Effects of citrated whole blood transfusion in response to hemorrhage.

BACKGROUND AND PURPOSE: Standard treatment for massive hemorrhage in dogs is infusion of whole blood or of packed red blood cells with fresh frozen plasma if whole blood is not available. Although most whole blood is collected using a citrate-based anticoagulant, knowledge of citrate's relevant non-anticoagulant effects is not widespread. Citrate's anticoagulant activity is achieved through chelation of divalent metal cations (e.g., magnesium, calcium), which may exacerbate cardiovascular and metabolic insults attributable to hemorrhage. METHODS: Blood pressures, gas tensions, metabolites, and electrolytes; myocardial metabolites, pressures, and contractility; cardiac output; and left cranial descending and circumflex coronary artery flows were measured in 21 anesthetized dogs after hemorrhage was induced by collection of blood into a citrated reservoir to mean arterial pressure of 45 mm Hg for approximately 60 min (until arterial lactate concentration was 7.0 mmol/L), followed by a 1-h transfusion and 2 h of maintenance. RESULTS: Arterial ionized calcium concentration, total peripheral resistance, and myocardial function decreased significantly during hemorrhage. All aforementioned responses but myocardial function continued to decrease during the initial 20 min of transfusion, then began to recover. Total peripheral resistance and end-systolic elastance were the only factors significantly related to calcium concentration. CONCLUSION: Transfusion with citrated whole blood may significantly alter calcium concentration, negatively affecting myocardial and vascular function.     

Effects of Chemical Speciation on the Mineralization of Organic Compounds by Microorganisms

The mineralization of 1.0 to 100 ng each of four complexing compounds—oxalate, citrate, nitrilotriacetate (NTA), and EDTA—per ml was tested in media prepared in accordance with equilibrium calculations by a computer program so that the H, Ca, Mg, Fe, or Al complex (chemical species) was predominant. Sewage microorganisms mineralized calcium citrate more rapidly than iron, aluminum, or hydrogen citrate, and magnesium citrate was degraded slowest. Aluminum, hydrogen, and iron oxalates were mineralized more rapidly than calcium oxalate, and magnesium oxalate was decomposed slowest. Sewage microorganisms mineralized calcium NTA but not aluminum, magnesium, hydrogen, or iron NTA or any of the EDTA complexes. Pseudomonas sp. mineralized calcium and iron citrates but had no activity on hydrogen, aluminum, or magnesium citrate. Pseudomonas pseudoalcaligenes mineralized calcium, iron, hydrogen, and aluminum citrates but had little activity on magnesium citrate. Pseudomonas alcaligenes used calcium, iron, hydrogen, and aluminum oxalates readily, but it used magnesium oxalate at a slower rate. Listeria sp. destroyed calcium NTA but had no effect on hydrogen, iron, or magnesium NTA. Increasing the Ca concentration in the medium enhanced the breakdown of NTA by Listeria sp. The different activities of the bacterial isolates were not a result of the toxicity of the complexes or the lack of availability of a nutrient element. NTA mineralization was not enhanced by the addition of Ca to Beebe Lake water, but it was enhanced when Ca and an NTA-degrading inoculum were added to water from an oligotrophic lake. The data show that chemical speciation influences the mineralization of organic compounds by naturally occurring microbial communities and by individual bacterial populations.     

Identifying the Ca(++) signalling sources activating chloride currents in Xenopus oocytes using ionomycin and thapsigargin

