Journal of applied biochemistry最新文献

Myosin was extracted from fresh rabbit liver using a solution with an ionic strength of 0.3 with two protease inhibitors and ATP. The myosin fraction was centrifuged at high speed in the presence of ATP and MgCl2. Its precipitate was dissolved in 0.6 M KCl, and the solution was treated with ammonium sulfate (30-60%). The fraction that precipitated was dissolved in 0.6 M KCl and put on a Sephacryl column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the myosin obtained was 90% pure. Its molecular weight was 220,000 by gel electrophoresis; by Sephacryl S-300 column chromatography, it was found to be a complex at high ionic strengths. The protein had high ATPase activity, comparable to that of the myosin prepared by D. L. Brandon (1976, Eur. J. Biochem. 65, 139-146). Electron micrographs of our myosin looked like myosins from nonmuscle cells purified by other workers. Slow preparation gave poor yields of myosin with low ATPase activity and extra bands on SDS-PAGE. Rapid handling of fresh materials is essential for obtaining myosin of satisfactory quality. Our simple method saves time and reagents.

A highly sensitive radioimmunoassay method has been established for the phosphomannosyl receptor using an antibody to the bovine receptor. The amount of the receptor in extracts from total membrane fractions varied remarkably in different tissues and species. The amount in liver was the highest and that in brain was the lowest among bovine tissues. Human tissues contained significant amounts of material cross-reacting to the antibody, the highest in spleen and the lowest in kidney. The physiological significance of the receptor is discussed in terms of the intracellular transport of lysosomal enzymes in human tissues.

Daunomycin interacts with canine and bovine erythrocytes making them permeable to [14C]sucrose, a nonpermeant. The drug causes the red blood cell (RBC) to swell and form an echinocyte-stomatocyte cell type. Defects in the RBC membrane were observed for murine RBC as well. Thus, daunomycin interacts with RBC such that its deleterious action would not make this drug a promising candidate for encapsulation in carrier erythrocytes. Contrary to previous reports, daunomycin easily permeates murine RBC and avidly binds to the cells; as much as 70% of the drug remains associated with normal murine RBC. Several different approaches were taken to unequivocally establish that daunomycin does not entrap in canine RBC in a classical manner. The extent of interaction of the drug with RBC does not appear to be RBC specific because similar results were obtained with canine, bovine, and murine RBC.

An enzymatic method for the determination of serum cholinesterase (ChE) activity is described. The method is based on the liberation of acetate from acetylcholine as a substrate by ChE and the conversion of the acetate to acetylphosphate and ADP in the presence of ATP by acetate kinase. The produced ADP is coupled with pyruvate kinase and lactate dehydrogenase in the presence of phosphoenolpyruvate and NADH. The amount of NADH consumed is determined by absorbance at 340 nm. The reaction proceeds stoichiometrically, and the dilution curve is linear up to 3300 U/liter. The results obtained by this method show a good correlation with those obtained by the usual methods.

The literature dealing with sulfite preservation of meat products is reviewed. Discussion is centered on three aspects: (i) the elective action of sulfite, whereby its presence in meat products encourages the development of an association of Gram-positive bacteria (Brochothrix thermosphacta and homofermentative lactobacilli) and yeasts. Unsulfited products tend to be dominated by Pseudomonas fragi at chill, and Enterobacteriaceae at ambient temperatures; (ii) the diminution of the preservative potential of a meat product, which is associated with the binding of sulfite by acetaldehyde-producing yeasts; and (iii) the sparing action that sulfite has on the carbohydrates contained in the meat or included as an ingredient.

A simple and reliable procedure for removal of AMP from NADP preparation is described. In this procedure, a mixture of AMP and NADP solution is first incubated with 5'-nucleotidase to hydrolyze AMP to adenosine and inorganic phosphate (Pi). The reaction mixture is then applied to a Dowex 1 (formate) column. Adenosine and 5'-nucleotidase are removed by washing the column with 20 mM HCOOH. NADP is finally eluted with 3.5 M HCOOH followed by precipitation and washing with acetone. The yield of salt-free NADP is about 80%. Although Pi is coeluted with NADP in the acid form (H3PO4), it is removed during the precipitation and repeated washing with acetone. A slight modification of this procedure for simultaneous removal of AMP, ADP, and ATP from NADP preparation has also been discussed.

Potentiometric as well as thermal titrations of phytic acid and its calcium complexes have been conducted using both the batch and titration microcalorimeters. For phytic acid, the experimental values by either method are in excellent agreement. For the calcium complexes, the total number of groups and the total heat evolved are in agreement but the placement of the curves is different due primarily to the differences in calcium concentration. Binding of calcium by phytic acid is endothermic for the pH range 2.0-12.0 while the heat of dilution of 1 M CaCl2 is exothermic. The binding at pH greater than 11 gives a value of 22.5 +/- 0.9 kcal mol-1. The same enthalpy of binding (22.3 +/- 0.6 kcal mol-1) could be calculated for the entire pH range studied which requires a knowledge of the observed heat of binding, the thermal titration curves of the acid and its calcium complex, and the change in the hydrogen ion environment. Inspection of the thermal binding curves at pH greater than 11 indicates that a number of step-binding constants are involved and that 5.2 mol calcium are bound/mole phytic acid. This value has been confirmed by atomic absorption spectroscopy. Both the thermal and the potentiometric curves are reversible either by the instantaneous injection of acid or base or by continuous titration. Values for the ionization constants (as pK') and the enthalpy of ionization (as delta Hi) have been estimated by computer assisted curve fitting.

A new rapid technique is developed for the determination of complement activity in a large number of samples. Following serial dilution of complement, hemolysis is performed in the same microtiter plate. After the reaction, the degree of hemolysis in wells of the plate is determined spectrophotometrically by measurement of "absorbance" (light scattering) at 630 nm, without additional procedures. This method can find application in clinical and experimental biochemistry for the analysis of a large (up to thousands) number of samples.

In a new strain of rat with inherited cataracts, a postnatal increase in serum lipid peroxide level was observed. It reached the maximum on the 13th day after birth. This seems to be a reflection of the increased lipid peroxides in the liver. In accordance with the increase in serum lipid peroxide level, the occurrence of vacuoles in the subcapsular fibers of the posterior region of the lens was observed by electron microscopy, suggesting a relationship between the increase in serum lipid peroxides and the provocation of cataract formation.

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) from the thermophilic bacteria, Bacillus stearothermophilus, was purified and its properties were examined. The enzyme was shown to consist of four identical subunits, each of about Mr 50,000. This enzyme utilized both NADP+ and NAD+ as cofactors, and the maximum velocity for both cofactors was similar. However, the Km values were quite different from each other, being 0.016 and 1.64 mM for NADP+ and NAD+, respectively. From the analysis of sulfhydryl groups it was shown that there is one sulfhydryl group and one disulfide bridge per subunit. This sulfhydryl group had no reactivity with 5,5'-dithiobis(2-nitrobenzoic acid) in the absence of guanidine hydrochloride. The enzyme showed a remarkable thermostability as well as storage stability.