Mol Of Calcium Research Articles

Preparation and properties of carp muscle parvalbumin fragments A ( [formula omitted]) and B ( [formula omitted

The calcium-binding protein (parvalbumin), isolated from carp (Cyprinus carpio) muscle, has been specifically fragmented into two polypeptides by tryptic hydrolysis at the single arginine residue at position 75. Fragment A contains residues 1 a ̊ 75 and fragment B is composed of residues 76 a ̊ 108 . The fragments have been characterized according to size, amino acid composition, carboxyl- and amino-terminal analysis. Both fragments appear to be homogeneous by these criteria. The intact protein is known to bind 2 mol of calcium per mol of parvalbumin, and although each fragment alone contains all of the essential ligands for the coordination of one Ca 2−, neither fragment displays calcium binding activity. Attempts to reconstitute the two fragments, under a variety of conditions, into a functional complex which can bind calcium have been unsuccessful. The side chain of Arg-75 is known to occupy an internal position in the crystalline structure of parvalbumin (Kretsinger, R.H. and Nockolds, C.E. (1973) J. Biol. Chem. 248, 3313), where it is stabilized by an intricate network of hydrogen bonding involving the side chain of Glu-81. Although this internal salt bridge is approx. 20 Å from either calcium binding site, it has been suggested that this structural feature of the molecule plays an essential role in the reversible binding of Ca 2+. That the side chain of Arg-75 likewise occupies an internal position in the solution structure is indicated by its unavailability for reaction with 1,2-cyclohexanedione under conditions of physiological pH and temperature. However in the presence of EDTA and at pH 8, it is readily modified by cyclohexanedione. This modification is accompanied by a concomitant loss in calcium binding activity. Reversal of the modification by treatment with hydroxylamine is accompanied by restoration of calcium binding activity. The sum of these data support the hypothesis that Arg-75 plays a critical role in the structural organization and calcium binding activity of the molecule, and in addition, suggests that the integrity of the peptide bond between Arg-75 and Ala-76 may be necessary for establishing the proper microenvironment required for formation of the internal salt bridge between Arg-75 and Glu-81.

Purification and characterization of a troponin-C-like protein from bovine adrenal medulla.

A low molecular weight protein found in the soluble extract of bovine adrenal medulla, and having a high affinity for calcium ions has been purified to apparent homogeneity. The purification requires three steps, including ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose, and gel filtration on Sephadex G-100. The protein was homogeneous by the criteria of both standard and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, sedimentation velocity analysis, and NH2-terminal analysis. The calcium-binding protein is very acidic and has an isoelectric point of 4.27. Aspartic and glutamic acid together account for 30% of the total amino acid composition. The ultraviolet absorption spectrum reveals a A280/A260 ratio of 0.83 and shows discrete maxima at 258, 264, 269, 278, and 284 nm. Two moles of calcium are bound per mole of protein. The apparent Kp is approximately 20 muM. The molecular weight was found to be 16,000 +/- 1,000 by both sodium dodecyl sulfate gel electrophoresis and sedimentation equilibrium ultracentrifugation. The protein was found to consist of a single polypeptide chain by the criteria of tryptic peptide mapping and NH2-terminal analysis. Amino acid analysis revealed the absence of tryptophan, single residues of cysteine and histidine, and 2 residues of tyrosine. The protein was void of carbohydrate and phosphate. The structural similarities and possible functional correlation between adrenal medulla calcium-binding protein and troponin-C from muscle tissue are discussed.

The Stimulation of Calcium Transport in Cardiac Sarcoplasmic Reticulum by Adenosine 3′:5′-Monophosphate-dependent Protein Kinase

Abstract The effects of an adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase on Ca2+-activated adenosine triphosphatase (ATPase) activity and calcium uptake of canine cardiac microsomes were examined. The cardiac microsomes, which represent an enriched preparation of fragmented sarcoplasmic reticulum, were preincubated with varying concentrations of cyclic AMP-dependent protein kinase or cyclic AMP or both in the presence of Mg2+ and ATP. Ca2+-activated ATPase activity and calcium uptake were determined in the presence of oxalate and various concentrations of Ca2+. Ionized Ca2+ concentrations were maintained with Ca2+ buffers containing CaCl2 and ethylene glycol bis (β-aminoethyl ether)-N,N'-tetraacetic acid. The initial rates of both Ca2+-activated ATPase and calcium uptake were increased 2 to 3 times following 10-min preincubation with protein kinase and 1 µm cyclic AMP. Pretreatment with protein kinase in the absence of cyclic AMP caused a lesser degree of stimulation, whereas pretreatment with cyclic AMP alone had no detectable effect. Stimulation of the Ca2+-activated ATPase and calcium uptake by protein kinase was dependent on cyclic AMP concentration: maximal stimulation was seen at approximately 10-6 m with apparent half-maximal stimulation at approximately 10-7 m. Marked stimulation of both Ca2+-activated ATPase activity and calcium uptake by cyclic AMP and protein kinase was seen at a Ca2+ concentration of approximately 1 µm. The stoichiometric coupling of Ca2+-activated ATPase and calcium uptake was maintained at 2 moles of calcium taken up per mole of ATP hydrolyzed, following stimulation by protein kinase and cyclic AMP. The stimulatory effects on Ca2+-activated ATPase and calcium uptake of pretreatment with protein kinase and cyclic AMP could be shown after microsomes were washed with buffered 50 mm KCl. These findings indicate that protein kinase can increase the rate of calcium transport by the cardiac sarcoplasmic reticulum without altering the efficiency of the calcium pump. This effect may account for the abbreviation of systole that is caused by agents, like epinephrine, which increase cyclic AMP production, and resulting alterations in the distribution of Ca2+ within the myocardial cell may be responsible, at least in part, for the augmentation of myocardial contractility.