The calcium ionophore, ionomycin (IM), and the sarcoplasmic/endoplasmic reticulum (SER) calcium pump inhibitor, thapsigargin (TG), were used to study the roles of Ca(++) from different sources in regulating Ca(++)-dependent Cl(-) currents in Xenopus oocytes. The Ca(++)-dependent Cl(-) currents, Ic, were measured in voltage-clamped oocytes (Vc = -60 mV). In the presence of extracellular Ca(++), both TG (0.1 to 10 microM) and IM (0.1 to 10 microM) induce release of Ca(++) from SER and activated capacitative Ca(++) entry (CCE) across the plasma membrane leading to activation of both "fast" and "slow" Cl(-) currents. The fast Ic was produced by Ca(++) release from SER while Ca(++) entry across the plasma membrane activated the slow Ic. Intracellular application of the calcium buffer, BAPTA, blocked activation of the slow Ic due to Ca(++) entry via CCE pathways, but not via IM-mediated movement across the plasma membrane. It is concluded that predominantly Ca(++) release from stores regulates a fast Ic while Ca(++) entry through CCE pathways regulates a slow Ic. Further, the CCE and slow Ic pathways must be located in spatially separated compartments since BAPTA can effectively abolish the effects of Ca(++) entry via the CCE pathway, but not by the IM-mediated entry pathway.     

High dietary sodium chloride and body temperature in the domestic fowl and the Glaucous-winged gull

Arad and Skadhauge (1986) correlated plasma sodium to calcium ratio and body temperature in domestic fowl (Gallus domesticus) during increased dietary sodium chloride intake which increased plasma sodium concentration. During acclimation to high dietary NaCl, body temperature should increase in proportion to the increase in plasma sodium concentration, and body temperature should increase less in gulls than in chickens because salt gland secretion of NaCl by gulls should prevent elevation of plasma sodium concentration. Plasma osmolality, plasma sodium concentration, plasma concentrations of total calcium and ionized calcium, and body temperature and panting threshold were measured in domestic roosters and Glaucous-winged gulls before and after exposure to high NaCl diets. Gull body temperature (40.4±0.2 °C) increased significantly (PM0.05) during salt acclimation. Rooster body temperature (41.0±0.2 °C) did not increase significantly. Plasma sodium concentration increased in gulls (5.4±0.5%, P<0.01) and was correlated with body temperature (r=0.497, P<0.05); the 3.8±1.0% increase in plasma sodium concentration in roosters (P<0.01) was not, suggesting that change in body temperature might be a response to the magnitude of increase in plasma sodium concentration. Plasma ionized calcium concentration increased by 12.9±4.6% (P<0.01) in gulls and by 5.3±1.0% (P<0.01) in roosters. Plasma sodium concentration was correlated with calcium ion concentration in both gulls (r=0.635, P<0.05) and roosters (r=0.664, P<0.05). In neither species were ratios of sodium to total calcium plasma concentration or sodium to ionized calcium concentration altered or related to body temperature. Panting threshold increased significantly in roosters following salt acclimation, but not in gulls due to high variability in response. The increase in gull plasma sodium concentration was small compared to previously reported (Saxena 1976; Denbow and Edens 1980, 1981; Maki et al. 1988) increases in hypothalamic and intraventricular sodium concentration following infusion of Na⁺, yet the effect on body temperature was similar in both types of studies. This suggests that sodium may have peripheral effects that augment the central effects imposed by altered hypothalamic interstitial sodium and calcium concentration.Abbreviations [Ca]_p1 total calcium concentration in plasma - [Ca²⁺] ionized calcium concentration in plasma - [Cl]_p1 chloride concentration in plasma - f respiratory frequency - Hct hematocrit - [K]_p1 potassium concentration in plasma - [Na]_p1 sodium concentration in plasma - osm_p1 plasma osmolality - PT panting threshold - T _a ambient temperature - T _b body temperature - V _t tidal volume     

Characterization and Distribution of Transferrin Receptors in the Rat Brain

The mechanism of calcium transport across the plasma membrane of chromaffin cells was studied using plasma membrane vesicles prepared from cells of adrenal medulla. Purification of the plasma membrane was about 30-fold, based on the alpha-bungarotoxin binding activity. The isolated membrane vesicles have both Na+/Ca2+ exchange and calcium pump activities. The Na+/Ca2+ exchange activity increased with the free calcium concentration and was not saturated at 1 mM, the highest concentration tried. The K1/2 of the calcium pump for calcium is 0.06 microM. Part of the Na+/Ca2+ exchange activity was inhibited by preincubation of the membrane vesicles with veratridine and the effect of veratridine was reversed by tetrodotoxin. The calcium taken up by the calcium pump was released by 0.005% saponin, but was not affected by oxalate. The calcium taken up by the calcium pump was released by exchanging with the external sodium, which suggests that the two calcium transport systems are located on the same population of membrane vesicles. The above evidence indicates that both calcium transport activities are located on the plasma membrane and not on contaminating organelle membranes. The significance of the two calcium transport systems in regulation of cytosolic calcium concentration of chromaffin cells is discussed.     

