A calcium-stat method for the measurement of calcium transport rates in isolated sarcoplasmic reticulum vesicles

Calcium transport by isolated sarcoplasmic reticulum vesicles has been measured by means of a calcium-stat method, utilizing a calcium-specific electrode as sensor. Free calcium ion levels were maintained between 10^?7 and 10^?4m during assay, without the use of calcium buffering agents. The method may be used at temperatures between 5 and 40°C and in the pH range 5.0 to 8.5. Measured initial rates of ATP-dependent calcium transport at 10^?5m free calcium, 20°C, pH 7.2, and 100 μg sarcoplasmic reticulum protein per milliliter were between 1.5 and 2.3 μmol min^?1 mg^?1, with a coefficient of variation of 2%.     

Purification of sarcoplasmic reticulum vesicles through their loading with calcium phosphate

A technique of purification of sarcoplasmic reticulum was devised through selective loading of the vesicular material with calcium phosphate. In presence of amount of disposable calcium lower than the maximum accumulation capacity of the total vesicular population, we have defined conditions of loading which allow the selection by centrifugation. The results described in this work show that about 30% of the starting material can be isolated as a vesicular population homogenous on the stand of the amount of accumulated cation. The purification is achieved by the removal of calcium by dissociation of the precipitate. Freeze-fracture electron microscopy studies show that the more active fraction when freed of calcium phosphate precipitate displays smooth convex (EF_s) and particulated concave (PF_p) fracture planes. It has been verified that the purification described in this work allows the removal of all the inactive material. The rate of calcium uptake of the selected preparation is about twice as large as that displayed by the starting material. The structural homogeneity of this material and the increase in the activity are good evidence for the purity of the selected sarcoplasmic reticulum vesicles.     

Ethanol increases calcium permeability of heavy sarcoplasmic reticulum of skeletal muscle

The effects of ethanol on both Ca2+ pump activity and Ca2+-induced Ca2+ release in sarcoplasmic reticulum (SR) of rabbit skeletal muscle were studied. In concentrations of 0.1-1.0%, ethanol conspicuously enhanced Ca2+ release from the heavy fraction of SR, whereas a much smaller effect was noted in the light fraction. When Ca2+-induced Ca2+ release was inhibited by 10 mM Mg2+, the Ca2+ pump activities of both the heavy and light SR were the same; the activities were not significantly influenced by 1% ethanol. Ethanol itself did not release Ca2+ from the heavy SR. However, it appeared to render the heavy SR more permeable to Ca2+, thereby decreasing the amount of storable Ca2+. This action of ethanol may be related to the mechanism of its negative inotropic effect.     

Phospholipid asymmetry in the isolated sarcoplasmic reticulum membrane

The total phospholipid content and distribution of phospholipid species between the outer and inner monolayers of the isolated sarcoplasmic reticulum membrane was measured by phospholipase A2 activities and neutron diffraction. Phospholipase measurements showed that specific phospholipid species were asymmetric in their distribution between the outer and inner monolayers of the sarcoplasmic reticulum lipid bilayer; phosphatidylcholine (PC) was distributed 48/52 +/- 2% between the outer and inner monolayer of the sarcoplasmic reticulum bilayer, 69% of the phosphatidyl-ethanolamine (PE) resided mainly in the outer monolayer of the bilayer, 85% of the phosphatidylserine (PS) and 88% of the phosphatidylinositol (PI) were localized predominantly in the inner monolayer. The total phospholipid distribution determined by these measurements was 48/52 +/- 2% for the outer/inner monolayer of the sarcoplasmic reticulum lipid bilayer. Sarcoplasmic reticulum phospholipids were biosynthetically deuterated and exchanged into isolated vesicles with both a specific lecithin and a general exchange protein. Neutron diffraction measurements directly provided lipid distribution profiles for both PC and the total lipid content in the intact sarcoplasmic reticulum membrane. The outer/inner monolayer distribution for PC was 47/53 +/- 1%, in agreement with phospholipase measurements, while that for the total lipid was 46/54 +/- 1%, similar to the phospholipase measurements. These neutron diffraction results regarding the sarcoplasmic reticulum membrane bilayer were used in model calculations for decomposing the electron-density profile structure (10 A resolution) of isolated sarcoplasmic reticulum previously determined by X-ray diffraction into structures for the separate membrane components. These structure studies showed that the protein profile structure within the membrane lipid bilayer was asymmetric, complementary to the asymmetric lipid structure. Thus, the total phospholipid asymmetry obtained by two independent methods was small but consistent with a complementary asymmetric protein structure, and may be related to the highly vectorial functional properties of the calcium pump ATPase protein in the sarcoplasmic reticulum membrane.     

