Purification and characterization of human mitochondrial creatine kinase. A single enzyme form

Purification of human mitochondrial creatine kinase has been difficult and procedures that were highly successful in purifying canine enzyme failed for human mitochondrial creatine kinase. In the present study, we employed ultracentrifugation to remove the lipid, urea to prevent aggregation, followed by a final step of chromatofocusing which yielded a preparation of human mitochondrial creatine kinase with a specific enzyme activity of greater than 400 IU/mg. Biochemical and immunological characterization showed the preparation to be highly pure and free of even trace amounts of other creatine kinase isoenzymes. Antiserum specific for mitochondrial creatine kinase was developed which exhibited no cross-reactivity to cytosolic creatine kinase and mitochondrial creatine kinase did not cross-react with antiserum to the cytosolic forms. Marked differences were noted, both biochemically and immunologically, between mitochondrial creatine kinase and the cytosolic forms. Human mitochondrial creatine kinase was shown to have a molecular weight of around 82,000 and to be composed of two subunits of equal molecular weights around 41,000. Aggregates of mitochondrial creatine kinase were observed with molecular weights of around 200,000 in the absence of urea or if isolated from material after having undergone proteolysis. Isolation from fresh material or in the presence of urea inhibited aggregate formation for both canine and human mitochondrial creatine kinase. Despite claims of several investigators that mitochondrial creatine kinase exhibits two to three forms with varying molecular weights, our data indicate a single enzyme form made up of a subunit with a molecular weight of 41,000 and the high molecular weight aggregates appear to be induced artifacts. A radioimmunoassay was developed for human mitochondrial creatine kinase which, with appropriate modifications, should detect mitochondrial creatine kinase in human plasma.     

Serum Albumin Washing Specifically Enhances Arachidonate Incorporation into Synaptosomal Phosphatidylinositols

Arachidonate incorporation into synaptosomal phospholipids was shown to be affected by factors including the procedure for preparation of the membrane fractions and preincubation of synaptosomes prior to assay of incorporation of arachidonate into both phosphatidylcholine (PC) and phosphatidylinositol (PI). However, the inhibition toward incorporation into PIs, but not PCs, was fully reversed when the membranes were washed with bovine serum albumin. A twofold increase in arachidonate incorporation into PIs was also observed when freshly prepared synaptosomes were washed with serum albumin immediately before assay of incorporation activity. The inhibitory action is thought to be due to an increase in polyunsaturated fatty acids and/or their oxidation products which may then elicit a special effect on the acyltransferase responsible for transferring arachidonate into phosphatidylinositols. The differences in fatty acid uptake and response to serum albumin also suggest the presence of different acyltransferase for acyl transfer to PIs and PCs.     

Detergent Effects on the Phosphatidylinositol-Specific Phospholipase C in Rat Brain Synaptosomes

In the presence of Ca2+ (2.5 mM) and using [14C]arachidonoyl phosphatidylinositol (PI) membrane as substrate, phosphatidylinositol-specific phospholipase C (PI-PLC) (EC 3.1.4.10) in rat brain synaptosomes was activated by deoxycholate but not taurocholate. Calcium stimulated enzymic hydrolysis by both detergents, but the stimulatory effect of taurocholate was less than that of deoxycholate. Peak stimulation for deoxycholate was observed at 1 mg/ml, whereas that for taurocholate was 4 mg/ml. When 1 mM EDTA was added to the taurocholate (4 mg/ml) and Ca2+ (3.5 mM) system, synaptosomal PI-PLC activity was greatly stimulated, to almost the same level as the deoxycholate + Ca2+ system. This system required the presence of all three factors, and EGTA could not effectively replace EDTA in the stimulatory action. The detergent-induced hydrolysis of synaptosomal PI by the deoxycholate + Ca2+ and the taurocholate + Ca2+ + EDTA systems was strongly inhibited by divalent metal ions such as Zn2+, Cu2+, Pb2+, and Fe2+, whereas Mg2+ and Ca2+ were ineffective. Nevertheless, only the deoxycholate + Ca2+ system was responsive to enzyme inhibition by membrane-perturbing agents such as lysophospholipids and free fatty acids. The specific requirement for EDTA in the taurocholate system may be due to the release of a pool of inhibitory divalent metal ions from the membranes.     

