Chiseko Noda

Primary structures of two homologous subunits of PA28, a γ-interferon-inducible protein activator of the 20S proteasome

The primary structures of two proteins that comprise PA28, an activator of the 20S proteasome, have been determined by cDNA cloning and sequencing. These protein subunits, termed PA28α and PA28β, are about 50% identical to one another and are highly conserved between rat and human. PA28α and PA28β are homologous to a previously described protein, Ki antigen, whose function is unknown. PA28α, but neither PA28β nor Ki antigen, contains a ‘KEKE motif’, which has been postulated to promote the binding of proteins having this structural feature. PA28α and PA28β were coordinately regulated by γ‐interferon, which greatly induced mRNA levels of both proteins in cultured cells. The mRNA level of the Ki antigen also increased in response to γ‐interferon treatment, but the magnitude of the increase was less than that for the PA28s, and the effect was transient. These results demonstrate the existence of a new protein family, at least two of whose members are involved in proteasome activation. They also provide the basis for future structure/function studies of PA28 subunits and the determination of their relative physiological roles in the regulation of proteasome activity.

cDNA cloning of a new putative ATPase subunit p45 of the human 26S proteasome, a homolog of yeast transcriptional factor Sug1p

The nucleotide sequence of a cDNA that encodes a new regulatory subunit, named p45, of the 265 proteasome of human hepatoblastoma HepG2 cells has been determined. The polypeptide predicted from the open reading frame consists of 406 amino acid residues with a calculated molecular weight of 45770 and isoelectric point of 8.35. The sequences of several fragments of bovine p45, determined by protein chemical analyses, spanning 27% of the complete structure, were found to be in excellent accord with those deduced from the human cDNA sequence. Computer analysis showed that p45 belongs to a family of putative ATPases which includes regulatory components of 26S proteasomes. The overall structure of p45 was found to be homologous to that of yeast Suglp, which has been identified as a transcriptional factor. It is closely similar, but not identical to the sequence reported for Tripl, a functional homolog of Suglp in human tissues. These results are consistent with the possibility that Sugl‐like proteins with distinct sequence function in transcription and protein degradation in human cells. However, the alternative hypothesis, that the same gene locus encodes both p45 and Tripl, cannot be excluded on the basis of such closely similar sequences. In either case, both proteins are likely to function equivalently well in either transcription or protein degradation.

Control of leucine metabolism with special reference to branched-chain amino acid transaminase isozymes

Abstract Branched chain amino acid transaminase has three types of isozyme (isozymes I–III) in rat. Isozyme I is found in all tissues, whereas isozymes II and III are present only in the liver and brain, respectively. Isozyme II is specific for leucine, while isozymes I and III transaminate valine, leucine and isoleucine at equal rates. Isozyme II is readily inducible by administration of glucocorticoid and a high protein diet. Hypophysectomy induces isozyme I in liver mitochondria. Ketone body formation from leucine is very active in slices of kidney and lactating mammary gland and less active in liver slice. Leucine metabolism in liver is controlled by the activity of the transaminase, while this enzyme is not rate limiting in kidney. Valine or isoleucine inhibited ketogenesis from leucine in the kidney, but not in the liver. Ketogenesis from leucine is also controlled by oxidative phosphorylation. Thus dinitrophenol inhibits ketone body formation and mitochondrial preparations should be fortified with ADP, MgCl2 and succinate to achieve maximal reaction rate. Isozymes I and II of this transaminase are present in normal adult rat liver, but only isozyme I was found in fetal liver. Rapidly growing Yoshida hepatomas contained isozymes I and III, but no isozyme II. Various Morris hepatomas showed a variety of isozyme patterns, such as normal, fetal or a combination of the two types. Cultured rat hepatocytes, containing only isozyme I, were transformed by chemical carcinogens and the transformed cells acquired isozyme III. There was a close correlation between the appearance of isozyme III and deviation of chromosomal numbers in various strains of cultured hepatocytes. These results indicate that change in the isozyme pattern of branched-chain amino acid transaminase is regulated by genetic expression, and not by mere selection of a cell population. It is suggested that the isozymes of this transaminase are good markers for determination of the grade of cell differentiation.

Two phase regulation of tyrosine aminotransferase activity by insulin in primary cultured hepatocytes of adult rats.