Ca2+-ATPase from sarcoplasmic reticulum: acylphosphatase activity

Fractionation of sarcoplasmic reticulum vesicles from rabbit skeletal muscle was performed by solubilization of the vesicles in the presence of deoxycholate, followed by sucrose density gradient centrifugation and gel filtration chromatography. This procedure permitted the isolation of essentially pure Ca²⁺-ATPase; this enzyme showed ATPase as well as acylphosphatase activity, both activities being clearly enhanced by deoxycholate. The acylphosphatase activity of the purified Ca²⁺-ATPase was characterized with regard to some kinetic properties, such as pH, Mg²⁺, Ca²⁺, and deoxycholate dependence, and substrate affinity, determined in the presence of acetylphosphate, succinylphosphate, carbamylphosphate, and benzoylphosphate; in addition, the stability of both activities was checked in time-course experiments. The main similarities between the two activities, such as the Mg²⁺ requirement, the deoxycholate activation, and the pH dependence, together with the competitive inhibition of the benzoylphosphatase activity by ATP, the inhibition of both activities by tris(bathophenanthroline)-Fe²⁺, and the relief of this inhibitory effect by carbonylcyanide-4-trifluoromethoxyphenyl hydrazone support the hypothesis that acylphosphatase and ATPase activities of sarcoplasmic reticulum vesicles reside in the same active site of the enzyme. With regard to possible relationships between acylphosphatase activity of the purified Ca²⁺-ATPase and “soluble” acylphosphatase present in the 100,000g supernatant fraction, comparison of some kinetic and structural parameters indicate that these two activities are supported by quite different enzymes.     

The influence of pH on the absorption spectrum of arsenazo III

The absorption spectrum of arsenazo III in media containing K⁺, Mg²⁺ and Ca²⁺ is sharply influenced by pH in the range of 7.5–5.0. The effect of pH is particularly pronounced in the wavelength range 532–602 nm due to the large pH dependence of the dissociation constant of Mg-arsenazo III complex. Therefore absorption changes at these wavelengths during muscle contraction cannot be used as reliable indicators of free ionized Ca²⁺ concentration in the cell. The effect of pH is less pronounced, but still noticeable at the wavelength pairs 575–650 or 660–685 nm.Multiple layers of muscle cells grown on polystyrene coils permit measurement of absorption changes of arsenazo III, introduced into the cells, by equilibration with 0.5 nM arsenazo III under routine culture conditions. The absorbance changes recorded at 660–685 nm are probably related to changes in intracellular free Ca²⁺ concentration.     

Influence of membrane fluidity on transport mediated by ubiquinones through phospholipid vesicles

Coenzymes Q₁₀ and Q₃ are incorporated into dipalmitoylphosphatidylcholine and egg yolk lecithin liposomes. Dithionite reduction of ferricyanide trapped inside these phospholipid vesicles is taken as a measure of ubiquinone-mediated transport of reducing equivalents. The reaction shows complex pattern with a high order for CoQ. The initial transport rates are very sensitive to the membrane physical state, being considerably reduced at temperatures below the phase transition of the pure dipalmitoylphosphatidylcholine, both for CoQ₁₀ and CoQ₃ reconstituted with this phospholipid. It is suggested that a different reaction mechanism operates in fluid and rigid membranes. This suggestion is related to the possible organization of CoQs in phospholipid membranes.     

