Yoshihiro Amaya

Cloning and sequence analysis of a full length cDNA encoding human mitochodrial 3-oxoacyl-CoA thiolase

The cDNA sequence of human mitochondrial 3-oxoacyl-CoA thiolase was determined. The nucleotide sequence contains an open reading frame of 1191 base pairs and encodes an amino acid sequence of 397 residues which exhibits 86.6% homology with that of the rat enzyme. Northern blot analysis gave a single mRNA species of 1.6 kb in the human liver, fibroblasts and intercostal muscle.

Cloning and expression in Escherichia coli of cDNA for arginase of rat liver

A cDNA expression library constructed in a plasmid pUC8 from poly(A)+ RNA of rat liver was screened immunologically, using an antibody against arginase of rat liver. A cDNA clone was isolated and identified by hybrid-selected translation. The clone contained an insert approximately 1.35 kilobase pairs in length. In the bacterial clone, we detected a specific protein of Mr = about 43,000 that is slightly larger than the purified arginase (Mr = about 40,000) and a high activity of arginase was expressed. The arginase mRNA species of about 1600 bases long was detected in the liver, but not in the small intestine, kidney, spleen and heart of the rats.

Cleavage of the ER-Targeting Signal Sequence of Parathyroid Hormone-related Protein is Cell-Type-Specific and Regulated in Cis by its Nuclear Localization Signal

Prepro-parathyroid hormone-related protein (ppPTHrP) has two targeting signals, an N-terminal signal sequence and a nuclear localization signal (NLS). In fact, the protein is not only secreted from the cell but also found in the nucleus and/or nucleolus. In order to understand the function of the PTHrP signal sequence for the dual localization, the signal sequence cleavage of a series of ppPTHrP deletion mutants fused to Escherichia coli leader peptidase was analysed in vitro and in several cell lines. Efficiency of the PTHrP signal sequence cleavage was intrinsically low in the in vitro reconstitution system. In cultured cells, cleavage efficiency of the PTHrP signal sequence varied significantly, being lowest in COS-1 cells, but rising in HeLa, HEK293 and CV-1 cells. However, virtually complete signal sequence cleavage was observed in CHO cells. In addition, the NLS of PTHrP had a negative effect on its own signal sequence cleavage, which could be enhanced by deletion of the spacer sequence between the signal sequence and the NLS. There was a roughly inverse relationship between the signal sequence cleavage and the nuclear localization of PTHrP. Thus, the final destination of PTHrP could be regulated at the ER membrane.

Molecular cloning of cDNAs for argininosuccinate lyase and arginase of rat liver

S. KAWAMOTO 1, Y. AMAYA 2, T. ODA 3, T. KUZUMI 4, T. SAHEKI 5, M. TATIBANA 6, S. KIMURA 1 and M. MORI 2 1Department of Bacteriology, Teikyo University School of Medicine, Kaga, ttabashi-ku, Tokyo 173, Japan; 2Institute for Medical Genetics, Kumamoto University Medical School, Kumamoto 862, Japan; 3Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu 43131, Japan; 4Department of Biochemistry, School of Medicine, Tokai University, Isehara 259-11, Japan; SDepartment of Biochemistry, School of Medicine, Kagoshima University, Kag3shima 890, Japan; 6Department of Biochemistry, Chiba University School of Medicine, Chiba 280, Japan

Evolution of a Urea Cycle Enzyme to δ-Crystallin

The urea cycle is closely related to the arginine biosynthetic pathway. It was thus expected that urea cycle enzymes evolved from arginine metabolic enzymes. cDNA clones for the mammalian urea cycle enzymes, ornithine trans- carbamylase, argininosuccinate lyase, and arginase, were isolated and sequenced, and the above view was confirmed. The amino acid sequences of the urea cycle enzymes are around 40% identical to those of the corresponding enzymes of the arginine metabolic pathways in microorganisms.