Reactive oxygen in skeletal muscle. II. Extracellular release of free radicals.

We have tested the hypothesis that diaphragm muscle fibers release superoxide anion radicals (O2-.) into the extracellular space. Fiber bundles were isolated from rat diaphragm and incubated in Krebs-Ringer solution containing cytochrome c (10(-5) M), a standard assay for O2-.. Bundles were either passive or active, i.e., directly stimulated to contract rhythmically. After 1 h, absorbance of reduced cytochrome c in the incubation medium was measured at 550 nm. Absorbance was greater in medium exposed to passive muscle than in medium without muscle (P < 0.01), indicating O2-. release by passive muscle. Absorbance was greater in medium exposed to active muscle than in that exposed to passive muscle (P < 0.01), an increase inhibited by superoxide dismutase (10(3) U/ml). Active bundles fatigued; bundles developing the lowest final stresses produced the greatest absorbance increases (P < 0.001), suggesting that the magnitude of fatigue was inversely related to O2-. release. We conclude that O2-. is released by diaphragm myocytes into the interstitium and surrounding medium, a process accelerated by fatiguing muscular contractions.     

Electron spin resonance studies of wild-type and mutant cytochromes P-450d: effects of mutations at proximal, aromatic and distal sites on g values

Low-temperature (6-40 K) electron spin resonance (ESR) spectra of cytochrome P-450d (P-450d) and its 17 mutants have been measured. The spectra of the wild-type and all mutant P-450ds showed signals at around g = 8, 3.7 and 1.7, while they didn't show any signal at around g = 2 up to 40 K. It was thus suggested that all of these P-450ds essentially take the ferric high-spin form. The g values of the proximal mutants were closer to those of the wild-type than those of the distal and aromatic mutants, suggesting that mutations at the distal and aromatic sites influence the electronic state of the heme more profoundly than those of the proximal site. The distal multiple mutants whose distal sequences are the same as those of the low-spin type P-450s such as rat P-450c, mouse P1-450 and P3-450 showed only high-spin ESR signals. Thus the spin state of P-450ds (the wild-type and all mutants) may not be solely due to specific characteristics of the distal site, but to the unique nature of the whole heme environment of P-450d. It is also suggested that the amino acids at the distal region of P-450d may be located close to the heme, so that the water molecule cannot bind to the heme, thus taking the high-spin state. Both the aromatic mutants showed rather large deviations of the g values from those of wild-type P-450d, suggesting that the aromatic region somehow interacts with the heme.     

Effect of EGF administration on EGF receptor mRNA in hCG producing tumor

In this study we examined the expression of EGF receptor mRNA after EGF administration in hCG producing tumor (choriocarcinoma). We transplanted the tissue of choriocarcinoma into female nude mice and investigated the effects of EGF on the growth of tumors, the binding activity of EGF receptor and the expression of EGF receptor mRNA in the tumor tissues. Two doses of EGF 5.0 micrograms, 50 micrograms and phosphate buffered saline as a control were injected subcutaneously every day for four weeks. Removed tumors were used for immunocytochemical studies and EGF receptor mRNA investigations. HCG and EGF receptors were detected immunocytochemically in the tumor. The low dose EGF employed stimulated the tumor growth while the high dose EGF inhibited the tumor growth compared with that of the control group. The binding activity of EGF receptor and the expression of EGF receptor mRNA also changed in accordance with the stimulation or inhibition of tumor growth. The growth of hCG producing tumor by EGF administration appeared to be dependent upon the binding activity of EGF receptor and the expression of EGF receptor mRNA.     

Potato and rabbit muscle phosphorylases: comparative studies on the structure,function and regulation of regulatory and nonregulatory enzymes

Summary Phosphorylases (EC 2.4.1.1) from potato and rabbit muscle are similar in many of their structural and kinetic properties, despite differences in regulation of their enzyme activity. Rabbit muscle phosphorylase is subject to both allosteric and covalent controls, while potato phosphorylase is an active species without any regulatory mechanism. Both phosphorylases are composed of subunits of approximately 100 000 molecular weight, and contain a firmly bound pyridoxal 5-phosphate. Their actions follow a rapid equilibrium random Bi Bi mechanism. From the sequence comparison between the two phosphorylases, high homologies of widely distributed regions have been found, suggesting that they may have evolved from the same ancestral protein. By contrast, the sequences of the N-terminal region are remarkably different from each other. Since this region of the muscle enzyme forms the phosphorylatable and AMP-binding sites as well as the subunit-subunit contact region, these results provide the structural basis for the difference in the regulatory properties between potato and rabbit muscle phosphorylases. Judged from CD spectra, the surface structures of the potato enzyme might be significantly different from that of the muscle enzyme. Indeed, the subunit-subunit interaction in the potato enzyme is tighter than that in the muscle enzyme, and the susceptibility of the two enzymes toward modification reagents and proteolytic enzymes are different. Despite these differences, the structural and functional features of the cofactor, pyridoxal phosphate, site are surprisingly well conserved in these phosphorylases. X-ray crystallographic studies on rabbit muscle phosphorylase have shown that glucose-1-phosphate and orthophosphate bind to a common region close to the 5-phosphate of the cofactor. The muscle enzyme has a glycogen storage site for binding of the enzyme to saccharide substrate, which is located away from the cofactor site. We have obtained, in our reconstitution studies, evidence for binding of saccharide directly to the cofactor site of potato phosphorylase. This difference in the topography of the functional sites explains the previously known different specificities for saccharide substrates in the two phosphorylases. Based on a combination of these and other studies, it is now clear that the 5-phosphate group of pyridoxal phosphate plays a direct role in the catalysis of this enzyme. Information now available on the reaction mechanism of phosphorylase is briefly described.     