The activation of phospholipase C from Clostridium Welchii by quinine: an absolute requirement for calcium ions.

Quinine activates the hydrolysis of phosphatidyl choline suspensions by phospholipase C (E.C. 3.1.4.3) obtained from Clostridium welchii. Low levels of calcium are an absolute requirement for this activation: Mg2+, Ba2+, Sr2+, and Zn2+ are ineffective. The induction period, or lag phase for this enzyme is dependent upon both calcium concentration and substrate interfacial surface area. At low concentrations (less then 50 muM) calcium ions affect the induction period but not the maximal rate of hydrolysis, whereas guinine predominantly affects the rate of hydrolysis by alterations in the surface charge carried by the substrate.     

Attempts to induce asymmetrical reconstitution of the sarcoplasmic reticulum calcium transporting system

The main purpose of this work was to explore the conditions allowing the most efficient reconstitution of the sarcoplasmic reticulum calcium transporting system, displaying the same morphology as in the corresponding native membranes. The method used was based on the centrifugation of the solubilized-in-Triton X-100 Ca²⁺-stimulated adenosine triphosphatase through a sucrose gradient containing Tween 80 and preformed small lipid vesicles. The morphology of the reconstituted material was followed by freeze-fracture electron microscopy. The results presented in this paper show that, under appropriate experimental conditions, a large part of the reconstituted material appears to be very similar to the native sarcoplasmic reticulum.     

1,25-Dihydroxyvitamin D3 metabolism. The effect of dietary calcium and phosphorus

Rats maintained on tritiated 1,25-dihydroxyvitamin D₃ as their sole source of vitamin D and placed on diets differing in calcium content had similar intestinal levels of tritiated 1,25-dihydroxyvitamin D₃. Since 1,25-dihydroxyvitamin D₃ administration eliminated adaptation of intestinal calcium transport, it appears that increased production of 1,25-dihydroxyritamin D₃ is responsible for the stimulation of calcium transport by low dietary calcium. When maintained on tritiated 1,25-dihydroxyvitamin D₃, rats fed a low-phosphorus diet had somewhat higher levels of tritiated 1,25-dihydroxyvitamin D₃ in the duodenum and plasma than rats on a normal-phosphorus diet. In addition to stimulating 1,25-dihydroxyvitamin D₃ synthesis, low dietary phosphorus may increase the accumulation of 1,25-dihydroxyvitamin D₃ in both intestine and plasma.     

Sea urchin egg cortical granule exocytosis is followed by a burst of membrane retrieval via uptake into coated vesicles

Using improved fixation procedures we have found that extensive endocytotic activity is turned on at fertilization in eggs of three species of sea urchins. Beginning after completion of cortical granule exocytosis and after exocytotic pits have completely smoothed over, the entire activated egg surface engages in a limited period of extensive removal of membrane via uptake into coated vesicles. This “burst phase” lasts about 3–5 min after which the number of invaginating coated vesicles decreases rapidly. At the end of this burst phase all the patches of cortical granule membranes have disappeared, and the egg surface is left uniformly covered by microvilli. For the remainder of the first cell cycle coated pits continue to form at a slower but steady rate. Endocytotic activity continues past the time of first cleavage. There is distinct overlap in onset and duration of the burst phase of endocytosis with the period of medium acidification during normal development. However, activation of eggs in choline sea water, which inhibits acid secretion, results in an endocytic burst whose timing and duration are similar to those in normal eggs. The endocytic burst is, therefore, independent of cytoplasmic alkalinization. These results suggest, in accord with the two-step model of egg activation (D. Epel, R. A. Steinhardt, and R. A. Humphreys, 1974; Dev. Biol.40, 245–255; D. Epel, 1978, Curr. Top. Dev. Biol.12, 185–246) that initiation of endocytosis is most likely a Ca²⁺-dependent event.     

