Binding of magnesium to myosin subfragment-1 ATPase

Tyr 180 of chicken breast muscle alkali light chain A1 was nitrated with tetranitromethane. The nitroA1 was incorporated into chicken breast muscle subfragment-1 (S-1) by exchange with the intrinsic alkali light chain. In the presence of adenylylimidodiphosphate (AMPPNP) or ADP, the S-1 containing nitroA1 showed a difference visible absorption spectrum by Mg2+ or Ca2+. The difference spectrum has a trough around 435 nm, indicating a blue shift of the absorption spectrum due to the nitrophenol chromophore of the modified A1. The plot of delta A at 435 nm versus concentration of free Mg2+ fitted a single binding curve, independent of the total concentration of AMPPNP. These results reveal that free Mg2+ binds to the active site of S-1 ATPase, but not as Mg-AMPPNP complex. The dissociation constants of magnesium from S-1 complex were different with the two nucleotides and were 1.25 X 10(-8) M and 1.24 X 10(-7) with AMPPNP and ADP, respectively. The difference spectrum was also obtained in the presence of ATP. The delta epsilon value after adding ATP changed with the ATPase reaction. The steady state rate of S-1 ATPase was measured at various concentrations of free Mg2+. The dissociation constant of magnesium from the steady state complex, EPADP(a), was estimated as 6 X 10(-8) M. These results suggest that the affinity of magnesium at the active site of ATPase changes with the intermediate states of ATPase reaction. The affinity of calcium was lower than that of magnesium.     

Structure of rice ferricytochrome c at 2.0 A resolution

The crystal structure of ferricytochrome c from rice embryos has been solved by X-ray diffraction to a resolution of 2.0 A, applying a single isomorphous replacement method with anomalous scattering effects. The initial molecular model was built on a graphics display system and was refined by the Hendrickson and Konnert method. The R factor was reduced to 0.25. Rice cytochrome c consists of III amino acid residues. In comparison with animal cytochromes c, the peptide chain extends for eight residues at the N-terminal end, which is characteristic for plant cytochromes c. These additional residues display a collagen-like conformation and an irregular reverse turn, and are located around the C-terminal alpha-helix on the surface or the rear side of the molecule. Two hydrogen bonds between the carbonyl oxygen of the N-terminal acetyl group and O eta of Tyr65, and between the peptide carbonyl oxygen of Pro-1 and O epsilon 1 of Gln89, are involved in holding these eight residues on the molecular surface, where Tyr65 and Gln89 are invariant in plant cytochromes c. Except for the extra eight residues, the main-chain conformations of both rice and tuna cytochromes c are essentially identical, though small local conformational differences are found at residues 24, 25, 56 and 57.     

Observations on microbial percent respiration values in arctic and subarctic marine waters and sediments

Percent respiration was measured in over 1,100 arctic and subarctic marine water and sediment samples using¹⁴C-labeled glucose and glutamate. These measurements were made at different times of the year in 4 regions. Percent respiration values were typically lower in regions where the waters of large rivers mixed with seawater. They were also lower in sediments and in waters collected near the bottom than in surface waters. They were higher in winter arctic waters than water samples collected in the summer; however, a similar seasonal trend was not observed in subarctic waters. There were a number of studies in which there were significant positive rank correlations between percent respiration and salinity and between percent respiration and temperature. From what is known about the range of temperature and salinity encountered in samples collected during these studies and the results of temperature and salinity effects experiments, it was concluded that changes in these 2 variables did not explain the variation observed in percent respiration. Correlations between percent respiration and the inorganic nutrients PO₄ ^–3, NH₄ ⁺ and NO₃ ^– showed that of the 3 variables, only NO₃ ^– showed relatively high correlations with all the same sign. From this it was concluded that there may be situations in which NO₃ ^– levels may influence percent respiration in nearshore marine waters. It is also likely that qualitative characteristics of the available organic nutrients may also influence percent respiration levels. Although no organic nutrient data is available for statistical analysis, the patterns of percent respiration near river plumes and the relatively strong negative correlation often observed between uptake rates (heterotrophic activity) and percent respiration suggests that organic nutrients may be a factor in controlling percent respiration. It is suggested that there are situations in which percent respiration measurements may be used to document stress in natural microbial populations due to nutrient deficiencies.     

Effects of nitrogen dioxide exposure on prostacyclin synthesis in lung and thromboxane A2 synthesis in platelets in rats

Effects of nitrogen dioxide (NO2) exposure on prostacyclin (PGI2) synthesis in the rat lung and thromboxane A2 (TXA2) synthesis in the platelets were studied. Male Wistar rats were exposed to 10 ppm NO2 for 1, 3, 5, 7 and 14 days. PGI2 synthesizing activity of homogenized lung decreased. The damage of PGI2 synthesizing activity reaches its maximum at 3 days. At 14 days, PGI2 synthesizing activity returned to the normal level. The activity of PGI2 synthetase decreased significantly. The formation of lipid peroxides due to NO2 exposure may cause the depression of PGI2 synthesizing activity of lung. On the other hand, platelet TXA2 synthesizing activity increased. This increased TXA2 synthesizing activity lasted at least till 3 days. Then, it returned to the normal level. The counts of platelet were decreased significantly by 1, 3, 5 and 7 days NO2 exposure. Then the decreased counts of platelet returned to the normal level at 14 days NO2 exposure. These results indicate that the depression of PGI2 synthesizing activity of lung by NO2 exposure cause an increase in TXA2 synthesizing activity of platelets. It may contribute to induce platelet aggregation and to the observed decrease in the number of platelets during NO2 exposure.     

