Ca2+,Mg2+-ATPase of microsomal membranes from bovine aortic smooth muscle. Identification and characterization of an acid-stable phosphorylated intermediate of the Ca2+,Mg2+-ATPase

An acid-stable phosphoprotein was formed in a microsomal membrane fraction isolated from bovine aortic smooth muscle in the presence of Mg2+ + ATP and Ca2+. The microsomes also showed Ca2+ uptake activity. The Ca2+ dependence of phosphoprotein formation and of Ca2+ uptake occurred over the same range of Ca2+ concentration (1-10 microM), and resembled similar findings from rabbit skeletal microsomes. The molecular weight of the phosphorylated protein, estimated by SDS-gel electrophoresis, was approximately 105,000. The phosphoprotein was labile at alkaline pH, and its decomposition was accelerated by hydroxylamine. Half-maximum incorporation of 32P in the presence of 10 microM Ca2+ occurred at 60 nM ATP. The calcium-dependent phosphoprotein formation was not affected by 5 mM NaN3, but was inhibited in a dose-dependent fashion by ADP with a 50% inhibition occurring at 180 microM. Fifty mM MgCl2 was required for the maximal phosphorylation. The rate of phosphoprotein decomposition after adding 2 mM EGTA was accelerated by varying the Mg2+ concentration from 10 microM to 3 mM. Alkaline pH (9.0) slowed the rate of phosphoprotein decay. Optimal Ca2+-dependent phosphoprotein occurred at 15 degrees C over a broad pH range (6.4 to 9.0). The activation energy of EGTA-induced phosphoprotein decomposition was 25.6 kcal/mol between 0 and 16 degrees C and 14.6 kcal/mol between 16 and 30 degrees C. The phosphoprotein formed by aortic microsomes was thus quite similar to the acid-stable phosphorylated intermediate of the Ca2+-transport ATPase of sarcoplasmic reticulum from skeletal and cardiac muscle. These data suggest that the Ca2+-dependent phosphoprotein is a reaction intermediate of the Ca2+,Mg2+-ATPase of the aortic microsomes.     

Stereospecific incorporation of hydrogens from NADPH in elongation of very long fatty acyl-CoA by swine cerebral microsomes

The elongation of arachidoyl-CoA by swine cerebral microsomes resulted in the production of behenic acid (22:0) and lignoceric acid (24:0) concomitantly. When 4S-[4-²H₁]NADPH was used for the elongation of arachidoyl-CoA, the incorporation of two deuterium atoms into 22:0 was observed by the technique of mass fragmentography. Furthermore, the incorporation of four deuterium atoms into 24:0 was also detected. On the other hand, when 4R-[4-²H₁]NADPH was used, no deuterium was incorporated into the elongated products.     

Lipoprotein lipase-like activity in the liver of mice with Sarcoma 180

The triglyceride lipase (TGL) activity of liver homogenates of mice with Sarcoma 180 was measured. The liver homogenate of normal or tumor-bearing mice was treated with 0.25% Triton X-100 and centrifuged at 100,000 g for 60 min, and the supernatant was applied to a heparin-Sepharose column. In normal mice, most of the TGL activities in the supernatant was eluted with 0.75 M NaCl from the column. In mice with Sarcoma 180, the TGL gave two peaks on heparin-Sepharose column chromatography, which were eluted with 0.75 M and 1.5 M NaCl, respectively. The activity in the first peak (0.75 M NaCl eluate) decreased; that in the second peak (1.5 M NaCl eluate) increased, and the ratio of the second peak to the first peak increased during tumor development. The livers of normal mice and mice on day 10 after tumor inoculation were perfused with heparin. The highest rate of the TGL release occurred within 1 min of heparin perfusion, and the bulk of heparin-releasable activity appeared within 2 min of perfusion in both normal and tumor-bearing mice. The TGL activity in liver perfusate of tumor-bearing mice, as well as that of liver homogenate, was resolved on a heparin-Sepharose column into two peaks, which were eluted with 0.75 M and 1.5 M NaCl, and most of the activity was eluted with 1.5 M NaCl. The nature of the TGL activity eluted from a heparin-Sepharose column was investigated. In both liver homogenates and liver perfusates, the first peak did not require serum for maximal activity and was relatively resistant to a high concentration of NaCl or protamine sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of cobalamin analogs to intrinsic factor-cobalamin receptor and its prevention by R binder

It is now known that nonphysiological cobalamin analogs exist in the gastrointestinal tract, but their metabolic behavior is unclear. In this study, [57Co]cobinamide was used to study its affinity to hog intrinsic factor-cobalamin (IF-Cbl) receptor which has no species specificity against human IF-Cbl receptor, and its relation to human saliva R binder. Cobinamide was prepared from [57Co]cyanocobalamin and separated by paper chromatography. Human IF-Cbl complex was bound to IF-Cbl receptor but free cyanocobalamin was not. Although R binder-cobinamide was not bound to the IF-Cbl receptor, free cobinamide was bound to the IF-Cbl receptor to a significant extent (about one-half of IF-cyanocobalamin binding to the IF-Cbl receptor). We then investigated the binding of cobinamide to R binder and trypsin-treated R binder. Association constant of cobinamide binding to the IF-Cbl receptor was 1.0 X 10(9) M-1 which was much lower than that of cobinamide binding to trypsin-treated R binder and to untreated R binder. Further study indicated that cobinamide binding to the IF-Cbl receptor was blocked by the addition of R binder and also by trypsin-treated R binder. We conclude that one of the roles of R binder is to prevent binding of free cobalamin analogs to the IF-Cbl receptor in the gut.     