The effects of autolysis on the structure of chicken calpain II.

Heparin catalyses the inhibition of two key enzymes of blood coagulation, namely Factor Xa and thrombin, by enhancing the antiproteinase activities of plasma antithrombin III and heparin cofactor II. In addition, heparin can directly inhibit the activation of Factor X and prothrombin. The contributions of each of these effects to the anticoagulant activity of heparin have not been delineated. We therefore performed experiments to assess how each of these effects of heparin contributes to its anticoagulant activity by comparing the effects of heparin, pentosan polysulphate and D-Phe-Pro-Arg-CH2Cl on the intrinsic pathway of coagulation. Unlike heparin, pentosan polysulphate catalyses only the inhibition of thrombin by plasma. D-Phe-Pro-Arg-CH2Cl is rapid enough an inhibitor of thrombin so that when added to plasma no complexes of thrombin with its inhibitors are formed, whether or not the plasma also contains heparin. Heparin (0.66 microgram/ml) and pentosan polysulphate (6.6 micrograms/ml) completely inhibited the intrinsic-pathway activation of 125I-prothrombin to 125I-prothrombin fragment 1 + 2 and 125I-thrombin. On the addition of thrombin, a good Factor V activator, to the plasma before each sulphated polysaccharide, the inhibition of prothrombin activation was demonstrable only in the presence of higher concentrations of the sulphated polysaccharide. D-Phe-Pro-Arg-CH2Cl also completely inhibited the intrinsic-pathway activation of prothrombin in normal plasma. The inhibitory effect of D-Phe-Pro-Arg-CH2Cl was reversed if thrombin was added to the plasma before D-Phe-Pro-Arg-CH2Cl. The inhibition of the activation of prothrombin by the three agents was also abolished with longer times with re-added Ca2+. Reversal of the inhibitory effects of heparin and pentosan polysulphate was associated with the accelerated formation of 125I-thrombin-antithrombin III and 125I-thrombin-heparin cofactor complexes respectively. These results suggest that the anticoagulant effects of heparin and pentosan polysulphate are mediated primarily by their ability to inhibit the thrombin-dependent activation of Factor V, thereby inhibiting the formation of prothrombinase complex, the physiological activator of prothrombin.     

Purification and characterization of a prothrombin activator from the venom of the Australian brown snake, Pseudonaja textilis textilis

A simple procedure, involving chromatography on concanavalin A-Sepharose and gel filtration, has been developed for the purification of a prothrombin activator from the venom of the Australian brown snake Pseudonaja textilis textilis. The prothrombin activator, which is a major venom component, is a high molecular weight protein (Mr greater than or equal to 200,000) which yields a number of subunits when examined by SDS-PAGE. It is related antigenically to the venom prothrombin activator of the taipan Oxyuranus scutellatus. P. textilis prothrombin activator is able to coagulate citrated plasma, warfarin plasma, and Factor V- and Factor X-deficient plasmas; to convert purified human prothrombin to thrombin; and to hydrolyse the peptide p-nitroanilide substrate S-2222. Calcium ions and phospholipids had little if any effect on the rates of coagulation of citrated plasma or S-2222 hydrolysis catalysed by this enzyme.     

