Isolation of cDNA for bovine stomach 155 kDa protein exhibiting myosin light chain kinase activity.

Two proteins with myosin light chain kinase activity and electrophoretic molecular weights of 155,000 and 130,000 were each isolated from bovine stomach smooth muscle [Kuwayama, H., Suzuki, M., Koga, R., & Ebashi, S. (1988) J. Biochem. 104, 862-866]. The 155 kDa component showed a much higher superprecipitation-inducing activity than the 130 kDa component, when compared on the basis of equivalent myosin light chain kinase activity. In this study, we isolated a cDNA for the entire coding region of the 155 kDa protein. The deduced amino acid sequence revealed a high degree of similarity to those of chicken and rabbit smooth muscle myosin light chain kinases. Multiple motifs, such as three repeats of an immunoglobulin C2-like domain, a fibronectin type III domain, and unusual 20 repeats of 12 amino acids were detected in the sequence. Part of the amino-terminal sequence was similar to that of the actin- and calmodulin-binding domain of smooth muscle caldesmon. These observations suggest that the 155 kDa protein has additional functions other than its enzymatic activity. Two mRNAs of 6.0 and 2.6 kb in length in the bovine stomach smooth muscle RNAs were hybridized with cDNA probes. The 2.6-kb RNA probably encodes telokin, which is the carboxyl terminus of smooth muscle myosin light chain kinase. mRNAs with identical lengths were also detected in bovine aorta.     

Settling-site selection and survival of two scale insects,Ceroplastes rubens andC. ceriferus,on citrus trees

We studied settling-site selection and the resulting survival of two sessile scale insects, Ceroplastes rubens and C. ceriferus, in the citrus tree, Citrus unshiu, in central Japan. C. rubens preferred 0-year-old twigs most as a settling-site; the density of nymphs settling on 0-year-old twigs was significantly higher than those on ≥1-year-old twigs, and few nymphs settled on ≥3-year-old twigs. The mean survival rates from settling until reproduction in the next year were significantly higher on more preferred twigs than on less preferred ones. In C. ceriferus, nymphs significantly preferred 1- and 2-year-old twigs to 0- and ≥3-year-old ones, and the mean survival rates on the more preferred 1- and 2-year-old twigs were significantly higher than those on less preferred ≥3-year-old twigs. However, the survival rate on less preferred 0-year-old twigs was slightly higher than those on 1- and 2-year-old ones. Thus, in both species of scale, it was the preferred twigs which were more profitable sites for survival after settling, except for less preferred 0-year-old twigs for C. ceriferus. In both scale species, most mortality was due to growth cessation, which is believed to be related to the twig quality as a food source. Predators and parasitoids were minor mortality factors. Both species showed constant survival rates until the density of settled nymphs exceeded double the “upper-limit” density, whereupon they decreased drastically. Nymphs of C. rubens settling on twigs of high scale density showed a spacing-out distribution, those of C. ceriferus did not. In C. rubens, an increase in preference for originally less profitable twigs at the later stage of the settling season was observed, but not in C. ceriferus. Accordingly, individuals of C. rubens showed a stronger tendency to avoid conspecifics than did C. ceriferus. Although nymphs of the two scales clearly preferred more profitable sites, their settling-site selection did not agree with the predictions from the ideal free distribution theory (Fretwell and Lucas, 1970). The discrepancies were (1) frequent settling on less profitable sites at the early stage of the settling season, (2) insufficient utilization of the most profitable twigs, and (3) virtually 100% mortality on overcrowded twigs under conditions where unoccupied profitable twigs still remained. These discrepancies are thought due to the limited dispersal time of nymphs.     

Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase.

Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl. The recent solution of the crystal structure of the alpha(1) catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 A resolution paved the way for a detailed examination of the molecular basis of substrate specificity in this enzyme. The crystal structure suggests that the side chains of Thr103, Leu48 and Leu194 are involved in substrate recognition. Three single site mutants (L48A, T103S and L194A) were overexpressed and their structures were solved to 2.3 A resolution or better by X-ray diffraction methods. Enzyme kinetics showed that, compared with wild-type protein, all three mutants have higher relative activity against phospholipids with sn-2 acyl chains longer than an acetyl. However, for each of the mutants we observed an unexpected and substantial reduction in the V(max) of the reaction. These results are consistent with the model in which residues Leu48, Thr103 and Leu194 indeed contribute to substrate specificity and in addition suggest that the integrity of the specificity pocket is critical for the expression of full catalytic function, thus conferring very high substrate selectivity on the enzyme.     

Biodiesel preparation from microalgae lipid by two-step lipase catalysis

To overcome the high energy-consuming process of microalgae drying, a two-step lipase catalysis technique for the preparation of biodiesel from microalgae lipid of Chlorella spp. was developed. In the first step, free fatty acids (FAAs) and triacylglycerols (TAGs) are released after cell disruption and extracted, while the TAGs were hydrolysed by free lipase in aqueous phase. In the second step, FAAs were esterified with ethanol in the catalysis of free suspended lipase. The maximum rate of hydrolysis and esterification was 93.6% and 91.3%, respectively. The effects of reaction parameters, such as reaction time, enzyme amount, water content and molar ratio of lipid to ethanol on hydrolysis or esterification, were investigated. The results indicated that two-step reaction process (hydrolyse esterify) for biodiesel production were feasible.