CO2 is the first species formed upon CO oxidation by CO dehydrogenase from Pseudomonas carboxydovorans

The active species of CO₂, i.e. CO₂ or HCO ₃ ^- , formed in the CO dehydrogenase reaction was determined using the pure enzyme from the carboxydotrophic bacterium Pseudomonas carboxydovorans. Employing an assay system similar to that used to test for carbonic anhydrase, data were obtained which are quite compatible with those expected if CO₂ is the first species formed. In addition, carbonic anhydrase activity was not detected in P. carboxydovorans.     

Photoreception in pineal organs of larval and adult lampreys,Lampetra japonica

A comparative study of the larval and adult pineal organs, which are sensitive to incident light, was carried out in the river lamprey Lampetra japonica, using intracellular recording from the pineal photoreceptors. The tissue overlying the larval pineal organ is transparent, whereas that over the adult pineal is translucent. The optical density of this oval pineal window in the adult lamprey was 1.2. In order to elucidate the early development of the larval pineal, the ratio r of the diameter (micron) of the pineal to the body-length (cm) was measured. The value of r was 62.5 in a small larva of 2.8 cm, 29.7 in a larger one of 14.3 cm, and 9.3 in an adult of 54 cm body-length. The intracellular response to light of the larval pineal was a hyperpolarization, showing fundamentally the same pattern as that of the adult pineal. It was possible to record a typical response even from the pineal of the smallest larva, 2.8 cm in body length, used in this study. The intensity-amplitude relationship was analysed after Naka-Rushton's hyperbolic equation. The value of sigma of isolated larval pineals was 0.88 log unit higher than that of adults. The value of n was larger in larvae, suggesting a sensitive reaction to changing photic stimulus. The spectral sensitivity was compared. The peak was at 505 nm in the larva, but 525 nm in the adult. A change of visual pigment in the pineal during metamorphosis is suggested.     

Electrophoretic heterogeneity and age-related change of serine proteinase fromAspergillus sojae

Five forms of serine proteinase (EC 3.4.21.14) have been purified fromAspergillus sojae. This paper reports heterogeneity of electrophoretic mobilities, isoelectric points, and kinetic parameters of multiple forms of serine proteinase fromAspergillus sojae, including agerelated changes in thek _cat /K _m of electrophoretic species.     

Construction and Characterization of Isogenic Series of Saccharomyces cerevisiae Polyploid Strains

Tetraploid cells of Saccharomyces cerevisiae are generated spontaneously in a homothallic MATa/MATα diploid population at low frequency (approximately 10⁻⁶ per cell) through the homozygosity of mating-type alleles by mitotic recombination followed by homothallic switching of the mating-type alleles. To isolate tetraploid clones more effectively, a selection method was developed that used a dye plate containing 40 mg each of eosin Y and amaranth in synthetic nutrient agar per liter. It was possible to isolate tetraploid clones on the dye plate at a frequency of 1 to 3% among the colonies colored dark red in contrast to the light red of the original diploid colonies. Isogenic series of haploid to tetraploid clones with homozygous or heterozygous genomic configurations were easily constructed with the tetraploid strains. No significant differences in specific growth rate or fermentative rate were observed corresponding to differences in ploidy, although the haploid clones showed a higher frequency of spontaneous respiratory-deficient cells than did the others. However, a significant increment in the fermentative rate in glucose nutrient medium was observed in the hybrid strains constructed with two independent homozygous cell lines. These observations strongly suggest that the polyploid strains favored by the brewing and baking industries perform well not because of the physical increment of the cellular volume by polyploidy but because of the genetic complexity or heterosis by heterozygosity of the genome in the hybrid polyploid cells.     

Effects of W-7 on catecholamine release and 45Ca2+ uptake in cultured adrenal chromaffin cells.

Effects of N-(6-aminohexyl)-5-chloro-1-naph-thalenesulfonamide (W-7), a calmodulin antagonist, on catecholamine (CA) release and ⁴⁵Ca²⁺ uptake were studied using cultured bovine adrenal chromaffin cells. W-7 inhibited the carbamylcholine (CCh)-evoked CA release and ⁴⁵Ca²⁺ uptake in a concentration-dependent manner. The inhibitory effect of W-7 on CCh-evoked CA release was not overcome either by an increase in extracellular calcium or CCh concentration. Although W-7 inhibited the high K⁺-evoked CA release and ⁴⁵Ca²⁺ uptake, potency of the drug was approximately 50–100 fold less than when inhibiting the CCh-evoked CA release and ⁴⁵Ca²⁺ uptake. The inhibitory effects of W-7 were observed both in norepinephrine release and epinephrine release. Moreover, W-7 inhibited the CCh-evoked ⁴⁵Ca²⁺ efflux. These results suggest that the inhibition of CA release by W-7 in adrenal chromaffin cells is mainly due to its inhibition of calcium uptake. W-7 may influence the linkage between acetylcholine-receptor and calcium uptake with higher potency than depolarization-dependent calcium entry.