Cloning of cDNA encoding rat TCP-1.

We have isolated and sequenced a cDNA encoding a rat homolog of the mouse t-complex polypeptide 1 (TCP-1). Its deduced gene product is a polypeptide of 556 amino acids, with a predicted Mr of 60,341. The similarity between mouse Tcp-1 and the rat homolog is about 94.0% at the nucleotide level and 97.1% at the amino acid level showing the evolutionary conservation of this protein. The similarity of the amino acid sequence of the rat TCP-1 is not significantly biased to any of those from wild (TCP-1B) or from t-haplotype mice (TCP-1A). From a comparison of deduced amino acid sequences of eukaryotic TCP-1 proteins, we found highly conserved domains. Southern blot analysis revealed that there are at least two similar sequences to Tcp-1 in the rat, one is a structural gene and the other seems to be a processed pseudogene.     

Formation and properties of smooth muscle myosin 20-kDa light chain-skeletal muscle myosin hybrids and photocrosslinking from the maleimidylbenzophenone-labeled light chain to the heavy chain.

Experimental conditions which permit the exchange of smooth muscle 20-kDa light chain into skeletal muscle myosin are described. The hybridization does not result in the regulation of actin-activated ATPase activity of the hybrid myosin by smooth light chain phosphorylation. Further, the KCl dependence of the Mg-ATPase activity of the hybrid was similar to that of skeletal muscle myosin. The dephosphorylation of the smooth light chain in the hybrid did not induce a conformational change in the hybrid from the 6 S to the 10 S state, thereby indicating that the conformational transition is dependent also on the nature of the heavy chain subunit. Exchange of the smooth light chain premodified at its Cys-108 by photolabile 4-(N-maleimido)benzophenone and photolysis resulted in crosslinking to the heavy chain subunit. Immunopeptide mapping using a monoclonal antibody against residues 1-23 at the N-terminus of the skeletal muscle myosin heavy chain identified the location of the photocrosslinking site to be beyond 92 kDa away from the N-terminus.     

Trace elements in human transitory milk

Multielement analysis was performed on human milk collected on 5-9-d postpartum from 51 Japanese females using inductively coupled plasma (ICP) mass spectrometry (MS), ICP atomic emission spectrometry (ICP-AES) and fluorometry. Thirty-one elements were detected by these analytical methods in milk. Twelve elements (Na, Mg, P, S, K, Ca, Cu, Zn, Se, Sr, Rb, and Mo) were detected in all of the samples. Al, Cs, and Ba were the elements detected by ICP-MS in more than half of the samples. Multiple regression analysis extracted biological attributes of mother and infant, such as maternal stature, maternal wt, or infant's birth wt, as statistically significant factors contributing to the variation in elemental concentration in milk. However, the rates of contribution were small in all cases. It was concluded that the biological attributes of mother and infant examined in this study were not the major factors that contribute to elemental variation in human milk.     

Spotted fever group rickettsia in dogs in Japan

Prevalence of antibody against spotted fever group-rickettsia in dogs (14/134) from the northern part of Shikoku Island, where spotted fever group rickettsia infection in human is endemic, is significantly higher than that in dogs (4/189) from nonendemic areas.     

Crystal structure analysis of omega-amino acid:pyruvate aminotransferase with a newly developed Weissenberg camera and an imaging plate using synchrotron radiation

The three-dimensional structure of omega-amino acid:pyruvate aminotransferase from Pseudomonas sp. F-126, an isologous alpha 4 tetramer containing pyridoxal 5'-phosphate (PLP) as a cofactor, has been determined at 2.0 A resolution. The diffraction data were collected with a newly developed Weissenberg camera with a Fuji Imaging Plate, using synchrotron radiation. The mean figure-of-merit was 0.57. The subunit is rich in secondary structure and comprises two domains. PLP is located in the large domain. The high homology in the secondary structure between this enzyme and aspartate aminotransferase strongly indicates that these two types of enzymes have evolved from a common ancestor.     

A novel blood coagulation factor IX/factor X-binding protein with anticoagulant activity from the venom of Trimeresurus flavoviridis (Habu snake): isolation and characterization

Using affinity chromatography on a column of factor X-Cellulofine, we have isolated a novel blood coagulation factor X-binding protein with anticoagulant activity from the venom of Trimeresurus flavoviridis (Habu snake). This anticoagulant protein was also purified by chromatography on Sephadex G-75 and S-Sepharose Fast Flow. The yield of the purified protein was approximately 16 mg from 400 mg of crude venom. The purified protein gave a single band on both analytical alkaline disc-gel electrophoresis and SDS-PAGE. This protein had a relative molecular weight (Mr) after SDS-PAGE of 27,000 before reduction of disulfide bonds and 14,000 after reduction of disulfide bonds. The protein prolonged the clotting time induced by kaolin or factor Xa. In the presence of Ca2+, it formed a complex with factor X, the molar ratio being 1 to 1. Similar complex formation was observed with factor Xa and factor IX/factor IXa, but not with other vitamin K-dependent coagulation factors, i.e., prothrombin, factor VII, protein C, protein S, and protein Z. The interaction of this anticoagulant protein with factor IX/factor X was dependent on gamma-carboxyglutamic acid (Gla) domains, since Gla-domainless derivatives of factor X and factor IXa beta' did not interact with this anticoagulant protein.