The oxygen uptake of Sarotherodon niloticus L. and the oxygen binding properties of its blood and hemolysate (Pisces: Cichlidae)

The oxygen consumption of Sarotherodon niloticus L. was found to decline below a critical oxygen concentration of about 2 mg O2/l. An important influence of CO2 on the oxygen affinity of whole blood was observed at all temperatures between 20 and 35 degrees C for gas mixtures containing 5.6% CO2. Purified hemolysate showed extremely high oxygen affinities (p50 = 1.08 mmHg at pH 8.2 and 20 degrees C). Low cooperativity was observed at all temperatures from 20 to 35 degrees C, and pH values between 6.5 and 8.2. The Bohr effect proved to be important at pH values lower than pH 7.5 (phi = delta log P50/delta pH = -0.58 between pH 6.5 and 7.0 at 35 degrees C). The oxygen affinities show high thermal sensitivity without a marked pH influence (delta H value for overall oxygenation at pH was -71.7 kJ/mol). The obtained results are interpreted as adaptations to diurnal variations in ambient temperature and oxygen availability.     

Temperature effects on hemocyanin oxygen binding in an antarctic cephalopod

The functional relevance of oxygen transport by hemocyanin of the Antarctic octopod Megaleledone senoi and of the eurythermal cuttlefish Sepia officinalis was analyzed by continuous and simultaneous recordings of changes in pH and hemocyanin oxygen saturation in whole blood at various temperatures. These data were compared to literature data on other temperate and cold-water cephalopods (octopods and giant squid). In S. officinalis, the oxygen affinity of hemocyanin changed at deltaP50/degrees C = 0.12 kPa (pH 7.4) with increasing temperatures; this is similar to observations in temperate octopods. In M. senoi, thermal sensitivity was much smaller (<0.01 kPa, pH 7.2). Furthermore, M. senoi hemocyanin displayed one of the highest levels of oxygen affinity (P50 < 1 kPa, pH 7.6, 0 degrees C) found so far in cephalopods and a rather low cooperativity (n50 = 1.4 at 0 degrees C). The pH sensitivity of oxygen binding (delta log P50/delta pH) increased with increasing temperature in both the cuttlefish and the Antarctic octopod. At low PO2 (1.0 kPa) and pH (7.2), the presence of a large venous oxygen reserve (43% saturation) insensitive to pH reflects reduced pH sensitivity and high oxygen affinity in M. senoi hemocyanin at 0 degrees C. In S. officinalis, this reserve was 19% at pH 7.4, 20 degrees C, and 1.7 kPa O2, a level still higher than in squid. These findings suggest that the lower metabolic rate of octopods and cuttlefish compared to squid is reflected in less pH-dependent oxygen transport. Results of the hemocyanin analysis for the Antarctic octopod were similar to those reported for Vampyroteuthis--an extremely high oxygen affinity supporting a very low metabolic rate. In contrast to findings in cold-adapted giant squid, the minimized thermal sensitivity of oxygen transport in Antarctic octopods will reduce metabolic scope and thereby contribute to their stenothermality.     

Hemoglobin heterogeneity and the oxygen affinity of the hemolysate of some Victoria cichlids

The hemoglobin patterns of ten cichlid species from Lake Victoria were characterized by polyacrylamide gel electrophoresis. In all tested species the hemoglobin bands display the same electrophoretic mobility. Oxygen equilibria of the purified hemoglobin solution of five species were determined under standardized conditions (pH 7.4 at 20 degrees C). The analysed hemolysates have a relatively high oxygen affinity and for all the tested species the Hill coefficient approached unity. The effect of temperature on the oxygen affinity of Haplochormis "velvet black" hemolysate was determined at 20, 25, 30 and 35 degrees C. The obtained results (delta H value-68 kJ/mol) at pH 8.2 is comparable with earlier published results for other African and South American Cichlidae. The Bohr effect (phi = delta log P50/delta pH = -0.18 between pH 6.6-7.4 at 25 degrees C) proved to be lower than so far reported in other Cichlidae.     