Structure of the vanadate-induced crystals of sarcoplasmic reticulum Ca2+-ATPase

The projected structure of the vanadate-induced crystalline aggregates of Ca2+-ATPase molecules in isolated sarcoplasmic reticulum membranes has been determined. The molecules form tubular crystals with an oblique surface lattice having cell dimensions a = 65.9 A, b = 114.4 A and gamma = 77.9 degrees. The space group is P2. The crystalline tubules are formed through lateral aggregation of chains made up of dimers of Ca2+-ATPase molecules.     

Manganese, calcium, and saccharide binding to concanavalin A, as studied by ultrafiltration

The binding of the ligands Mn2+, Ca2+, and methyl alpha-D-glucopyranoside to concanavalin A, purified as described (A.J. Sophianopoulos and J.A. Sophianopoulos (1981) Prep. Biochem. 11, 413-435), was studied by ultrafiltration in 0.2 M NaCl, pH 5.2 and pH 6.5 to 7, and at 23 to 25 degrees C. The association constant (Ka) of methyl alpha-D-glucopyranoside to concanavalin A was (2 +/- 0.2) X 10(3) M-1, both at pH 5.2 and 7. At pH 5.2 and in the absence of Ca2+, the Ka of Mn2+ to concanavalin A was (5 +/- 1) X 10(3) M-1, and in the presence of 1 mM Ca2+, the Ka was (9.1 +/- 2.1) X 10(5) M-1. At pH 6.5 Mn2+ bound to concanavalin A with a Ka of (7.3 +/- 1.8) X 10(5) M-1, and the binding affinity was virtually independent of the presence of Ca2+. Experiments of binding of 4-methylumbelliferyl alpha-D-mannopyranoside to concanavalin A indicated that at pH 5.2, binding of a single Mn2+ per concanavalin A monomer was sufficient to induce a fully active saccharide binding site. Ca2+ is not necessary for such activation, but rather it increases the affinity of concanavalin A for binding Mn2+.     

Differences in the cation sensitivity of adenylate cyclase from heart and skeletal muscle: modification by guanyl nucleotides and isoproterenol

Low concentrations of Mn²⁺ supported the basal adenylate cyclase activity in crude and purified sarcolemmal membranes from cardiac muscle more effectively than did relatively high concentrations of Mg²⁺; at saturating concentrations the cyclase activities obtained with Mg²⁺ or Mn²⁺ were similar. In contrast, Mg²⁺ supported the basal cyclase activities of crude membrane fractions and purified sarcolemmal membranes from skeletal muscle far more effectively than did Mn²⁺; at saturating concentrations of either metal ion the Mg²⁺-supported cyclase activities were 5- to 10-fold greater than Mn²⁺-supported activities. Further, compared to Mg²⁺, Mn²⁺ supported the cyclase activities very poorly in all the primary subcellular fractions of skeletal muscle, whereas this cation was at least as effective as Mg²⁺ in all fractions of cardiac muscle. The apparent affinities of the cyclase for Mn²⁺ in heart as well as skeletal muscle appeared to be greater compared to those for Mg²⁺. The skeletal muscle cyclase displayed greater apparent affinity for MnATP^2? (app. K_m 0.10 mm) compared to MgATP^2? (app. K_m 0.32 mm) whereas the heart enzyme displayed greater apparent affinity for MgATP^2? (app. K_m 0.07 mm) compared to MnATP^2? (app. K_m 0.19 mm). Following preactivation with guanyl-5′-yl imidodiphosphate and isoproterenol, Mn²⁺ (0.15 to 2 mm) supported the cyclase activity of skeletal muscle even more effectively than did optimally effective concentrations of Mg²⁺. With the heart enzyme the relatively greater potency of Mn²⁺ persisted following preactivation. Significant enhancement in the Mn²⁺-sensitivity of skeletal muscle cyclase was also observed when assayed in the presence of GTP and isoproterenol or in the presence of NaF. Preactivation of both heart and skeletal muscle cyclases caused selective enhancement in the enzyme's apparent affinity for free Me²⁺ (Mg²⁺ or Mn²⁺) without influencing the apparent K_m for MeATP^2? (MgATP^2? or MnATP^2?). Evidences were obtained to show that the poor effectiveness of Mn²⁺ in supporting the basal activity of skeletal muscle cyclase is not related to (a) potentiation by Mn²⁺ of adenosine-mediated inhibition of the cyclase, (b) Mn²⁺-induced lability of the cyclase, (c) indirect effects of Mn²⁺ on ATP-regenerating system, or (d) the presence of different cation-specific molecular forms of the cyclase. It is also shown that the onset of enhanced Mn²⁺ sensitivity of the skeletal muscle enzyme following preactivation is not accompanied by a general loss of cation specificity of the cyclase. These results suggest that cations support the catalytic activity of adenylate cyclase by interacting with an enzymeregulatory free metal binding site and that the differential cation sensitivity of nonactivated (basal) cyclases from heart and skeletal muscle is likely due to differences in the properties of such an allosteric metal site. Furthermore, the metal site appears to undergo a conformational change following interaction of the cyclase system with the guanyl nucleotide and isoproterenol since the cation sensitivity of the cyclase and the relative potency of cations depend on the conformational status of the enzyme.     