Transplacental 45Ca and 32P flux in the guinea pig: effect of maternal hypercalcaemia and hypocalcaemia

Transplacental 45Ca and 32P flux was measured across the in situ perfused guinea-pig placenta under conditions of acute maternal hypocalcaemia and hypercalcaemia. Maternal hypercalcaemia induced acutely by calcium gluconate infusion caused an increase in maternal-to-fetal 45Ca flux which was proportional to the increase in maternal plasma ionized calcium concentration. Acute maternal hypocalcaemia was induced by EGTA infusion and resulted in a decrease in maternal plasma ionized calcium concentration proportional to a corresponding decrease in transplacental 45Ca transfer. A bolus of calcium gluconate caused a transient decrease in 32P flux, whereas EGTA administration was without significant effect on transplacental 32P transfer. Calcium transport across the placenta is not saturated under conditions of maternal normocalcaemia and may be altered according to acute changes in maternal plasma calcium concentration. Thus, control of maternal-to-fetal calcium transfer does not appear to be at the placental level. This suggests that fetal calcium homeostasis may be regulated by the fetus itself.     

Isolation and characterization of an anticoagulant protein from human placenta

An anticoagulant protein was purified from the EDTA extract of human placental tissue. The purified protein had a molecular weight of 73,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis under both reducing and non-reducing conditions. Because this protein had the ability to bind phospholipids such as phosphatidylserine, phosphatidylinositol, and cardiolipin in the presence of Ca2+, this protein was designated as calphobindin II (CPB-II). CPB-II prolonged the clotting time of normal plasma when coagulation was induced by tissue factor, cephalin and ellagic acid or recalcification, but did not affect thrombin-initiated fibrin formation. CPB-II also inhibited the activation of prothrombin by the complete prothrombinase complex or factor Xa-phospholipid-Ca2+ but not that by phospholipid-free factor Xa. In addition, CPB-II had an inhibitory activity against phospholipase A2.     

Human placental anticoagulant protein: isolation and characterization

An anticoagulant protein was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephadex G-75, and Mono S (Pharmacia). The yield of the purified protein was approximately 20 mg from one placenta. The purified protein gave a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 36,500. This protein prolonged the clotting time of normal plasma when clotting was induced either by brain thromboplastin or by kaolin in the presence of cephalin and Ca2+. It also prolonged the factor Xa induced clotting time of platelet-rich plasma but did not affect thrombin-induced conversion of fibrinogen to fibrin. The purified placental protein completely inhibited the prothrombin activation by reconstituted prothrombinase, a complex of factor Xa-factor Va-phospholipid-Ca2+. The placenta inhibitor had no effect on prothrombin activation when phospholipid was omitted from the above reaction. Also, it neither inhibited the amidolytic activity of factor Xa, nor did it bind to factor Xa. The placenta inhibitor, however, did bind specifically to phospholipid vesicles (20% phosphatidylserine and 80% phosphatidylcholine) in the presence of calcium ions. These results indicate that the placental anticoagulant protein (PAP) inhibits coagulation by binding to phospholipid vesicles. The amino acid sequences of three cyanogen bromide fragments of PAP aligned with those of two distinct regions of lipocortin I and II with a high degree of homology, showing that PAP is a member of the lipocortin family.     

Increased Calcium Absorption in Nephrolithiasis Explained by Uptake Studies in Ileal Brush Border Membrane Vesicles

We previously showed that recurrent calcium renal stone formers have enhanced urinary excretions of calcium and oxalate resulting from malabsorption of citrate. In the present investigation, the mechanism of the citrate-induced increased calcium uptake was studied using guinea pig ileal brush border membrane vesicles. In this model, calcium is absorbed in a concentration dependent, single mechanism uptake with a K_m of 275 ± 30 umol/liter (SD) and a V_max of 4.0 ± 0.5 nmol/min · mg protein. Under conditions of maximal calcium uptake, both citrate and phosphate inhibited calcium absorption into brush border membrane vesicles (BBMVs). In contrast, when phosphate and citrate were added together, calcium absorption normalized. Citrate inhibition of calcium absorption appeared to be due to free citrate ions, and phosphate ions overcame this inhibition. Phosphate inhibition was mostly due to decreased concentrations of ionized calcium and partly to precipitation of insoluble calcium phosphate. These studies confirm that the effects of citrate in humans in enhancing calcium absorption occur in the lumen of the gut and are not related to further biochemical conversions of citrate by the gut cells, to effects of citrate on calcium-related hormones, or to the renal handling of calcium. Also, the effects of citrate on increasing calcium absorption should be increased or attenuated in patients who malabsorb citrate, and this explains the increased urinary calcium and oxalate excretions reported for recurrent calcium stone formers.     