Oxygen binding and acid base status of the blood from the freshwater turtle, Phrynops hilarii

Oxygenation studies with the whole blood of Phrynops hilarii show a P50 of 38 torr at extracellular pH (pHe) of 7.4 which corresponds to an intracellular pH (pHi) of 7.05 at 25 degrees C. The blood CO2 Bohr effect was -0.56 when related to pHi. pHi is related to pHe by the following equation: pHi = 0.75.pHe + 1.54 (r = 0.99); pHi = 0.72. pHe + 1.72 (r = 0.96) at 10 and 25 degrees C respectively. Blood pHe, for 25 degrees C, was 7.519 +/- 0.254 (n = 6). Blood gas partial pressures were: pCO2 = 25.8 +/- 3.8 torr (n = 6); pO2 = 61.7 +/- 21.2 torr (n = 6). The major red cell phosphates, in mmole/l erythrocytes, n = 6, were: ATP (3.66 +/- 0.86); GTP (0.53 +/- 0.28); 2.3-DPG (0.32 +/- 0.12) and inorganic phosphates (2.00 +/- 0.35). The plasma inorganic ion composition, n = 6, was, in mEq/l: K+ (3.04 +/- 0.40); Na+ (148.4 +/- 12.6); Ca2+ (4.75 +/- 1.32); Cl- (106.6 +/- 5.0). Additional blood parameters of interest (n = 6) were: lactate (2.07 +/- 1.72 mM in plasma); erythrocytes/mm3 (416 X 10(3) +/- 4.6 X 10(3)); leucocytes/mm3 (44636 +/- 2618); haematocrit (%) (14.5 +/- 3.6); haemoglobin, g/dl (3.2 +/- 0.5); plasma protein g/dl (4.4 +/- 0.4); osmolarity (293 +/- 10 mOsm/l). The non-bicarbonate buffer value was -22.6 mmol/kg H2O/pH. For a constant CO2 content, delta pHe/delta t = 0.0141 +/- 0.002 (n = 18) and delta pHi/delta t = 0.0157 +/- 0.003 (n = 18).     

Red blood cell function and haematology in two tropical freshwater fishes from Australia

Salmon catfish and tarpon occur in habitats that periodically become deficient in oxygen resulting in high mortalities of other fish species. The water-breathing catfish, Arius leptaspis, and the facultative air-breathing tarpon, Megalops cyprinoides, both have high haemoglobin and haematocrit, and the oxygen carrying capacity in the air-breather is exceptionally high (15.6+/-1.2 vol%). Iso-pH oxygen equilibria of the red blood cells at 25 degrees C revealed high affinity (P(50)=9 mmHg, pH 7.4) and co-operativity (n(50)>2.2, pH 7.4) in the catfish, and contrasted with low affinity (P(50)=32 mmHg, pH 7.4) and co-operativity (n(50) approximately 1) in the air-breathing tarpon. Oxygen binding was further distinguished by relative pH insensitivity (Bohr factor, ?=Deltalog P(50)/Deltalog pH=-0.22) in the catfish, compared with a significant Bohr effect in the tarpon (?=-0.96). The potential for modulation of haemoglobin-oxygen affinity was indicated by a high ratio of GTP to ATP in the erythrocytes of the catfish, whereas regulation in the tarpon appeared due to ATP alone. Differences in blood respiratory functions between the two species are likely to reflect reduced opportunity for activity under extreme hypoxia in the catfish.     

