The antioxidant action of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-α]pyrazin-3-one (MCLA), a chemiluminescence probe to detect superoxide anions

The antioxidant effect of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-α]pyrazin-3-one (MCLA), a Cypridina luciferin analog that acts as a chemiluminescence probe to detect O^⋅−₂, was investigated. MCLA produced a lag in oxygen consumption induced by cumene hydroperoxide in microsomes or by 2,2′-azobis (2-amidinopropane) dihydrochloride in liposomes and disappeared during the duration of the lag. MCLA profoundly inhibited the propagation reaction in Fe²⁺-dependent lipid peroxidation in liposomes, and MCLA disappearance accompanied by suppression of oxygen consumption markedly occurred in liposomes susceptible to peroxidation. Thiobarbituric acid-reactive substances in all systems used were also suppressed by MCLA dose dependently. These results indicate that MCLA has an antioxidant property through scavenging free radicals.     

Regulation of creatine phosphokinase B activity by protein kinase C

We previously reported that topical application of 12-o-tetradecanoylphorbol-13-acetate to mouse skin causes phosphorylation of epidermal proteins with molecular weights of 40,000 (p40) and 34,000 (p34). In the accompanying paper, p40 was identified as creatine phosphokinase B. Here we report that both in intact cells and in a cell-free system, phosphorylation of creatine hosphokinase B by protein kinase C resulted in an increase in its ability to catalyze the transfer of the high-energy phosphate of phosphocreatine to ADP, thereby producing ATP. H-7, a specific inhibitor of protein kinase C was found to abolish the increase in enzyme activity. Lineweaver-Burk plot analysis indicated that the increased activity was mostly due to a decreased Km for phosphocreatine. Phosphorylation and activation of creatine phosphokinase B may be a physiological response to maintain ATP balance when a protein kinase C pathway is stimulated.     

Purification and identification of creatine phosphokinase B as a substrate of protein kinase C in mouse skin in vivo

We previously described epidermal proteins with molecular weights of 40,000 (p40) and 34,000 (p34) as target proteins of protein kinase C in mouse skin carcinogenesis in vivo. In the present work, p40 was purified from mouse brain by the use of 32P-labeled p40 of BALB/MK-2 cells as a tracer. Following four lines of evidence indicate that p40 is creatine phosphokinase B. 1) The amino acid sequences of all peptide fragments of p40 from mouse brain were located in the primary structure of creatine phosphokinase B. 2) p40 of BALB/MK-2 cells was immunoprecipitated with goat antibody against human creatine phosphokinase B. 3) p40 of BALB/MK-2 cells was absorbed to and eluted from a creatine affinity column. 4) Purified creatine phosphokinase B was phosphorylated in vitro by purified protein kinase C, but not by cAMP-dependent kinase or casein kinase II.     

Human complex II (succinate-ubiquinone oxidoreductase): cDNA cloning of iron sulfur (Ip) subunit of liver mitochondria

Complex II (succinate-ubiquinone oxidoreductase) is an important enzyme complex of both the tricarboxylic acid cycle and of the aerobic respiratory chains of mitochondria in eukaryotic cell and prokaryotic organisms. In this study, the amino acid sequence of iron sulfur-subunit in human liver mitochondria was deduced from cDNA which was isolated by immunoscreening a human liver lambda gtll cDNA library. An isolated clone contains an open reading frame of 786 nucleotides and encodes a mature protein of 252 amino acids with a molecular weight of 28,804. The amino acid sequence was highly homologous with that of bovine heart (94.1%) which has been determined from the purified peptide and that of Escherichia coli sdh B product (50.8%). Striking sequence conservation was found around the three cysteine-rich clusters which have been thought to comprise the iron-sulfur centers of the enzyme. This is the first report on the cDNA sequence of mitochondrial complex II.     