Properties of human neutral bronchial mucins after modification of the peptide or the carbohydrate moieties.

Trypsin and pronase treatment of purified human neutral bronchial mucins released small fragments from the C-terminal end of these molecules and resulted in slight increases in their sedimentation coefficient presumably reflecting conformational changes. The antigenic determinant of neutral bronchial mucins which appears to be located on this C-terminal fragment is destroyed by pronase or by treatments such as periodate oxidation or galactose oxidase-bromine oxidation which modify the carbohydrate moieties. Thus, both amino acid and carbohydrate residues are involved in the structure of the antigenic determinant.     

Effects of several calcium antagonists and dipyridamole in the isolated perfused guinea pig heart

Three calcium (Ca) antagonists and dipyridamole were examined in the isolated perfused guinea pig heart at perfusate Ca concentrations of 1.25 and 3.75 mM. The Ca antagonists: FR 7534, nifedipine and D600 produced similar dose-dependent decreases in left ventricular dp/dt and myocardial oxygen consumption (MV?O₂) at both Ca concentrations. However, dose response curves were shifted significantly to the right by increased perfusate Ca requiring six to ten times more Ca antagonist to produce equivalent effects. Dipyridamole produced only slight negative inotropic effects which appeared to be less dependent on external Ca concentration. All four agents significantly increased coronary blood flow at 1.25 mM Ca but not at 3.75 mM Ca. The Ca antagonists decreased heart rate at 3.75 mM Ca whereas dipyridamole had strong negative chronotropic effects at both perfusate Ca concentrations. These experiments provide evidence that FR 7534 acts as a Ca antagonist. In addition, Ca antagonists of different structure had similar effects on the isolated heart distinct from those of dipyridamole.     

O-methylhydroxylamine as a reagent for NAD+ modification in urocanase.