Blood oxygen transport in common map turtles during simulated hibernation

We assessed the effects of cold and submergence on blood oxygen transport in common map turtles (Graptemys geographica). Winter animals were acclimated for 6-7 wk to one of three conditions at 3 degrees C: air breathing (AB-3 degrees C), normoxic submergence (NS-3 degrees C), and hypoxic (PO2=49 Torr) submergence (HS-3 degrees C). NS-3 degrees C turtles exhibited a respiratory alkalosis (pH 8.07; PCO2=7.9 Torr; [lactate]=2.2 mM) relative to AB-3 degrees C animals (pH 7.89; PCO2=13.4 Torr; [lactate]=1.1 mM). HS-3 degrees C animals experienced a profound metabolic acidosis (pH 7.30; PCO2=7.9 Torr; [lactate]=81 mM). NS-3 degrees C turtles exhibited an increased blood O2 capacity; however, isoelectric focusing revealed no seasonal changes in the isohemoglobin (isoHb) profile. Blood O2 affinity was significantly increased by cold acclimation; half-saturation pressures (P50's) for air-breathing turtles at 3 degrees and 22 degrees C were 6.5 and 18.8 Torr, respectively. P50's for winter animals submerged in normoxic and hypoxic water were 5.2 and 6.5 Torr, respectively. CO2 Bohr slopes (Delta logP50/Delta pH) were -0.15, -0.16, and -0.07 for AB-3 degrees C, NS-3 degrees C, and HS-3 degrees C turtles, respectively; the corresponding value for AB-22 degrees C was -0.37. The O2 equilibrium curve (O2EC) shape was similar for AB-3 degrees C and NS-3 degrees C turtles; Hill plot n coefficients ranged from 1.8 to 2.0. The O2EC shape for HS-3 degrees C turtles was anomalous, exhibiting high O2 affinity below P50 and a right-shifted segment above half-saturation. We suggest that increases in Hb-O2 affinity and O2 capacity enhance extrapulmonary O2 uptake by turtles overwintering in normoxic water. The anomalous O2EC shape and reduced CO2 Bohr effect of HS-3 degrees C turtles may also promote some aerobic metabolism in hypoxic water.     

Hemoglobin-oxygen affinity in anemia

In blood of 21 anemic patients and 8 normal subjects (N) three oxygen dissociation curves each were measured at different pH values to calculate Bohr coefficients after acidification with CO2 (BCCO2) or fixed acid (BCFA), and other important parameters of oxygen affinity. The patients had either low hemoglobin or red cell production (L: n = 11, 7.3 g/dl Hb) or high erythrocyte production combined with high loss (H: n = 10, 7.8 g/dl Hb). The standard half saturation pressure P50 (pH 7.4, 37 degrees C) was equally elevated in both anemic groups (L: 30.5, H: 30.8, N: 26.7 mmHg), as well as the diphosphoglycerate concentration (DPG) (L: 18.7, H: 18.6, N: 12.7 mumol/g Hb). The red cell pH of the anemics was lower than for the N (approximately 0.045 units) causing part of the difference in P50. Hill's "n" tended to high values in the anemics except at low O2-saturation in the H. For BCCO2 no significant difference among the groups was observed. BCFA, however, increased in the H at low SO2 compared to the N and L. The cause for most of the changes in hemoglobin oxygen affinity in anemics was the high [DPG]. The combination of high P50 and high "n" value as in the L seems to be most advantageous for tissue oxygenation.     

Low P50 in deer mice native to high altitude

Whereas it is widely believed that animals native to high altitude show lower O2 partial pressures at 50% hemoglobin saturation (P50) than do related animals native to low altitude, that "fact" has not been well documented. Consequently, P50 at pH 7.4, PCO2(7.4), the CO2 Bohr effect, and the buffer slope (delta log PCO2/delta pH) were determined via the mixing technique in Peromyscus maniculatus native to a range of altitudes but acclimated to 340 or 3,800 m. PCO2(7.4) and buffer slope were substantially lower at high altitude. The change in P50(7.4) between acclimation altitudes was minimal (0.8% increase at 3,800 m), because of counterbalancing changes in PCO2, 2,3-diphospho-D-glycerate concentration, and perhaps other factors. At both acclimation altitudes there was a highly significant negative correlation between P50(7.4) and native altitude. Since pH in vivo probably increases slightly at high altitude, the data on P50 corrected to pH 7.4 are probably underestimates of the difference in in vivo P50 at low vs. high altitude. Hence these results corroborate theoretical predictions that low P50 is advantageous under severe hypoxic stress.     

Physiology of hibernation in Rana pipiens: metabolic rate, critical oxygen tension, and the effects of hypoxia on several plasma variables

Rates of O(2) consumption (.VO(2)) were determined for adult northern leopard frogs (Rana pipiens) submerged at 3 degrees C at water PO(2)s (P(w)O(2)) ranging from 0-160 mmHg. The critical O(2) tension (P(c)) was 36.4 mmHg. Hematocrit and blood levels of PO(2), glucose, lactate, pH, [Na(+)], [K(+)], and osmolality were determined for frogs submerged for two days. Above a P(w)O(2) of 50 mmHg, blood PO(2) ranged from 1-7 mmHg, which was sufficient to allow the frogs to function entirely aerobically. Plasma [lactate] increased as P(w)O(2) fell below 50 mmHg, the increase preceding significant changes in any other variable, and apparently preceding a fall in .VO(2). Most other variables showed little or no change from those of air-breathing control animals, even during anoxia. We present an analysis of the importance of a large decrease in P(c) in permitting frogs to successfully overwinter in icebound ponds and of the factors that contribute to that decrease.     

