Incorporation of methionine by a soluble enzyme system from Escherichia coli

A soluble fraction from Escherichia coli B was found to incorporate methionine into 95°C CCl₃COOH-insoluble fraction. The incorporation required methionyl-tRNA synthetase, methionine tRNA, ATP, Mg²⁺ and bovine milk casein. The casein could be replaced by arginylated bovine serum albumin and arginylated bovine α-lactalbumin. A mixture of 19 amino acids other than methionine and GTP had no effect on the incorporation. KCl was rather inhibitory. Puromycin, RNase A and trypsin inhibited the incorporation, while DNase I did not. The soluble fraction also incorporated the methionyl moiety of methionyl-tRNA. This incorporation was not affected by the addition of free methionine.     

Heterogeneous mutations in the glycogen-debranching enzyme gene are responsible for glycogen storage disease type IIIa in Japan

Glycogen storage disease type IIIa (GSD IIIa) is an autosomal recessive disorder caused by deficiency of the glycogen-debranching enzyme (AGL). Recent studies of the AGL gene have revealed the prevalent mutations in North African Jewish and Caucasian populations, but whether these common mutations are present in other ethnic groups remains unclear. We have investigated eight Japanese GSD IIIa patients from seven families and identified seven mutations, including one splicing mutation (IVS 14+1G-->T) previously reported by us, together with six novel ones: a nonsense mutation (L124X), a splice site mutation (IVS29-1G-->C), a 1-bp deletion (587delC), a 2-bp deletion (4216-4217delAG), a 1-bp insertion (2072-2073insA), and a 3-bp insertion (4735-4736insTAT). The last mutation results in insertion of a tyrosine residue at a putative glycogen-binding site, and the rest are predicted to cause synthesis of truncated proteins lacking the glycogen-binding site at the carboxyl terminal. Thirteen novel polymorphisms have also been revealed in this study: three amino acid substitutions (R387Q, G1115R, and E1343 K), one silent point mutation (L298L), one nucleotide change in the 5'-noncoding region, and eight nucleotide changes in introns. Haplotype analysis with combinations of these polymorphic markers showed L124X, IVS14+1G-->T, and 4216-4217delAG to be on different haplotypes. These results demonstrate the importance of the integrity of the carboxy terminal domain in the AGL protein and the molecular heterogeneity of GSD IIIa in Japan.     

A sensitive and reproducible procedure for the assay of arginyl-tRNA: Protein arginyl transferase

The activity of arginyl-tRNA: protein arginyl transferase was found to be enhanced four- to sevenfold by substituting bovine α-lactalbumin for bovine serum albumin, the standard acceptor protein used thus far. With α-lactalbumin as the acceptor protein in place of serum albumin, a sensitive and reproducible procedure for the transferase assay was established.     

PURIFICATION AND CHARACTERIZATION OF CARNOSINE SYNTHETASE FROM MOUSE OLFACTORY BULBS

Carnosine synthetase was purified about 500-fold from mouse olfactory bulb to a specific activity of approx 25 nmol/min/mg. This is an increase of 800-fold over that previously reported for this enzyme from rat brain and 11 times higher than the most highly purified enzyme from chicken pectoral muscle. ATP was essential for activity and could not be replaced by ADP. NAD had no effect on the synthesis of carnosine. Of the β-alanine analogues tested, the purified mouse enzyme incorporated only γ-aminobutyric acid and β-amino-n-butyric acid into peptide linkage with histidine. Synthesis of carnosine by the mouse olfactory bulb enzyme was competitively inhibited by the histidine analogues, 1-methyl histidine and 3-methyl histidine, with K_i values which were at least 40 times the K_m value for histidine (16 μM). Ornithine and lysine were more efficient β-alanine acceptors than 1-methyl histidine for the mouse enzyme. Enzyme from olfactory epithelium and leg skeletal muscle of mice also showed higher K_i values for 1–methyl histidine than the K_m value for histidine. In contrast, carnosine-anserine synthetase from chicken pectoral muscle gave K_m values for histidine, 1-methyl histidine and 3-methyl histidine, which were all in the range of 4–12 μM. The differences in substrate specificity between the enzyme from mouse and chicken implies alternate routes of anserine synthesis in these species and predicts the occurrence of certain novel peptides in mouse brain.     

Flexibility in the phosphorylase catalytic reaction. Glucosyltransfer from pyridoxal (5')-triphospho(1)-alpha-D-glucose to glycogen catalyzed by phosphorylase

When rabbit muscle phosphorylase reconstituted with pyridoxal (5')-diphospho(1)-alpha-D-glucose is incubated with glycogen, its glucosyl moiety is transferred to the nonreducing end of glycogen with the formation of a new alpha-1,4-glucosidic linkage. This finding provided the first evidence for the direct phosphate-phosphate interaction between the coenzyme pyridoxal 5'-phosphate and the substrate alpha-D-glucose 1-phosphate in the phosphorylase catalytic reaction (Takagi, M., Fukui, T., and Shimomura, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3716-3719). We have examined whether pyridoxal(5')triphospho(1)-alpha-D-glucose can act in a similar manner to the diphospho compound or not. In the absence of glucan the enzyme-bound triphospho compound was stable for 1 day at pH 6-9. In the presence of glucan, however, its glucosidic linkage was cleaved, and the glucosyl moiety liberated was transferred to glycogen with the formation of a new alpha-1,4-glucosidic linkage. Allosteric activator AMP accelerated the reaction and allosteric inhibitor glucose 6-phosphate showed the reverse effect. The pH optimum of the reaction was pH 8.1-8.4. Mg2+ slightly but significantly accelerated the reaction, whereas Mn2+ and Ca2+ inhibited the reaction. These results indicate that the glucosyltransfer from the triphospho compound occurs in an identical manner to that from the diphospho compound. Based on the present and previous data, we discuss the catalytic mechanism of phosphorylase, especially in comparison with that of phosphoryltransferases.