(Na+ + K+)-ATPase : phosphorylation-dependent cross-linking of the alpha-subunits in the presence of Ca2+ and o-phenanthroline

In previous studies we had demonstrated that in the presence of 0.25 mM Cu2+ and 1.25 mM o-phenanthroline, cross-linking of the alpha-subunits of Na+ + K+)-dependent adenosine triphosphatase was induced by the addition of Na+ + ATP, and that the formation of the alpha,alpha-dimer was preceded by that of phosphoenzyme. The purpose of the present studies was the further evaluation of the role of phosphoenzyme in the process of cross-linking. Na+ + UTP did not induce cross-linking unless Mg2+ was also added. In contrast, Na+ + ATP-induced cross-linking did not require the addition of Mg2+. The different effects of ATP and UTP in the absence of added Mg2+ could be accounted for by the presence in the enzyme preparation of bound Mg2+ which supported enzyme phosphorylation by ATP but not by UTP. When the enzyme was phosphorylated by Pi, in the presence of Mg2 and ouabain, and the exposed to Cu2+ and o-phenanthroline, the alpha,alpha-dimer was obtained. Under these conditions, Na+ blocked both phosphorylation and cross-linking. These results indicate that it is the formation of phosphoenzyme per se that leads to conformational transitions favorable to cross-linking. They also suggest that Cu2+ and o-phenanthroline participate in the cross-linking reaction, but not in the phosphorylation reactions. In the digitonin-treated enzyme, Na+ and ATP induced the formation of phosphoenzyme, but not that of alpha,alpha-dimer. These findings indicate that in addition to phosphorylation, a proper orientation o alpha-subunits in an oligomer is also necessary for cross-linking.     

Isolation of P700-chlorophyll-protein complex from a blue-green alga by a nondetergent method

A procedure has been developed for the isolation of a P700-chlorophyll-protein complex from a blue-green alga by a nondetergent method. The use of a Sepharose 4B column in conjunction with sucrose density-gradient centrifugation allows the separation of this complex from three other chlorophyll a-containing fractions which have higher ratios of carotenoids to chlorophyll a. Isoelectric focusing results in a single band with an isoelectric point at 4.5. Analysis of the major (green) fraction reveals the presence of P700. The absorption and emission spectra of this complex are reported.     

Structure of valinomycin-K+ complex in solution by extended x-ray absorption fine structure.

We used synchrotron radiation to measure the K-edge absorption spectra of the potassium ion in valinomycin-K+ complexes dissolved in ethanol and methanol. Our motivation is to study the structure of valinomycin around the potassium ion and the effect of solvents. From the extended x-ray absorption fine structure, we found that the mean distance from potassium to its coordination atoms, oxygen, is the same for both solvents, 2.79 +/- 0.02 A, compared with 2.76 A in crystal. The K-edge threshold spectra of the two solutions are almost identical but have a small difference in their relative peak intensities. The coincidence of their corresponding peak positions indicates that the strength of ligand field is about the same in these two samples. This agrees with the known binding energies of potassium ion to valinomycin in solutions. The difference in the relative peak intensities suggests a perturbation of ligand symmetry by solvents.     

Dephosphorylation of rabbit skeletal muscle glycogen synthase (phosphorylated by cyclic AMP-independent synthase kinase 1) by phosphatases

Phosphorylation of rabbit skeletal muscle glycogen synthase by cyclic AMP-independent synthase kinase 1 results in the incorporation of 4 mol of PO4/subunit. Incubation of the phosphorylated synthase with rabbit muscle phosphoprotein phosphatase brings about the hydrolysis of phosphates from all four major tryptic peptides and an increase in the synthase activity ratio from 0.01 to 0.85. Incubation of the phosphorylated synthase with calf intestinal alkaline phosphatase brings about the preferential hydrolysis of phosphates from three of the four major tryptic peptides and a slight increase in the four major tryptic peptides and a slight increase in the synthase activity ratio from 0.01 to 0.1. The phosphorylation site which is resistant to hydrolysis by calf intestinal alkaline phosphatase can be dephosphorylated by subsequent incubation with rabbit muscle phosphoprotein phosphatase. This dephosphorylation is accompanied by an increase in the synthase activity ratio to approximately 0.9. Measurements of the changes in the kinetic properties of the synthase samples dephosphorylated by alkaline phosphatase reveal that the phosphorylation sites susceptible to hydrolysis by alkaline phosphatase mainly affect the binding of glucose-6-P to the synthase. Comparison of the kinetic properties of the synthase samples dephosphorylated by alkaline phosphatase and by phosphoprotein phosphatase we find that the phosphorylation site resistant to hydrolysis by alkaline phosphatase affects both the binding of UDP-glucose and glucose-6-P to the synthase.     