Fractionation of plasma globulin for prothrombin,thrombokinase, and accessory thromboplastin

1. Crude globulin from more than 1,000 liters of citrated bovine plasma has been used in developing a procedure for moderately large scale separation of clotting factors. Fraction A, prothrombin, kinase, and thrombin fractions were prepared. Fraction A contained both kinase and accessory thromboplastin, the latter predominating when fraction A was diluted. 2. When prothrombin was activated by kinase, the rate of thrombin production was enhanced by the addition of platelets, or brain lipid, or dilute fraction A. These accessory thromboplastins caused this acceleration only when calcium chloride was added. Even with calcium, they were not effective unless kinase was present. 3. In contrast, the action of kinase was not entirely dependent on either ionic calcium or accessory thromboplastin. The concentrated kinase fraction activated prothrombin in the presence of excess oxalate. Although kinase often contaminates highly purified thrombins, it is probably distinct from thrombin. The ratio of kinase to thrombin was 100 times as great in the kinase fraction as in the thrombin fraction. 4. The kinase fraction, diluted 45,000-fold, to protein-nitrogen concentrations as low as 0.02 microgram per ml., accelerated the conversion of crude prokinase in three-stage tests. 5. The findings are consistent with the following concept of the basic enzymatic mechanism: See PDF for Structure It is now added that calcium and accessory thromboplastin exert their effects by impinging on the basic mechanism, in a chemically secondary or indirect manner.     

Potential anticoagulant activity of lipocortins and other calcium/phospholipid binding proteins

Thrombin generation was determined in the presence of phospholipids, coagulation factors Xm, Vm and II (prothrombin), calcium, and various calcium/phospholipid bindings proteins, including lipocortins I and II, 35 kDa calelectrin, and 32.5 kDa endonexin. All of these proteins induced a dose-dependent inhibition of thrombin generation similar to the inhibition of pig pancreas phospholipase A2. It is suggested that the ability of lipocortins and other related proteins to interact with anionic phospholipids in the presence of Ca++ is responsible for both their anticoagulant and anti-phospholipase A2 activity.     

Developmental implications of differential effects of calcium in tail and body skin of Anuran tadpoles

The effects of external Ca(++) on metamorphosis of Rana catesbeiana tadpoles were assessed. Treatment of tadpoles with Ca(++) (0.05 mM) during early prometamorphic stages induced precocious metamorphic events such as tail regression, shortening of the intestine, forelimb emergence, and keratinization of body epidermis within 23 days of treatment compared to control tadpoles still in mid-prometamorphic stages. These effects of Ca(++) are probably mediated by the thyroid gland, as indicated by histological features of the gland at the light and electron microscopic levels. Calcium levels of tail and body skin were measured at various stages of development by atomic absorption spectrophotometry. In control and experimental groups, body skin had significantly higher Ca(++) concentrations than tail skin. There were no statistically significant effects of developmental stage on Ca(++) levels of tail or body skin. Experimental Ca(++) treatment significantly increased Ca(++) concentration in tail but not body skin. Ultrastructure studies and gel electrophoresis indicated that calcium induced keratinization of body skin, but not tail epidermis. Ca(++)-treated tail epidermis showed various autolysing figures in apoptotic cells. In summary, calcium treatment accelerated metamorphosis and induced the following region-dependent cellular events: keratinization of body skin-a characteristic of adult epidermis-and programmed cell death in the tail. Whatever signal elicited by calcium in this experimentally induced accelerated metamorphosis is probably mediated via the thyroid gland.     

