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Biomedical subjects

R H Wasserman

Publications and source records attributed to R H Wasserman.

At least 55 records · Page 3Linked to original sources
PubMed1983

Proton-NMR studies of the solution conformations of vitamin-D-induced bovine intestinal calcium-binding protein.

1H NMR is used to study the solution structure of vitamin-D-induced bovine intestinal calcium-binding protein. The study of the native protein is aided by the recently published crystal structure; it is shown that the conformations of the molecule in the crystal and in solution are very similar. The effect of pH and temperature on the native structure is described. The structure of the apo protein is then described, and the effect of pH and temperature on its fold is outlined. A comparison between apo and native protein folds is made which indicates that the folds are very similar. The two folds are related by a calcium titration, which indicates that the protein binds two calcium ions sequentially. Both steps in the Ca2+ titration occur under conditions of slow exchange (kex 80 s-1). The effect of binding Ca2+ ions is to cause twisting motions of helices, with the helices acting as rods, relaying the conformational change induced by Ca2+ binding to the linker regions of the protein.

Animals↗
PubMed1982

Calcium and 24,25-dihydroxyvitamin D: inverse relation in cows with parturient paresis.

Recent work suggests a role for 24,25-dihydroxyvitamin D in inhibiting mobilization of bone. This study was undertaken to investigate its possible role in the etiology of parturient paresis, a hypocalcemic condition of dairy cows occurring at the onset of lactation. This metabolic disease was chosen to serve as a model of impaired mineral homeostasis. The animals examined were parturient Holstein cows with (N = 6) and without (N = 7) parturient paresis. Determinations of serum 1,25-dihydroxyvitamin D, serum 24,25-dihydroxyvitamin D, and serum calcium were used to evaluate the 2 groups. The hormones were isolated using methylene chloride:methanol extraction, Sephadex LH-20 chromatography, and high-pressure liquid chromatography (HPLC). Quantitation was by competitive protein binding assays. Serum 1,25-dihydroxyvitamin D levels of affected cows were not significantly different from those of normal cows. The 24,25-dihydroxyvitamin D levels of paretic cows (3.48 +/- 0.27 ng/ml) were significantly higher than in the normal cows (2.03 +/- 0.34 ng/ml) (p less than 0.01). Linear regression analysis of the data from the paretic cows revealed an inverse relationship between serum calcium and 24,25-dihydroxyvitamin D (r = -0.94). This negative correlation between serum 24,25-dihydroxyvitamin D and serum total calcium in a naturally occurring hypocalcemic disease of dairy cattle may provide evidence that this metabolite is of significance in the etiology and pathogenesis of this syndrome.

24,25-Dihydroxyvitamin D 3↗
PubMed1982

Evidence for multiple effects of vitamin D3 on calcium absorption: response of rachitic chicks, with or without partial vitamin D3 repletion, to 1,25-dihydroxyvitamin D3.

The effects of vitamin D3 or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], or both, on the relationship among calcium absorption, vitamin D-induced calcium-binding protein (CaBP), and phospholipid metabolism were examined. When 1,25(OH)2D3 was injected intracardially into D3-deficient chicks, both the stimulation of calcium absorption and the induction of the synthesis of CaBP occurred 2-4 hr later. When 1,25(OH)2D3 was injected into chicks partially repleted with D3, an earlier increase in calcium absorption was observed without a significant change in the concentration of CaBP already present in the duodenal mucosa. Other early events were an increased uptake of calcium by the intestinal tissue and an alteration in phospholipid metabolism. These and other observations support the proposal that at least two phases of calcium absorption are influenced by 1,25(OH)2D3--permeation of calcium across the brush border, and transfer of calcium through and out of the cell. The first phase responds more rapidly to 1,25(OH)2D3 than does the second phase, correlates with changes in phospholipid metabolism, and might not be dependent on de novo protein synthesis. The second phase correlates with CaBP synthesis and therefore is dependent on protein synthesis. Either the first phase or the second phase can constitute the limiting step in calcium absorption.

Alkaline Phosphatase↗
PubMed1982

Analysis of cytoskeletal proteins and Ca2+-dependent regulation of structure in intestinal brush borders from rachitic chicks.

We have investigated several structural aspects of the intestinal epithelial brush border from rachitic chicks. At both the light and electron microscope levels, rachitic brush borders are indistinguishable from controls. Although several of the prominent periodic acid-Schiff-positive proteins of the brush border membrane have slightly slower mobilities on sodium dodecyl sulfate/polyacrylamide gels than do corresponding proteins from control brush borders, the major components of the microvillus core, including subunits of 105, 95, and 68 kilodaltons, actin, and calmodulin, are not detectably different. As assayed by a (125)I-labeled calmodulin gel overlay technique, the same calmodulin-binding proteins are present in rachitic and control brush borders. Two proteins, the 105-kilodalton subunit of the microvillus core and an approximately 30-kilodalton membrane protein, bind calmodulin in a calcium-independent manner. Four cytoskeletal proteins (250, 190, 180, and 150 kilodaltons) and one membrane protein (35 kilodaltons) bind calmodulin only in the presence of calcium. Calcium-dependent solation of microvillus core proteins and calcium-dependent phosphorylation of the 20-kilodalton light chain of brush border myosin both occur as in controls. Our results show that rachintic chicks have brush borders that are quite similar to controls with respect to their ultrastructural organization, constituent contractile proteins, and calcium-dependent regulation of contractility and microvillus core structure. Therefore, the decreased absorption of calcium by intestinal epithelial cells in rachitic chicks is probably not due to gross structural or chemical differences in the brush border cytoskeleton.