Oxygen transport function of the blood in hyperthermia in rabbits

After 30-min or longer incubation of rabbit blood under 42 degrees C the blood O2 affinity was increased. During governed hyperthermia of conscious rabbits PO2, pH and the oxygen content in the mixed venous blood were decreased. Similar events were observed after 1 hour from the ceasing of hyperthermia. In the real blood circulation the oxygen affinity of haemoglobin was decreased because of the higher temperature. But after the recalculation of P50 to the standard conditions (t = 37 degrees C, pH 7.4, PCO2 = 40 Torr) it's value was below the initial one by 4.0 +/- 0.68 Torr. The mechanism of the increase of the oxygen affinity of haemoglobin is discussed.     

Effects of temperature and oxygen availability on circulating catecholamines in the toad Bufo marinus.

The release of catecholamines during hypoxia has received limited attention in amphibians and the adrenergic regulation of cardio-pulmonary functions is, therefore, not well understood at the organismic level. To describe the changes in plasma catecholamine concentrations, we exposed toads (Bufo marinus) to different levels of hypoxia at two temperatures (15 and 25 degrees C). In addition, blood oxygen binding properties were determined in vitro at 15 and 25 degrees C at two different pH values. Hypoxia elicited a significant increase in plasma catecholamines (adrenaline and noradrenaline) at both temperatures, in spite of a respiratory alkalosis. At 15 degrees C, the increase was from 2.6+/-1.0 in normoxia to 4.8+/-1.4 ng ml(-1) at an inspired oxygen fraction of 0.05. At 25 degrees C, the hypoxic release of catecholamines was significantly higher (maximum levels of 44.8+/-11.6 ng ml(-1)). Plasma noradrenaline concentration was elevated at the most severe hypoxic levels, suggestive of an adrenal release. The arterial oxygen threshold for catecholamine release were approximately 1.0 mmol O(2) l(-1) blood or a PaO(2) of 30 mmHg. The P(50) values at 15 degrees C were 23.5+/-0.7 and 28.9+/-1.0 mmHg at pH 7.98+/-0.01 and 7.62+/-0.02, respectively, and increased to 36.5+/-0.6 and 43.0+/-1.1 mmHg at pH 8.04+/-0.04 and 7.67+/-0.05, respectively, at 25 degrees C. The oxygen equilibrium curves were linear when transformed to Hill-plots and Hills n (the haemoglobin subunit co-operativity) ranged between 2.24 and 2.75. The in vitro blood O(2) binding properties corresponded well with in vivo data.     

Fat energy use and plasma lipid changes associated with exercise intensity and temperature

The effect of 60 min of exercise at two intensities (50 and 60% VO2max) and temperatures (0 and 22 degrees C) on changes (delta) in plasma lipids [triglycerides (TG), glycerol (GLY), total cholesterol (TC), and HDL-cholesterol (HDL-C)] was examined. Subjects were 10 men aged 27 +/- 7 years (VO2max = 3.81 +/- 0.45 1 min, % fat = 12.2% +/- 7.1%). VO2 and respiratory exchange ratio results indicated that total energy and fat energy use were similar at the two temperatures. Changes in plasma volume (%delta PV) were different (P less than 0.05) at the two temperatures (22 degrees C: -2.3% vs 0 degrees C: 1.1%). Combining the data at each temperature revealed that the increases in concentrations were greater (P less than 0.05) at 22 degrees C (delta TG = 0.22, delta GLY = 0.20, delta TC = 0.14, delta HDL-C = 0.05 mmol l-1) vs 0 degrees C (delta TG = 0.10, delta GLY = 0.12, delta TC = 0.05, delta HDL-C = 0.02 mmol l-1). Combining the data for each intensity revealed that the increases in concentration were greater (P less than 0.05) at 60% VO2max for delta TG and delta HDL-C. The 60% VO2max/22 degrees C bout produced greater changes (P less than 0.05) than all other bouts for delta TC and delta HDL-C (0.21 and 0.08 mmol l-1, respectively). Only delta TG and delta GLY were greater at 22 degrees C when adjusted for %delta PV. These metabolic and plasma lipid results indicate that cold exposure does not act synergistically with exercise to further stimulate fat metabolism.     