Rapid Test for Urease and Phenylalanine Deaminase Production

A rapid urea-phenylalanine medium was effective for the identification of Proteus and, with one exception, Providencia. Most Klebsiella and a few Enterobacter were urease-positive with this method.     

A combination rapid and standard method for identification of Candida albicans

A medium consisting of an aqueous extract of zein, lactose, and Tween 80 is used together with an overlay of 1 % Tween 80 and coverslipping to provide a combined rapid (germ tube) and standard (chlamydospore) method for diagnosis ofCandida albicans. The method is exquisitely sensitive for diagnosis ofC. albicans but lumps chlamydospore-producing strains ofC. tropicalis withC. albicans.     

THE METABOLISM OF PALMITIC ACID IN THE PHOSPHOLIPIDS, NEUTRAL GLYCERIDES AND GALACTOLIPIDS OF MOUSE BRAIN

Abstract— Following intracerebral injection, [¹⁴C]palmitic acid was rapidly incorporated into a variety of brain lipids. After 12 hr, 78 per cent of the lipid radioactivity was in phospholipids, 15 per cent was in triacylglycerols, 1 per cent each was in free fatty acids and galactolipids, and the remainder was in other neutral glycerides. Over 65 per cent of the phospholipid radioactivity was found in the choline phosphoglycerides but this proportion decreased substantially with time. At later times, increasing portions of the radioactivity were present in the monounsaturated acyl groups and the alkenyl groups but no radioactivity was detected in cholesterol or polyunsaturated acyl groups. These results indicate that most of the extensive recycling of radioactivity took place without oxidative degradation of the palmitoyl groups. The relative rates of incorporation of radioactivity were compared at 12 hr after injection. The specific radioactivities of the serine, ethanolamine, and choline phosphoglycerides had ratios of 6:3:2 based on the palmitoyl group content and 1:2:4 based on their phosphorus content. The specific radioactivities of galactolipids with O -acyl groups were higher than the specific radioactivitiesof cerebrosides or cerebroside sulphates. A new solvent mixture for thin-layer chromatography of brain galactolipids was described (chloroform-acetone-methanol-water, 60:20:20:1, by vol.).     

Sex-related Resistance in Hamsters to Adenovirus-12 Oncogenesis II. Influence of Virus Dose

A significantly higher proportion of female hamsters developed tumors than did males given the same dose of adenovirus-12 (Huie) at birth over a dose range from 10(5.0) to 10(7.0)tcd(50) for human embryonic kidney cells. The 50% tumor dose (td(50)) was calculated to be 10(5.90)tcd(50) for females and 10(6.27) for males. Tumor response patterns induced with approximate td(50) inocula, 10(6.0) for females and 10(6.3) for males, were quite similar. The greater susceptibility of females was not found to be characteristic of a single strain of hamsters; nor was it attributable to a single lot of virus, to a single type of human cell used to produce the virus, nor to the degree of purification of the virus inoculum. The inoculation route did not appear to be of importance. Inasmuch as the foregoing extrinsic factors were of little influence, it was concluded that the mechanism is host-mediated, presumably hormonally controlled. The possibility that female cells, independent of host control, are more susceptible to adenovirus-12 oncogenesis than male cells has not been explored. Tumor regression occurred in 20% of the 211 tumors in males and in 15% of the 355 tumors in females. Adenovirus-12 T-antibody was detected in all but six of 473 sera tested from tumor-bearing hamsters and in 50% of 94 sera tested from non-tumor-bearing animals given virus at birth. Antibodies in the latter group were detected almost exclusively by indirect immunofluorescence. This technique appears to be extremely sensitive for detection of low levels of adeno-12 T-antibodies. The implications of T-antibody in nontumor-bearing virus-injected hamsters are discussed. Sera from normal hamsters were free of T-antibody.