An endogenous inhibitor of Ca++-ATPase from human placenta

Intracellular free calcium is regulated by Ca(++)-ATPase, one form present on the plasma membrane (PM Ca(++)-ATPase) and the other on sarcoplasmic (endoplasmic) reticulum (SR/ER Ca(++)-ATPase). An endogenous inhibitor of SR Ca(++)-ATPase from human placenta was shown to be present in normal placenta and the activity was not detectable in placenta from preeclamptic patients. The inhibitor was distributed in cytosol and microsomes. The inhibition of Ca(++)-ATPase by this inhibitor was concentration- and time-dependent. The inhibitor neither bound to DEAE- nor CM-sepharose resins at pH 7.5 and 8.5. Furthermore, it was heat stable for 15 min up to 55 degrees C and completely destroyed at 80 degrees C in a few minutes. It was also observed to be stable at room temperature for at least 3 months. The purification and characterization of this inhibitor would be valuable in achieving an understanding of the normal regulation of Ca(++)-ATPase in the placenta during pregnancy.     

Effects of citrate, heparin and cation supplementation on mortality and egg production of laboratory-reared horn flies

Abstract. The effects of the blood anticoagulants sodium citrate and sodium heparin on horn fly, Haematobia irritans L., egg production were tested. Sodium citrate was added to freshly collected bovine blood to give final concentrations of 5-100mM while sodium heparin was used in concentrations of 10–70 USP units/ml blood. Small cages containing five male and ten female newly emerged laboratory-reared horn flies were maintained for 8–10 days on these blood samples, and mortality and egg production recorded daily. Results showed that as blood citrate concentration was increased, egg production decreased logarithmically. At sodium citrate concentrations of 50 mM and above, severe impacts on egg production and adult horn fly survival occurred. Although no dose-related response of egg production to increasing heparin concentrations was noted, the 25 USP units heparin/ml blood treatments gave the largest egg production, yielding approximately 28% more eggs than any other treatment. Since citrate is a known chelator of divalent metal cations, the effects of supplemental cation additions to citrated blood were tested for their ability to reverse the egg production decrease seen at 50 mM sodium citrate. Blood samples containing 50mM sodium citrate were supplemented with CaCl₂, calcium lactate, CuCl₂, cupric acetate, FeCl₃, ferric citrate, MgCl₂, magnesium acetate, MnCl₂, ZnSO₄, EGTA or EGTA plus calcium lactate, each at 1 mM except EGTA which was used at 2.5 mM. The magnesium acetate supplement and the combination of calcium lactate plus EGTA resulted in a statistically significant increase in egg production ( P < 0.05).     

Subcellular calcium localization and AT0-dependent Ca2+-uptake by smooth endoplasmic reticulum in an invertebrate photoreceptor cell. An ultrastrucutral, cytochemical and X-ray microanalytical study.

In Hirudo medicinalis an extensive and highly elaborate three dimensional network of smooth endoplasmic reticulum cisternae is found in very close structural relationship to the receptive (microvillar) membrane, as reported for many other invertebrates. A variant of the potassium pyroantimonate technique showed that these submicrovillar endoplasmic reticulum cisternae (SMC) and mitochondria are major intracellular calcium stores. Furthermore, using saponine-skinned photoreceptors for an in situ accumulation experiment, calcium oxalate precipitates in SMC demonstrate that this organelle is able to accumulate Ca2+ from a concentration of 2 x 10(-5) M, when ATP, Mg2+, and oxalate ions are present in the accumulation medium. This result provides direct evidence for the hypothesis that SMC may play a particularly important role in the regulation of intracellular ionized calcium in invertebrate photoreceptor cells. Morphological evidence supports this view.     