Temperature-induced changes in blood acid-base status: Donnan rCl and red cell volume.

The Donnan ratio for chloride ion (rCl) was determined for human red cells in plasma utilizing 36Cl. The effect of altered PCO2 and pH on rCl was followed in two ways. CO2 partial pressure was varied (1-1.5% CO2 in O2; pH range 7.1-7.9) at 37.5 degrees C (isothermal); PCO2 and pH were also changed by altering temperature (range 5-45 degrees C) at constant CO2 content (temperature induced). At pH 7.4 and 37.5 degrees C, rCl was 0.631 +/- 0.0269 (SE, N = 5); isothermal drcl/dpH = -0.306 +/- 0.0234. When measured under conditions of variable temperature at constant CO2 content (pH range 7.3-7.9), drcl/dpH = .018 +/- 0.0232, significantly different from isothermal response (P less than 0.001). Hematocrit (H) changes with pH for conditions of initial H(7.4) of 0.45, under these conditions were also determined: isothermal dH/dpH = -0.031 +/- 0.0019; temperature induced, -0.004 +/- 0.0009. Temperature change alone at constant carbon dioxide content produces no significant change in distribution of chloride ions or water between erythrocyte and plasma compartments.     

Hemoglobin and oxygen: different affinities in two species of rodents (Mus musculus and Pitymys duodecimcostatus)

Five different hemoglobins have been demonstrated by polyacrylamide-gel disk electrophoresis in the species Mus musculus. Oxygen affinities of hemoglobin (P50) from Mus musculus and Pitymys duodecimcostatus hemolysates were determined at pH 7.4 and 37 degrees C. Values obtained for delta log P50/delta pH in hemolysates from both species point out a more pronounced Bohr effect in Pitymys duodecimcostatus.     

The relationship between initial creatine phosphate breakdown and recovery oxygen consumption for a single isometric tetanus of the frog sartorius muscle at 20 degrees C

A previous paper (Mahler, M. 1978 J. Gen. Physiol. 71:559--580) describes the time-course of the suprabasal rate of oxygen consumption (delta QO2) in the sartorius muscle of R. pipiens after isometric tetani of 0.1--1.0 s at 20 degrees C. To test whether these were the responses to impulse changes in the rate of ATP hydrolysis, we compared the total suprabasal oxygen consumption during recovery (delta[O2]) with the amount of ATP hydrolyzed during a contraction, measured indirectly as the decrease in creatine phosphate (delta[CP]O). If suprabasal ATP hydrolysis during recovery is negligible in comparison with that during contraction, delta[CP]0/delta[O2] should approximate the P:O2 ratio for oxidative metabolism, which has an expected value of 6.1--6.5. We found: formula; see text. We conclude that in this muscle at 20 degrees C: (a) after a tetanus of 0.2--1.0 s, delta QO2(t) can be considered the response to an impulse increase in the rate of ATP hydrolysis; (b) the reversal during recovery of unidentified exothermic reactions occurring during the contraction (Woledge, R. C. 1971. Prog. Biophys. Mol. Biol. 22:39--74) can be coupled to an ATP hydrolysis that is at most a small fraction of delta[CP]0; (c) the pooled mean for delta[CP]0/delta[O2], 6.58 +/- 0.55, sets an experimental lower bound for the P:O2 ratio in vivo.     

Respiratory adaptations in burrowing pocket gophers from sea level and high altitude.

