using pCB12 as a probe yielded a single positive designated CL-1 which contains a DNA insert of 13.5 kilobase pairs. Partial sequencing of CL-1 has confirmed the presence of sequences which code for calmodulin. A comparison of the restriction maps of CL-1 and pCB12 and pCB15 indicates that CL-1 contains at least 3 intervening sequences.

Amino Acid Sequence↗
PubMed1983

Vitamin D. Its effect on the protein composition and core material structure of the chick intestinal brush-border membrane.

Vitamin D3 is known to stimulate the absorption of calcium across the asymmetric intestinal epithelial cells. Efforts to elucidate the mechanism of stimulation of intestinal calcium transport by vitamin D are now focused on evaluating the protein composition and topology of the brush-border membrane and its associated core material. Intestinal brush-border membranes were isolated from vitamin D-replete and vitamin D-deficient chicks. Core material proteins were isolated, by sedimentation, from brush-border membranes which were solubilized with Triton X-100. As determined by polyacrylamide gel electrophoresis, dietary vitamin D3 treatment caused no change in the relative amounts of five major core material proteins with Mr = 101,000, 94,000, 67,000, 42,000 (actin), and 17,000. In contrast, dietary vitamin D3 treatment caused a significant reduction in the levels of two proteins with Mr = 111,000 (sucrase) and 83,000, and an increase in the levels of a protein with Mr = 78,000 (possibly a subunit of alkaline phosphatase). The Mr = 111,000, 83,000, and 78,000 proteins are readily solubilized by Triton X-100 and are located on the extracellular surface of the brush-border membrane, as judged by [125I]diazoiodosulfanilic acid and lactoperoxidase 125I labeling. A significant vitamin D-dependent difference was found with respect to iodination of isolated core material as evidenced by the 125I labeling patterns of the Mr = 42,000 protein (actin). The Mr = 42,000 protein was labeled two to three times more extensively when associated with core material derived from vitamin D-deficient chicks as compared to vitamin D-replete chicks. Increasing the salt concentration (0-125 mM KCl) present during core material isolation from either vitamin D-replete or vitamin D-deficient chicks yields core material actin which is more susceptible to iodination by both [125I]diazoiodosulfanilic acid and lactoperoxidase. This increase in the extent of actin iodination is coupled to a salt-induced decrease in the stability of the core material which is evidenced by a decrease in the percentage of total brush-border membrane actin which is Triton-insoluble. This strongly suggests that the vitamin D-induced decrease in the accessibility of actin to iodination reagents results from a vitamin D-dependent change in the structure of the core material. Collectively, these results implicate a role for dietary vitamin D3 in maintaining a specified composition and topology of both the brush-border membrane proteins as well as its associated cytoskeletal core proteins, which is possibly important for intestinal calcium transport.

Animals↗
PubMed1983

Vitamin D-mediated alterations in the topography of intestinal brush border proteins: effect of papain on hydrolase release and calcium uptake.

The effect of vitamin D status on the topography of intestinal cell membranes was studied in isolated brush borders, as well as their purified membranes, by limited proteolysis. Addition of papain to brush borders isolated from vitamin D3-treated and deficient chicks resulted in a differential solubilization of leucine aminopeptidase, maltase, and sucrase activities (114, 195, and 79%, respectively, of appropriate control levels) but not alkaline phosphatase activity. In comparison, proteolysis of purified membranes exhibited vitamin D3- and 1,25-dihydroxycholecalciferol [1,25(OH)2D3]-dependent differences in release of all four marker hydrolases monitored. Calcium uptake studies revealed that preincubation with papain yielded vesicles with a calcium content that was 125% of corresponding native vesicles, in preparations from vitamin D3-treated, as well as deficient birds. Membrane vesicles prepared from 1,25(OH)2D3-treated chicks initially accumulated calcium to a greater extent than those from rachitic birds, but thereafter exhibited a decline in calcium content to basal levels. Preincubation with papain, however, abolished this loss of calcium. The combined results indicate that vitamin D mediates alterations in brush border protein topography and raise the possibility that this action of the seco-steroid might be involved in calcium absorption. However, if vitamin D-stimulated calcium transport across the brush border is dependent on a protein carrier, the molecular entity is not sensitive to inactivation by papain.

Animals↗
PubMed1982

Vitamin D-mediated intestinal calcium transport. Effects of essential fatty acid deficiency and spin label studies of enterocyte membrane lipid fluidity.