Two new subspecies of Bacillus thuringiensis isolated in Japan: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) and Bacillus thuringiensis subsp. tochigiensis (serotype 19)

Bacteriological and serological characteristics of three Bacillus thuringiensis isolates obtained in Japan were investigated. They formed typical rhomboidal parasporal inclusions but flagellar (H) antigens of these isolates were different from those of the known 17 H serotypes of B. thuringiensis. The three isolates were divided into two new serotypes (serotypes 18 and 19). The serotype 18 isolate (3–71) produced thermostable exotoxin and the inclusions of this isolate were toxic to larvae of the silkworm, Bombyx mori, but nontoxic to larvae of the mosquito, Aedes aegypti. The other isolate (119-72) belonging to serotype 18 produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. However, other bacteriological properties of the isolate 119-72 were similar to those of the isolate 3–71. The serotype 19 isolate (117-72) produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. Acid production from saccharose and the production of brownish purple pigment were observed in the two serotype 18 isolates, while neither of them was observed in the serotype 19 isolate. In other 29 biochemical properties tested, there was no difference among the three isolates. Based on these characteristics, the following two subspecies names are proposed: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) for the type strain 3–71 and Bacillus thuringiensis subsp. tochigiensis (serotype 19) for the type strain 117-72.     

Inhibition of the positive inotropic effect of anthopleurin-A (AP-A) by dantrolene

The effect of dantrolene on the positive inotropic effects (PIE) of three cardiotonic agents was assessed on rat and rabbit atria. Dantrolene (10^?5M) had no effect on contractile tension or on the PIE to isoproterenol (10^?10?10^?7M) or ouabain (10^?6?10^?4). The dose-response curve for the PIE of anthopleurin-A (AP-A) was shifted to the right in rat and rabbit atria by dantrolene (10^?4?10^?6M). The maximum effect and the concentration of AP-A required for it remained the same. The results suggest that the PIE of AP-A involves intracellular translocation of calcium, unlike those of isoproterenol and ouabain which require increased transmembrane calcium flux. This conclusion is supported by the observation that the exchange and distribution of the labile calcium involved in excitation-contraction coupling was unaffected by AP-A (10^?8M), by dantrolene (10^?6M) or by the combination. Therefore, AP-A may produce its cardiotonic effect by a action at an intracellular site, a mechanism unlike that of isoproterenol or ouabain.     

Electrophoretic and biochemical studies of human aldehyde dehydrogenase isozymes in various tissues

Four major ALDH isozymes have been identified in human tissues using starch gel electrophoresis and isoelectric focusing. The isozyme bands have been termed as ALDH I, II, III and IV according to their decreasing electrophoretic migration and increasing isoelectric point. The isozymes have been partially purified via preparative isoelectric focusing. Kinetic characteristics of ALDH I and II were found to be quite similar to ALDH enzyme 2 and enzyme 1 described earlier by Greenfield and Pietruszko (Biochem Biophys Acta, $\text{483}$ 35–45 1977). ALDH III and IV showed a very high Km for propionaldehyde (1.0–1.5 mM at pH 9.5) and were not inhibited by disulfiram at pH 9.5. A variant phenotype of ALDH which lacked in isozyme I was detected in various tissues from Japanese individuals. Comparative kinetic properties of normal and variant enzyme are given.     

Soluble and bound forms of intracellular acid carboxypeptidase inAspergillus saitoi

The enzymological, physical, and immunological properties of soluble and bound forms of intracellular acid carboxypeptidase isolated from fresh mycelia ofAspergillus saitoi are reported. In the broken mycelia, about 60% of the total activity was found in the 2,000×g precipitate, with most of the remainder in the 100,000×g supernantant. The highly purified enzymes, I_a and I_b, from the 100,000×g supernatant were found to be homogeneous by such criteria as disc gel electrophoresis at pH 9.4 The bound enzyme, II, was solubilized from the 2,000×g precipitate by self-digestion at pH 6.4 and was highly purified by chromotography. The two forms of intracellular enzymes, the soluble enzymes (I_a and I_b) from the 100,00×g supernatant and the solubilized enzyme (II) from the 2,000×g precipitate, were closely related to, but not completely identical with, the extracellular acid carboxypeptidase.