To examine the adaptations to low O2 and high CO2 among fossorial and nonfossorial rodents, hematological parameters were determined for laboratory rats, the valley pocket gopher (Thomomys bottae) from 250 m, and the mountain pocket gopher (T. umbrinus melanotis) from 3150 m. Hematocrit, hemoglobin concentration, and O2 capacity were higher in pocket gophers than in rats. Blood PO2 at 50% saturation and pH 7.4 was 33 mmHg for both gophers and 39 mmHg for rats. Bohr factors for all three rodents were similar (-0.55 to -0.61) but buffer value, delta log PCO2/delta pH, was -2.54 for T. umbrinus, -1.97 for T. bottae, and -0.98 for Rattus. Concentrations of total acid-soluble phosphates were 50-75% higher in gophers than in rats, while bicarbonate values were within the normal mammalian range. All three rodents had similar myoglobin concentrations in cardiac muscle. Myoglobin concentrations were significantly higher in skeletal muscles (diaphragm, gastrocnemius) of T. umbrinus when compared to T. bottae, and significantly higher in both gophers when compared to rats. These differences may constitute important adaptations to the hypoxia and hypercapnia in burrows; certain of these factors in pocket gophers respond to the additional stress of high altitude hypoxia.     

Effect of cadmium ions on dioxygen affinity and polyphosphate activity of human red blood cells

The effect of cadmium ions on the dioxygen affinity, the time-dependent depletion of intracellular polyphosphates, and the elongation of human red blood cells (RBC's) was examined. The incubation of RBC's in the presence of 1 mM Cd2+ at 37 degrees C for more than one hour results in a decrease of the p50 value by 2.5-3.0 mmHg in comparison to controls. The p50 of stripped (phosphate-free) hemoglobin is not affected by the presence of 1 mM Cd2+ (p50 = 4.8 mmHg at pH 7.2 and 37 degrees C). Experiments with RBC cryolysates demonstrate an apparently competitive effect of 2.3-bisphosphoglycerate (DPG) with cadmium ions on the dioxygen affinity. From 31P NMR spectra, 31P T1 relaxation, and 31P T2 relaxation behavior a more direct evidence for DPG-Cd2+ complexation is obtained. 31P NMR spectra of RBC cryolysates also indicate DPG-Cd2+ complexation. The hydrolysis of free polyphosphates in RBC's incubated at 37 degrees C as monitored by 31P NMR spectra can be noticed after a three-hour lag phase (constant polyphosphate level). This lag phase is lengthened from three hours to four hours in the presence of Cd2+ ions. RBC elongation, as a measure of deformability, decreases slightly upon incubation with 1 mM Cd2+.     

血红蛋白携氧-释氧动力学研究

本文研究了鸡、家兔、鲤鱼、蟾蜍4种实验动物血红蛋白(hemoglobin,Hb)携氧-释氧动力学过程,初步建立Hb携氧-释氧动力学研究方法,并探讨Hb携氧-释氧动力学过程与动物生存环境之间的关系.结果显示:4种动物Hb携氧动力学曲线均呈"S"形曲线特征,与传统的Hb氧解离曲线(oxygen dissociation curve,ODC)相似;同时不同动物Hb携氧-释氧动力学曲线也有各自特点,如鸡Hb释氧时间长达(1 411±6)S;在Hb携氧.释氧曲线I阶段,鲤鱼上升斜率远大于家兔等.提示Hb携氧-释氧动力学曲线可反映不同动物Hb携氧效率的差异.与传统ODC参数P50相对应,由动力学曲线可得到Hb携氧动力学参数T50°T50是Hb达到50%氧饱和度所需时间,可直观反映Hb携氧效率的差异.4种实验动物Hb均有较稳定的T50,从大到小依次为:鸡、家兔、鲤鱼和蟾蜍.对Hb携氧动力学曲线与ODC综合分析,可得到Hb携氧效能参数E50,表示Hb达到50%氧饱和度所用时间与环境氧分压之间的关系,即E50(50% Sat,Xeo2,yr).E50有可能成为全面评价Hb携氧效能的综合指标.     

Studies on (Na+ + K+)-activated ATPase. XXXVIII. A 100 000 molecular weight protein as the low-energy phosphorylated intermediate of the enzyme.