Purification of a Calcium-binding Phosphoprotein from Beef Adrenal Medulla: IDENTITY WITH ONE OF TWO CALCIUM-BINDING PROTEINS OF BRAIN

Abstract An acidic calcium-binding phosphoprotein has been isolated from beef adrenal medulla and purified to apparent homogeneity. The purification procedure employed ammonium sulfate fractionation, DEAE-cellulose chromatography, and gel filtration on Sephadex G-100 and G-50. This protein, which appears to be the only soluble calcium-binding protein in adrenal medulla, shows high affinity calcium binding (Kd = 1.7 x 10-5 m) and binds 1 mole of calcium per mole of protein. Each mole of calcium-binding protein contains 1 mole of phosphate. The molecular weight was determined to be 11,900 by equilibrium ultracentrifugation. An acidic calcium-binding phosphoprotein was also isolated from beef brain and compared with the protein from beef adrenal medulla. The two proteins appear to be identical on the basis of electrophoretic migration in 15% acrylamide gels and 10% acrylamide gels containing sodium dodecyl sulfate, molecular weight, amino acid composition, and peptide maps. The protein is not found in other tissues including liver, kidney, lung, and parotid gland. It is suggested that this calcium-binding protein of brain may be associated with adrenergic neurons.

Purification of a Calcium-binding Phosphoprotein from Pig Brain

Abstract An acidic phosphoprotein has been isolated from adult pig brain and purified to apparent homogeneity. The purification procedure employed ammonium sulfate precipitation, ECTEOLA - cellulose chromatography, gel filtration on Sephadex G-75, and chromatography on hydroxylapatite. This protein exhibits high affinity calcium binding (Kd = 2.5 x 10-5 m), binds 1 mole of calcium per mole of protein, and is one of two high affinity calcium-binding proteins found in pig brain. This calcium-binding protein is not present in either liver or kidney, suggesting that it may play a role in calcium function within nervous tissue. The calcium binding properties, phosphorus content and amino acid composition of the protein have been determined. The molecular weight of the protein, as determined by ultracentrifugation, is 11,500. The protein aggregates in the presence of calcium with the formation of multimers. Treatment with phosphatase results in loss of calcium binding activity, suggesting that calcium binds to the phosphate group of the protein. Each mole of protein contains 10 moles of phosphate.

The calcium binding characteristics of a salivary gland mucoid

The binding of calcium by a mucoid isolated from the submaxillary salivary glands of cattle was studied by use of the Toribara ultrafiltration apparatus. In general, the results showed that the amount of calcium bound by a given weight of mucoid remained constant over a range of mucoid concentration from 0 to 1.25%, but increased with the pH and with the calcium concentration, at least up to a calcium concentration of approximately 10 mM/l. Above this point the amount of bound calcium remained constant, indicating that maximum binding had occurred. Based on the molecular weight of the mucoid it is estimated that at saturation each mole of mucoid is able to bind 10.8 moles of calcium at pH 6, 15.9 moles at pH 7, 22.0 moles at pH 8, and 35.4 moles at pH 9.

Ultraviolet Phosphors and Fluorescent Sun Tan Lamps

An investigation was undertaken of phosphors capable of converting primary ultraviolet radiation (Hg line, 2537 angstroms) to radiation in the erythemal or sun tan ultraviolet region (2900–3200 angstroms). Several types of alkaline earth silicates and phosphates activated with lead or thallium were found to be practical. Among these was a calcium phosphate activated with thallium and containing about 1.3 moles of calcium per mole of phosphate. This phosphor not only showed a good initial erythemal emission, but when used in an experimental fluorescent lamp maintained a high percentage of this emission for the several hundred hours tested. The lamp was constructed of special glass and coated with the above phosphor, but otherwise was similar to commercial fluorescent lamps. In addition, the investigation showed some similar types of phosphors to be efficient in the neighboring “black-light” ultraviolet region (approximately 3400–3800 angstroms), and to give more emission in that region than the cerium-activated phosphors commonly used at present. Some discussion of sun tan lamp and fixture design is also included.