The inhibition of urocanase from Pseudomonas putida by O-methylhydroxylamine has been characterized as being due to the formation of an adduct between CH₃ONH₂ and NAD⁺, the latter of which has been recently shown to be a tightly bound coenzyme for this urocanase. Inhibition is maximal at pH 8.5 and is blocked by the presence of the substrate analog imidazole propionate. Loss of catalytic activity corresponds directly with the binding of 1 mol of ¹⁴CH₃ONH₂ per mole of enzyme, and partial reversibility of the modification, achieved by dialysis at pH 7.5, is accompanied by concomitant restoration of enzymatic activity. No incorporation of ¹⁴CH₃ONH₂ into urocanase is seen when enzyme-bound NAD⁺ is first converted to NADH or when NAD⁺ is removed by prior treatment of urocanase with 8 m urea. Stability and spectral properties of the CH₃ONH · NAD adduct are consistent with previous data reported for the product of the hydroxylamine reaction with NAD⁺. It is concluded that other urocanases which exhibit inhibition by hydroxylamine may likewise contain NAD⁺ as an essential coenzyme and that the use of ¹⁴CH₃ONH₂ as a reversible modification reagent for NAD⁺ should prove helpful for studies on the role of NAD⁺ in the urocanase catalytic process.     

Sedimentation characteristics of subcellular vesicles associated with internalized insulin and those bound with intracellular glucose transport activity

Comparative studies were made on the sedimentation characteristics of microsomal vesicles associated with internalized [¹²⁵I]iodoinsulin and those bound with intracellular glucose transport activity. Upon linear sucrose density gradient centrifugation, the internalized hormone formed a peak slightly, but significantly, on the higher density side of the peak of intracellular glucose transport activity. After a long centrifugation, the peak of ¹²⁵I activity became lower and broader than that of glucose transport activity. Internalized ¹²⁵I activity was also found in the medium-density microsomal fraction, which had little glucose transport activity. Accumulation of ¹²⁵I activity in the medium-density fraction and that in the low-density fraction were both completed in approximately 10 min. Under basal conditions, little, if any, insulin binding activity was detectable in either the medium- or low-density microsomal fractions; in contrast, some glucose transport activity was always present in the low-density fraction. These results indicate that the subcellular distribution of internalized insulin and of intracellular glucose transport activity are different, suggesting that the pathways of intracellular processing of the insulin receptor and the glucose transport mechanism are different.     

Adaptation of intestinal calcium absorption: parathyroid hormone and vitamin D metabolism.

It has already been demonstrated that the adaptation of intestinal calcium absorption of rats on a low calcium diet can be eliminated by thyroparathyroidectomy plus parathyroid hormone administration. This treatment elevates intestinal and plasma levels of 1,25-dihydroxyvitamin D₃ in rats on a high calcium diet while producing no change in rats on a low calcium diet. It therefore appears likely that the modulation of intestinal calcium absorption by dietary calcium is mediated by the parathyroid glands and the renal biogenesis of 1,25-dihydroxyvitamin D₃. Changes in the other unknown vitamin D metabolite levels as a result of dietary calcium are also modified by thyroparathyroidectomy and parathyroid hormone administration, but the effect of these metabolites on intestinal calcium transport is unknown.     

Chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum

The chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum has been examined relative to enzymatic activity and reactivity of these groups in the native protein. 4,4′-Dipyridyl disulfide, dansylaziridine, and fluorescein mercuric acetate all reacted with just one of six sulfhydryls per enzyme subunit, resulting in activities of 100, 95 and 70%, respectively. The K_m values for MgATP, formate, and tetrahydrofolate were unaltered in the modified enzymes. ATP did produce a 2.5-fold reduction in the rate of reaction between the enzyme and 4,4′-dipyridyl disulfide. Tetranitromethane reacted most rapidly with a single sulfhydryl group per subunit to produce a 20–30% loss in activity. Subsequent additions of tetranitromethane modified 2.2 tyrosines per subunit which was proportional to the loss of the remaining enzymatic activity. Folic acid, a competitive inhibitor, protected against modification of the tyrosines and the associated activity losses; however, the oxidation of the single sulfhydryl group and the initial 20–30% activity loss were unaffected. In the presence of folic acid, higher concentrations of tetranitromethane produced a loss of the remaining activity proportional to the modification of 1.2 tyrosines per subunit. It is proposed that at least 1 tyrosine critical for enzymatic activity is located at or near the folic acid/tetrahydrofolate binding site.