Vitamin D-3 and its metabolites regulate the transport of calcium across the intestinal epithelial cell via a mechanism which is as yet unknown. The purpose of this study was to evaluate the effect of an essential fatty acid deficiency on vitamin D-stimulated intestinal calcium transport as measured by both in vivo and in vitro techniques. We also describe in this report a procedure for the isolation of chick intestinal epithelial cell brush border and basal lateral membranes and an assessment of the effect of dietary vitamin D on the lipid fluidity of these membranes. An essential fatty acid deficiency in both vitamin D-replete and deficient chicks resulted in a decrease in intestinal mucosal levels of linoleic acid, with a compensatory increase in the levels of the short chain fatty acid, myristic acid, and the unsaturated fatty acids, palmitoleic and eicosatrienoic acids. An essential fatty acid deficiency did not affect the ability of vitamin D-deficient chicks to respond to vitamin D with a 2-fold increase in serum calcium and a 4-5-fold increase in intestinal calcium transport, measured in vivo. However, an essential fatty acid deficiency resulted in an inability of vitamin D to increase calcium efflux in vitamin D-deficient chick ileum as measured under in vitro conditions. Dietary vitamin D resulted in no detectable change in the protein composition in either the brush border or basal lateral membranes as evidenced by SDS-polyacrylamide electrophoresis. In addition, vitamin D did not alter the levels of brush border membrane cholesterol or lipid phosphorus (0.27 +/- 0.03 and 0.19 +/- 0.01 mumol/mol protein, respectively). Brush border and basal lateral membranes were labeled with the 5-nitroxide stearate spin probe I(12,3). The polarity of the environment of the probe in the brush border membranes is much greater than that of the basal lateral membranes. In addition, the lipid environment of the brush border membrane is much less fluid (S = 0.650) that that of the basal lateral membrane (S = 0.583). The data concerning membrane lipid fluidity is qualitatively similar to fluorescence polarization studies of rat intestinal epithelial cell membranes and confirms the concept that a given cell may contain plasma membrane regions having discrete lipid structures/fluidities. Dietary vitamin D had no detectable effect on the lipid fluidity or polarity in either the brush border or basal lateral membranes. The results do not support a role for an alteration in essential fatty acid composition or gross changes in the lipid fluidity of the brush border or basal lateral membranes as mechanisms by which vitamin D regulates intestinal calcium transport.

Animals↗
PubMed1982

Intestinal calcium transport: pleiotropic effects mediated by vitamin D.

The primary biological response to vitamin D is an increase in intestinal calcium transport. Efforts to elucidate the molecular mechanism by which this increased transport occurs has revealed that vitamin D mediates pleiotropic effects that are most likely a result of both genomic and nongenomic mechanisms of action of vitamin D. The spectrum of intestinal responses to vitamin D must now include an alteration in the topography of intestinal epithelial cells' brush border membrane (BBM). Initial indications of such an event were obtained with the polyene antibiotic filipin. Filipin induces active calcium transport in vitro in the small intestine of vitamin D-deficient chicks by specifically interacting with cholesterol and thereby altering membrane organization. Alterations in BBM topography in response to vitamin D were detected with the use of membrane-impermeable protein-labeling reagents and limited proteolysis. Administration of vitamin D to vitamin D-deficient chicks induces specific changes in accessibility of BBM proteins to labeling reagents. In addition, an alteration is induced in the rate of solubilization of BBM hydrolases during limited proteolysis. These vitamin D-mediated alterations in membrane topography offer new possibilities for regulation of intestinal calcium transport.

Animals↗
PubMed1978

The interaction of 1,25-dihydroxyvitamin D3 with its intestinal mucosa receptor: kinetic parameters and structural requirements.

Vitamin D3 and its metabolites comprise an endocrine system which plays a critical role in calcium homeostasis. The active form of vitamin D3 is 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Chromatin localization of 1,25(OH)2D3 and sucrose density gradient centrifutation have demonstrated the presence of an intestinal mucosa cytosol receptor which specifically binds 1,25(OH)2D3. The kinetic parameters of 1,25(OH)2D3 binding to its receptor have been determined by hydroxylapatite and reconstituted chromatin cytosol assays. Utilization of these assays has also permitted a determination of the precise structural requirements of the vitamin D ligand for the intestinal receptor. Furthermore, it has been possible to propose two receptor-ligand models which are capable of accommodating the conformationaly modile A ring of the vitamin D seco-steroids.

Animals↗