Actins↗
PubMed1982

Effect of vitamin D on the intestinal absorption of 203Pb and 47Ca in chicks.

The transfer of 203Pb and/or 47Ca across the intestinal epithelium of the chick was investigated, with emphasis given to the functional role of cholecalciferol (vitamin D-3). 203Pb, after introduction in the intestinal lumen, is rapidly accumulated by the intestinal tissue, and only a fraction of 203Pb is translocated parenterally (absorbed). Cholecalciferol did not significantly affect the accumulation of 203Pb by intestinal tissue but did accelerate 203Pb movement across the basal-lateral membrane. In contrast, cholecalciferol both decreased 47Ca tissue levels and increased 47Ca absorption. In rachitic chicks, the rate of absorption of 203Pb was greater in the distal than in the proximal segments of the intestine; after cholecalciferol repletion, the degree of absorption in al segments was similar, indicting the order of cholecalciferol effectiveness as duodenum greater than or equal to jejunum greater than ileum. An acute dose of 1,25(OH)2D3 to rachitic chicks also enhanced both 203Pb and 47Ca absorption, but the time course and pattern of absorption of these metal cations differed. The time at which the absorption of 203Pb peaked and returned to base-line occurred sooner than for 47Ca. Also the back-flux (blood leads to intestinal lumen) of 47Ca was enhanced by cholecalciferol, whereas no effect on the back-flux of 203Pb was noted. These studies show that cholecalciferol and 1,25(OH)2D3 affects both the 203Pb and 47Ca absorptive processes, but the nature of these responses are not identical, suggesting differences in the transport path or the macromolecular interactions of these metal ions during the course of absorption, or both.

Animals↗
PubMed1981

The amino acid sequence of bovine intestinal calcium-binding protein.

The complete amino acid sequence of the vitamin D-dependent bovine intestinal calcium-binding protein (minor A component) has been determined: Lys-Ser-Pro-Glu-Glu-Leu-Lys-Gly-Ile-Phe-Glu-Lys-Tyr-Ala-Ala-Lys-Glu-Gly-Asp-Pro-Asn-Gln-Leu-Ser-Lys-Glu-Glu-Leu-Lys-Leu-Leu-Leu-Gln-Thr-Glu-Phe-Pro-Ser-Leu-Le u-Lys-Gly-Pro-Ser-Thr-Leu-Asp-Glu-Leu-Phe-Glu-Glu-Leu-Asp-Lys-Asn-Gly-Asp-Gly-Glu-Val-Ser-Phe-Glu-Glu-Phe-Gln-Val-Leu-Val-Lys-Lys-Ile-Ser-Gln-OH. It is a 75-residue protein (computed Mr = 8501), contains a single Tyr, and is devoid of Cys, Met, Trp, His, and Arg. The bulk of the sequence was determined by automated sequencing of: (i) the intact protein for 20 cycles; (ii) a large N-bromosuccinimide peptide for 37 cycles; (iii) a tryptic peptide (29 cycles), isolated by high performance liquid chromatography. Also described is a highly sensitive and rapid procedure for peptide mapping by high performance liquid chromatography.

Amino Acid Sequence↗
PubMed1981

Gastrointestinal absorption of lead (203Pb) in chicks: influence of lead, calcium, and age.

The present study was designed to investigate, in more detail, the mechanism of lead transport by the gastrointestinal tract and particularly the similarities or dissimilarities between lead and calcium in this process. The absorption of these metals was determined in 3-week-old white Leghorn cockerels, raised on a commercial diet or special diets, using an in vivo ligated loop procedure. The dose administered into the loop usually contained 0.5 microCi 203Pb (and/or 0.1 microCi 47Ca), 0.01 mM lead acetate (and/or 1 mM CaCl2) in 0.5 ml of 0.15 M NaCl, pH 6.5. It was shown that lead is rapidly taken up by the intestinal tissue, and only slowly transferred into the circulation whereas calcium, also accumulated rapidly by the tissue, is rapidly released from the tissue in the serosal direction. The absorption processes of these cations show similar responses to various experimental conditions (low calcium intake, age of the animal, pH of the dosing solution). However, increasing luminal stable lead concentration from 0.01 to 1 mM Pb, significantly reduced the percentage of radiolead absorbed, but did not inhibit the absorption of radiocalcium, Also, luminal Ca (0-25 mM) did not significantly affect the absorption of 203Pb. These data imply that, in spite of the similarities in the response of the lead and calcium absorptive processes to various treatments, there is no direct interaction between these cations in the intestine of the chick.