Ca(2+) -induced binding of anticoagulation factor II from the venom of Agkistrodon acutus with factor IX

Anticoagulation factor II (ACF II), a coagulation factor X- binding protein from the venom of Agkistrodon acutus has both anticoagulant and hypotensive activities. Previous studies show that ACF II binds specifically with activated factor X (FXa) in a Ca(2+) -dependent manner and inhibits intrinsic coagulation pathway. In this study, the inhibition of extrinsic coagulation pathway by ACF II was measured in vivo by prothrombin time assay and the binding of ACF II to factor IX (FIX) was investigated by native polyacrylamide gel electrophoresis and surface plasmon resonance (SPR). The results indicate that ACF II also inhibits extrinsic coagulation pathway, but does not inhibit thrombin activity. ACF II also binds with FIX with high binding affinity in a Ca(2+) -dependent manner and their maximal binding occurs at about 0.1 mM Ca(2+) . ACF II has similar binding affinity to FIX and FX as determined by SPR. Ca(2+) has a slight effect on the secondary structure of FIX as determined by circular dichroism spectroscopy. Ca(2+) ions are required to maintain in vivo function of FIX Gla domain for its recognition of ACF II. However, Ca(2+) at high concentrations (>0.1 mM) inhibits the binding of ACF II to FIX. Ca(2+) functions as a switch for the binding between ACF II and FIX. ACF II extends activated partial thromboplastin time more strongly than prothrombin time, suggesting that the binding of ACF II with FIX may play a dominant role in the anticoagulation of ACF II in vivo.     

Purification and properties of chicken prothrombin

Prothrombin was isolated from citrated chicken plasma. The isolation depends upon the elimination of an interfering substance closely adherent to chicken prothrombin by treatment with SrCO₃. Subsequent to this, the classical adsorption to barium citrate, chromatography on DEAE-cellulose, and gel filtration on Sephadex G-200 was carried out. Prothrombin purified by this method was found to have a specific activity of 1050 Iowa units (850 N.I.H. thrombin units) per mg. Recovery from plasma averaged 40%. Molecular weight by Sephadex G-200 chromatography was 73,000 ± 5,000 and by dodecyl sulfate sodium salt acrylamide gel electrophoresis 70,000 ± 5,000. A stable dimer of M_r 138,000 was observed in some preparations. The isoelectric pH in both acetate and phosphate buffers (μ = 0.1) was 3.95. Rabbit antibody to chicken prothrombin evidenced a single line by immunoelectrophoresis against purified antigen and chicken plasma.     

Calcium distribution in islets of Langerhans: a study of calcium concentrations and of calcium accumulation in B cell organelles.

Calcium concentrations of various pancreatic B cell organelles have been determined by X-ray microanalysis of areas of frozen sections of unfixed rat islets of Langerhans. Highest concentrations were detected in storage granules and in mitochondria, although calcium was also present in nuclei, in areas of endoplasmic reticulum and of cytoplasm. Accumulation of 45Ca by isolated organelles has been studied in homogenates and isolated subcellular fractions of rat islets of Langerhans. In the presence of a permeant anion (oxalate or phosphate), accumulation of 45Ca into mitochondria and microsomes was strongly stimulated by ATP. This net uptake was diminished during incubation of homogenates or of a mitochondria plus storage granule-rich fraction in the presence of cyclic AMP, dibutyryl cyclic GMP; 2:4-dinitrophenol or of ruthenium red. Investigations of the characteristics of 45Ca accumulation by homogenates prepared from storage granule-depleted islets showed no differences from those of normal islets, suggesting that the granules do not represent an important labile pool of calcium. With the exception of cyclic AMP and cyclic GMP none of the insulin secretagogues tested (glucose, leucine, arginine, adrenalin, noradrenalin, theophylline, glibenclamide) altered calcium accumulation by islet homogenates. On the basis of absolute calcium levels and of 45Ca uptake studies it is concluded that islet B cells contain a readily exchangeable mitochondrial calcium pool, and an endoplasmic reticulum pool containing a lower concentration of calcium which is also readily exchangeable. The storage granules, despite their high calcium content, do not appear to constitute a labile pool. It seems likely that the labile mitochondria and endoplasmic reticulum pools play a predominant role in the regulation of cytoplasmic free calcium levels, which may in turn be important in the regulation of rates of insulin secretion.