Phosphorylation of NaI-treated bovine brain cortex microsomes by inorganic phosphate in the presence of Mg2+ and ouabain has been studied at 0 degrees C (pH 7.4) and 20 degrees C (pH 7.0). Nearly maximal (90%) and half-maximal phosphorylation are achieved at 20 degrees C within 2 min with 50--155 and 5.6--17 muM 32Pi, respectively, and at 0 degrees C within 75 s with 300--600 and 33--66 muM 32Pi, respectively. Maximal phosphorylation yields 146 pmol 32P - mg-1 protein. Without ouabain (20 degrees C, pH 7.0) less than 25% of the incorporation observed in the presence of ouabain is reached. Preincubation of the native microsomes with Mg2+ and K+, in order to decompose possibly present high-energy phosphoryl-bonds prior to ouabain treatment, does not affect the maximal phosphate incorporation. This indicates that the inorganic phosphate incorporation is not due to an exchange with high-energy phosphoryl-bonds, which might have been preserved in the microsomal preparations. Phosphorylation of the native microsomes by ATP in the presence of Mg2+ and Na+ reaches 90 and 50% maximal levels within 15--30 s at 0 degrees C and pH 7.4 at concentrations of [gamma-32P]ATP of 5--32 and 0.5--3.5 muM, respectively. The maximal phosphorylation level is 149 pmol 32P-mg-1 protein, equal to that of ouabain-treated microsomes phosphorylated by inorganic phosphate. Both inorganic phosphate and ATP phosphorylate on site per active enzyme subunit of 135 000 molecular weight. From the equilibrium constants for the phosphorylation of ouabain-treated microsomes by inorganic phosphate at 0 degrees C and 20 degrees C standard free-energy changes of --5.4 and --6.8 kcal/mol, respectively, are calculated. These values yield a standard enthalpy change of 14 kcal/mol and an entropy change of 70 cal/mol - degree K. This characterizes the reaction as a process driven by an entropy change. The intermediate formed by phosphorylation with Pi has maximal stability at acidic pH, as is the case for the intermediate formed with ATP. Solubilization in sodium dodecyl sulfate stabilizes the phosphoryl-bond in the pH range of 4--7. The non-solubilized preparation has optimal stability at pH 2--4, the level of which is equal to that of detergent-solubilized intermediate. Sodium dodecyl sulfate gel electrophoresis of the microsomes at pH 3, following incorporation of 32Pi yields 11 protein bands, only one of which (mol. wt 100 000--106 000) carries the radioactive label. This protein has the same molecular weight as the protein, which is phosphorylated by ATP in the presence of Mg2+ and Na+.     

Blood oxygen transport in rats under hypothermia combined with modification of the L-Arginine-NO pathway.

Nitric oxide (NO) has high affinity to heme and by interaction with oxyhemoglobin (HbO2) is converted into nitrate to form methemoglobin (MetHb) as a side product. In combining with deoxy-Hb NO yields a stable molecule of nitrosyl-hemoglobin (HbFe(II)NO) that can further be converted into nitrate and hemoglobin (Hb). In addition, Hb was shown to transport NO in a form of S-nitrosohemoglobin (SNO-Hb). These features of the Hb and NO interaction are important for blood oxygen transport including hemoglobin-oxygen affinity (HOA). The present investigation was aimed to study the blood oxygen transport indices (pO2, pCO2, pH, HOA, etc.) in rats under hypothermia combined with a modification of L-arginine-NO pathway. To modify the L-arginine-NO pathway, rats were administered with N(G)-nitro-L-arginine methyl ester (L-NAME), L-arginine, or sodium nitroprusside (SNP) intravenously before cooling. A substantial impairment of oxygen delivery and development of hypoxia, with an important contribution of HOA into the latter accompanied the deep hypothermia in rats. All the experimental groups developed metabolic acidosis, less pronounced in rats treated with L-arginine only. In the experiments with a modification of the L-arginine-NO pathway, an enhanced cold resistance, attenuated oxygen deficiency, and a weaker oxyhemoglobin dissociation curve (ODC) shift leftwards were observed only after the administration of L-arginine. Neither SNP nor L-NAME had not any protective effects. L-Arginine lowered the value of standard P50 (pO2, corresponding to 50% Hb saturation with oxygen at 37 degrees C, pH 7.4, and pCO2 = 40 mmHg). The actual P50 (at actual pH, pCO2 and temperature) decreased by approximately 15 mmHg and was significantly higher than that under hypothermia without the drug treatment (21.03 +/- 0.35 vs 17.45 +/- 0.60 mmHg). NO also can contribute to this system through different mechanisms (HOA modification, vascular tone regulation, peroxynitrite formation, and effects).