Aging↗
PubMed1981

Intestinal absorption of calcium and phosphorus.

The intestinal absorption of calcium and phosphorus has received considerable attention in recent years. The evidence has clearly indicated that calcium is absorbed by two processes: active transport and diffusion. Vitamin D appears to affect both processes, and has a significant effect at the brush border of the intestinal cell. Vitamin D also affects the release of calcium from the intestinal cell to the lamina propria. Several proposed models to account for the transmural movement of calcium are discussed. The active transport of phosphate is under the control of vitamin D and is located at the brush border region of the intestinal cell. This transport system, like several others, appears to be sodium-dependent and inhibited by ouabain. In-transit phosphate does not mix with the cellular phosphate pool, implying that phosphate is moving through the cell as a distinct packet or through specialized channels, or possibly a phosphorylated derivative. Emphasized in the presentation is current knowledge of the transport mechanisms and macromolecular changes that potentially account for the stimulatory effect of vitamin D on calcium and phosphate transport.

Adenylyl Cyclases↗
PubMed1980

Enhanced absorption of calcium by casein phosphopeptides in rachitic and normal chicks.

The present studies were undertaken to investigate the effect of casein phosphopeptides on calcium absorption in normal and rachitic chicks. Phosphopeptides were produced by tryptic hydrolysis of casein and the crude peptide mixture was further purified, using gel filtration. The chromatographic fractions were pooled according to their protein and phosphorus content. Some of these fractions, when introduced into the ligated duodenal loop at a concentration of 1 mg/ml or added to the mucosal solution in vitro at a concentration of 0.1 mg/ml, were capable of increasing the intestinal transport of radiocalcium. This effect was seen both in the normal and rachitic chicks, indicating that the stimulatory response was not dependent upon molecular changes induced by vitamin D. Furthermore, this response was not shared by peptides from albumin, not likely due to phosphate generated from the phosphopeptides, nor related to the phosphate content of the fractions. These experiments demonstrate that a component of milk, in addition to lactose, might account for the high availability of calcium in milk.

Animals↗
PubMed1980

Effect to cholecalciferol and 1,25-Dihydroxycholecalciferol on the intestinal absorption of zinc in the chick.

The effect of cholecalciferol on the intestinal absorption of 65Zn was assessed in zinc-deficient and zinc-replete rachitic chicks, using the in situ ligated loop techniques. Cholecalciferol did not significantly affect 65Zn absorption in either group, although the synthesis of the intestinal calcium-binding protein (CaBP) in both groups was similar. In an analogous study, 1,25-dihydroxycholecalciferol increased 47Ca absorption and induced the synthesis of CaBP but exerted on effect on 65Zn absorption in zinc-deficient rachitic chicks. When fed a diet adequate in cholecalciferol, more CaBP was present in the intestine of the zinc-adequate group than in the zinc-deficient group, possibly due to the greater rate of growth and therefore the greater need for calcium by the former group. These results suggest that cholecalciferol and its most active metabolite do not directly affect zinc absorption and, by inference, that the vitamin D-dependent transport mechanism is not involved in zinc homeostasis, or in the interaction between calcium and zinc.

Animals↗
PubMed1979

Analytical peptide mapping by high performance liquid chromatography. Application to intestinal calcium-binding proteins.

Peptide mapping of underivatized tryptic digests of bovine and chick intestinal calcium-binding proteins has been accomplished by high performance liquid chromatography (HPLC). High precision analysis of nanomolar quantities of peptides were achieved in less than 1 h (recycle time). Peak resolution and definition are superior compared to conventional techniques and recoveries of both small (4-residue) hydrophilic and large (30-residue) hydrophobic peptides are excellent. The total amino acid composition of the bovine intestinal calcium-binding protein has been accounted for on the basis of two tryptic maps of 20 microgram of protein each.

Amino Acids↗
PubMed1979

Studies on the subcellular localization of the membrane-bound fraction of intestinal calcium-binding protein.

Sorbitol density gradient centrifugation applied to intestinal mucosa homogenates resulted in a complete separation of soluble calcium-binding protein from the bound fraction of calcium-binding protein, providing further documentation of the bound pool of calcium-binding protein. The peak of the bound calcium-binding protein was not associated with the major peaks of any of the markers used, but was associated with minor peaks of alkaline phosphatase, RNA, and glucose-6-phosphatase. Lack of association of bound calcium-binding protein with (Na+ + K+)-ATPase indicated that the bound calcium-binding protein is not on the basolateral membrane. Differential centrifugation fractionation indicated that the bound calcium-binding protein is not associated with nuclei or mitochondria. The bound calcium-binding protein also could not be detected in partially purified brush borders. Exclusion of the brush border and basolateral membranes as the location of the bound calcium-binding protein suggests an intracellular locale.

Alkaline Phosphatase↗