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The phosphotransacetylase gene ( pta ) from Escherichia coli strain K-12 1100 was identified in a cloned fragment of chromosomal DNA (Yamamoto-Otake, H., Matsuyama, A. and Nakano, A. (1990) Appl. Microbiol. Biotechnol. 33, 680–682). Overexpression in E. coli confirmed the presence of the pta gene within the cloned fragment. DNA sequence analysis of the cloned pta gene indicates that the predicted phosphotransacetylase polypeptide chain is 713 amino acids in length. The carboxyterminal region of the E. coli phosphotransacetylase shows 42.6% sequence identity with the corresponding enzyme from Methanosarcina thermophila (142 out of 333 residues in corresponding positions are identical). Several short regions of high sequence identity may be structurally or functionally important for enzymic activity.

The neutral proteinase II from Aspergillus oryzae (NpII) is a zinc proteinase with three intramolecular disulfide bonds. NpII is most unstable after 10 min at about 75 degrees C, but regains stability beyond this temperature and is relatively stable at 100 degrees C. We analyzed the thermal stability of wild-type NpII and apo NpII. The results suggested that NpII unfolds reversibly upon incubation up to 100 degrees C, and that the irreversible inactivation observed is mainly due to autoproteolysis. To further understand the stability, a mutant NpII (Cys78-->Ala) lacking one of the disulfide bonds, was produced in a heterologous yeast expression system. The mutant NpII showed a similar stability profile, but the most unstable temperature and the most catalytically active temperature decreased to the same extent (around 10 degrees C), confirming that autoproteolysis is the main cause of the irreversible inactivation. Several lines of evidence presented in this study demonstrated that the thermal stability of o++NpII is attributed to reversible thermal unfolding and autoproteolysis.

We have cloned a cDNA encoding Luciola lateralis (a common firefly in Japan) luciferase from a cDNA library of lantern poly(A) + RNA, using a cDNA of L. cruciata (another common firefly in Japan) luciferase as a probe. The primary structure of L. lateralis luciferase deduced from the nucleotide sequence was shown to consist of 548 amino acids with a molecular weight of 60 132. Sequence comparison indicates that L. lateralis luciferase has significant sequence identity (94%) to L. cruciata luciferase, and that it has less sequence similarity (67%) to Photinus pyralis (a North American firefly) luciferase. The isolated cDNA clone, when introduced into Escherichia coli , directed the synthesis of enzymatically active luciferase under the control of the lacZ promoter.

Luciferases of Luciola cruciata and Luciola lateralis, LcL and LlL, were purified to homogeneity by ammonium sulfate precipitation, gel-filtration column chromatography, and hydroxyapatite HPLC. The molecular masses of the enzymes determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were both 62 kDa, almost identical to that of Photinus pyralis (PpL). LcL was found to be similar to PpL in thermal stability, pH stability, and the wavelength of maximum light intensity. LlL was superior to LcL and PpL in thermal and pH stability, and the reaction catalyzed by LlL emits green light with a peak intensity at 552 nm, which is 10 nm shorter in wavelength than those of PpL and LcL.

1. 1. The complex formation of calf thymus DNA with pancreatic ribonuclease A (ribonucleate pyrimidinenucleotido-2′-transferase (cycling), EC 2.7.7.16) was studied through ultracentrifugation, competitive inhibition of ribonuclease activity and gel filtration on Sephadex G-200. Ultracentrifugation data indicated that under low ionic strength ribonuclease formed a soluble complex with DNA at least in the range of pH 7.5 to pH 8.5 and this ability to form the complex was strongly affected by the presence of 0.05 M NaCl. 2. 2. The activity of ribonuclease was competitively inhibited by DNA when assayed in 0.01 M phosphate buffer (pH 7.5). The denatured DNA was much more effective than native DNA in inhibiting ribonuclease activity. 3. 3. Through gel filtration on Sephadex G-200, ribonuclease was found to form a complex with DNA in a definite combining ratio in 0.01 M phosphate buffer (pH 7.5). The combining ratios were calculated to be about one ribonuclease molecule per 500 000–1000 000 daltons of native DNA and 8000–10 000 daltons of denatured DNA. In the presence of 0.1 M NaCl the complex dissociated into free enzyme and DNA. The combining ratio of ribonuclease to native DNA was about 100 times smaller than that of denatured DNA and the discrepancy is discussed in connection with specific sites on DNA. The thermal denaturation profile of DNA was not affected by ribonuclease in the amount of this combining ratio.