Specific inactivation of glutathione S-transferases in class Pi by SH-modifiers

Treatment of Class Pi glutathione S-transferases (GST) such as rat GST P (7-7), human GST pi and mouse GST MII with 0.05-0.1 mM N-ethylmaleimide (NEM) in 0.1 M Tris-HCl (pH 7.8) resulted in almost complete inactivation of these forms, whereas no or less inactivation occurred for GSTs in Class Alpha and Mu under the same conditions. Inactivated GST P lost its S-hexyl-GSH-Sepharose column affinity. About 0.8 mol of [14C]NEM was found to be covalently bound to 1 mol of GST P subunit when 80% of the activity was lost. Similar treatment with N-dimethyl-amino-3,5-dinitrophenyl maleimide, a colored analogue of NEM, followed by trypsin digestion, HPLC and amino acid sequence analysis revealed that one cysteine residue at the 47th position from the N-terminal of the GST P subunit was preferentially modified. Subunits of GST P and GST pi are known to have 4 cysteine residues at the same corresponding positions. The present results suggest that the 47th cysteine residue may be located in the vicinity of the active site of Class Pi GSTs.     

Structures of the asparagine-linked sugar chain of glucose transporter from human erythrocytes

The asparagine-linked sugar chain of glucose transporter from human erythrocytes was quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. They were converted to radioactive oligosaccharides by NaB3H4 reduction after N-acetylation and fractionated by anion-exchange column chromatography and Bio-Gel P-4 column chromatography after sialidase treatment. Structural study of each oligosaccharide by exo- and endoglycosidase digestion and methylation analysis indicated that the glycoprotein contains a high-mannose-type oligosaccharide, Man9.GlcNAc.GlcNAc, and biantennary complex-type oligosaccharides with Man alpha 1----6(+/- GlcNAc beta 1----4)(Man alpha 1----3) Man beta beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc as their cores and the poly-N-acetyllactosamine composed of about 16 N-acetyllactosaminyl units as their outer chains. These structural features of the sugar moiety of glucose transporter are quite different from those of two major intrinsic glycoproteins of human erythrocytes, glycophorin A and band 3.     

Different effects of two proteinase inhibitors on insulin-induced cellular responses in rat adipocytes

Among various proteinase inhibitors, N-acetyl-L-tyrosine ethyl ester (ATEE), a chymotrypsin substrate analog, and N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), a trypsin inhibitor, showed significant inhibitory effects on insulin stimulated glucose transport in rat adipocytes. ATEE did not affect insulin binding, but inhibited insulin internalization. In intact adipocytes, ATEE inhibited tyrosine phosphorylation of the beta-subunit of the insulin receptor, a 170 kDa protein and a 60 kDa protein at almost the same concentration (ID50 = 0.24 +/- 0.05 mM, n = 4, mean +/- S.E.), but in a plasma membrane fraction, ATEE did not appreciably inhibit the tyrosine phosphorylation of the beta-subunit of the insulin receptor, TLCK did not inhibit insulin binding. At 0.25 mM, TLCK did not inhibit insulin internalization, but inhibited 70% of the insulin-stimulated glucose transport (ID50 = 0.19 +/- 0.02 mM, n = 7). TLCK inhibited insulin internalization at more than 0.25 mM. TLCK did not inhibit the tyrosine phosphorylation of the beta-subunit of the insulin receptor in intact cells or in the plasma membrane fraction. In intact cells, TLCK inhibited the phosphorylation of the 60 kDa protein and simultaneously it stimulated the phosphorylation of the 170 kDa protein more than 3-fold. These results indicate that there are at least two sites in the insulin-induced signal transduction pathway where proteinase inhibitors act to suppress the insulin signal transduction. A major ATEE site is very close to phosphorylation of the beta-subunit of the insulin receptor. On the other hand, TLCK inhibits a step(s) in the signal transduction pathway after the insulin receptor but before the glucose transporter.     

Multiple forms of rat calpastatin cDNA in the coding region of functionally unknown amino-terminal domain.

Partial mouse and rat calpastatin cDNAs containing functionally unknown amino-terminal regions were cloned by the polymerase chain reaction (PCR) method. Two types of clones were obtained for the rat calpastatin; one had a sequence similar to mouse and human calpastatins, while the other had a deletion of 38 amino acid residues in this region. Both types of the rat sequence differed from the previously reported rat calpastatin which had additional deletions. The occurrence of multiple forms of calpastatin suggests alternative splicing in the functionally unknown domain.