Changes in RNA polymerase II in a cell cycle-specific temperature-sensitive mutant of hamster cells

tsAF8 cells are a temperature-sensitive mutant of BHK cells that arrest at the nonpermissive temperature in the G₁ phase of the cell cycle. The activity of solubilized RNA polymerase II and its ability to bind [³H]-γ-amanitin decrease in tsAF8 cells at 40.6°, with a half-life of ～ 10 hr. No appreciable changes occur in these two parameters in tsAF8 cells at 34° or in BHK cells at either 34° or 40.6°. Protein synthesis is not appreciably affected for at least 24 hr after tsAF8 cells are shifted to 40.6°. These results indicate that in tsAF8 cells at the nonpermissive temperature, there is a defect in either the synthesis, the assembly, or the stability of RNA polymerase II, and that the loss of RNA polymerase II molecules is not due to widespread cellular damage.     

A novel method for converting apolipoprotein B, the major protein moiety of human plasma low density lipoproteins, into a water-soluble protein.

We have developed a new technique to solubilize apolipoprotein B (ApoB) in aqueous solutions. The procedure involves stirring ApoB in 6 M guanidine/20 mM NH4Cl/NH4OH in the presence of cupric ammonia complexes at pH 9.7 for 20 h in N2, and then removing these reagents by a series of dialysis in N2. The resulting Cu(NH3)4(2)+-treated (Cu2+-treated) ApoB is soluble in aqueous buffers of pH above 8.3 or below 3. Parallel experiments carried out on control proteins, human albumin, alpha-lactalbumin, and insulin, indicated no change in molecular weight and no creation of a new NH2-terminal amino acid after Cu2+-treatment. By Edman degradation, the Cu2+-treated ApoB showed no detectable NH2-terminal amino acid. These results showed that the mechanism of Cu2+-solubilization of ApoB was not due to the cleavage of peptide bonds. Electrophoresis on urea-polyacrylamide gel, Cu2+-treated ApoB showed the same number of bands as the non-treated ApoB in the separating gels (7%) near the cathode, suggesting the heterogeneity of ApoB. In SDS-polyacrylamide gel (10%), the reduced and Cu2+-treated ApoB migrated with the similar mobilities to the monomer or dimer of human albumin. Antibodies raised against Cu2+-treated ApoB gave at least two immunoprecipitin lines against the Cu2+-treated ApoB as well as the non-treated guanidine-HCl-soluble ApoB, suggesting the presence of non-identical subunits.     

Production of potent antivenin against cobra venom with conjugated-cobrotoxin.

Cobra venom (Naja naja atra) and its fractions obtained by ammonium sulfate precipitation were subjected to chromatography on CM-Cellulose colum. A highly purified cobrotoxin obtained by the repeated chromatography on preparative CM-Cellulose column was 6.7 times more toxic than the original cobra venom. The toxin was detoxified by a bifunctional reagent, glutaraldehyde, to about 99.8% and utilized for immunization in animals. Mice received 4 weekly immunization with detoxified cobrotoxin and challenged one week after the last injection showed 60% protection in rabbits by immunization with detoxified cobrotoxin reached 360 LD50 neutralizing level against the cobra venom within 30 days. The results indicate that it is feasible and promising to prepare potent antivenin in animals by glutaraldehyde-treated cobrotoxin.     

Murine antigen H-2.7: In vitro phenotypic conversion of erythrocytes

The H-2.7 antigen in normal mouse serum can be passively adsorbed to H-2.7^– erythrocytes in 10 percent sucrose (low ionic strength) solution. This antigen can also be stripped off the H-2.7⁺ erythrocytes under the same conditions provided the H-2.7⁺normal serum is absent. The stripped red blood cells can regain the H-2.7 antigen upon reincubation with H-2.7⁺ normal serum. The attachment of the H-2.7 antigen to erythrocytes probably occurs via a specific receptor.Abbreviations used in this paper BSA bovine serum albumin - B10 C57BL/10Sn - HA hemagglutination - LIS low ionic strength solution - NMS normal mouse serum - PBS phosphate-buffered saline - PVP polyvinylpyrrolidone - RBCs red blood cells     

A radioimmunoassay method for the rapid detection of Candida antibodies in experimental systemic candidiasis

Rabbits were employed as experimental models to evaluate a solid-phase radioimmunoassay (RIA) method for the diagnosis of systemic candidiasis. Ten rabbits were inoculated subcutaneously to mimic superficial candidiasis and were found to produce no antibodies to Candida as determined by both immunodiffusion and RIA procedures. However, 94 per cent of 18 rabbits systemically infected by intravenous injection of Candida cells were observed to produce antibody as assessed by the RIA technique. These data encourage further tests with human sera and the continued development of this RIA procedure as a useful tool in the early serodiagnosis of systemic candidiasis.