Abstract In primary cultures of adult rat hepatocytes, dexamethasone (10 −5 M) induced tyrosine aminotransferase (TAT) 24 h after its addition. Glucagon (10 −7 M) alone had no effect, but strongly enhanced the induction by dexamethasone. Glucagon could be replaced by butyryl cyclic-AMP (10 −4 M), which caused about 20-fold increase in activity. In contrast to many previous reports that insulin induced TAT activity in vivo and in vitro , it inhibited the inductions of TAT by dexamethasone and dexamethasone plus glucagon 24 h after its addition. However, insulin significantly induced TAT activity in the early pahse, 4 h after its addition. Dose-response curves of the effect of insulin on TAT activity showed reverse relations to activity in early and late phase. These results show that TAT activity is regulated by insulin in a two phase fashion.

Developmental and growth-related regulation of expression of serine dehydratase mRNA in rat liver

In rat liver, serine dehydratase mRNA is undetectable in the late prenatal period, but its level increases rapidly after birth to a transient peak, and then after decrease gradually increases again to a maximum 2 weeks after birth that is slightly higher than that of adult liver. To determine whether mature quiescent hepatocytes proliferate without loss of differentiated functions, we measured the serine dehydratase mRNA contents in regenerating liver and primary cultured hepatocytes from adult rats. Partial hepatectomy resulted in a dramatic decrease in the mRNA content within 24 h and then its recovery within a week. In subconfluent cultures of adult rat hepatocytes that did not grow even in the presence of mitogens, serine dehydratase mRNA was maintained at a high level. However, when the hepatocytes were cultured at low cell density without added mitogens, their serine dehydratase mRNA content decreases to a quarter of that of subconfluent cultures. The possibility that the expression of serine dehydratase mRNA is regulated in G0/G1 transition before entry into the S phase and the relationship of the mRNA with growth are discussed.

Hormonal regulation of serine dehydratase activity in primary cultures of adult rat hepatocytes

Summary The basal activity of serine dehydratase in primary cultured rat hepatocytes decreased during culture. Addition of dexamethasone (1 × 10 −5 M) plus glucagon (2.5 × 10 −7 M) not only prevented the decrease, but also caused 3–8 fold increase in the activity. Glucagon could be replaced by dibutyryl cyclic AMP (1 × 10 −4 M). Neither dexamethasone alone nor glucagon alone had any effect. Insulin (1 × 10 −7 M) strongly inhibited the induction by glucagon plus dexamethasone. The induction was completely blocked by cycloheximide (5 × 10 −6 M) or puromycin (1 × 10 −6 M). Of the other hormones tested, testosterone (1 × 10 −7 M) enhanced the induction by dexamethasone plus glucagon, whereas epinephrine (1 × 10 −5 M) and dibutyryl cyclic GMP (1 × 10 −4 M) inhibited the induction. Epinephrine seems to cause inhibition via α-adrenergic receptor, because its effect was completely blocked by simultaneous addition of phenoxybenzamine (1 × 10 −5 M), an α-adrenergic blocker. These results suggest that this enzyme activity is regulated by several hormones, such as pancreatic, adrenal, sexual and adrenergic hormones.

Purification and properties of l-lysine-α-ketoglutarate reductase from rat liver mitochondria

l-Lysine-α-ketoglutarate reductase (N5-(1,3-dicarboxypropyl)-l-lysine: NADP+ oxidoreductase (l-lysine-forming, EC 1.5.1.8) was purified from rat liver mitochondria to a homogeneous state judged by SDS polyacrylamide gel electrophoresis, and its molecular weight was estimated as 52 000. On Sepharose 4B filtration it has a molecular weight of 230 000 and it is suggested that the active enzyme is a tetramer of subunits of similar size. The purified enzyme was clearly separated from saccharopine dehydrogenase (N5-(1,3-dicarboxypropyl)-l-lysine:NAD+ oxidoreductase (l-glutamate-forming, EC 1.5.1.9). The reaction of purified l-lysine-α-ketoglutarate reductase favored the forward reaction (saccharopine formation) and the rate of the reverse reaction (lysine formation) was only 3–5% that of the forward reaction. The forward reaction was specific for l-lysine, α-ketoglutarate and NADPH and followed Michaelis-Menten kinetics, whereas the dose vs. response curve of the reverse reaction was sigmoidal with saccharopine. Among the amino acids examined, ornithine, leucine and trytophan inhibited the forward reaction competitively. These results are different from earlier reports on human and yeast enzymes. The fact that rats fed on lysine-deficient diet do not lose weight much is discussed in relation to the properties of this enzyme.

Expression of GATA-binding transcription factors in rat hepatocytes

Recently, we demonstrated that a DNA‐binding protein(s) is involved in transcriptional repression of the rat serine dehydratase gene in fetal liver. Here, we report that a GAT(A/T) motif is the target sequence for the DNA‐binding protein. By screening a fetal liver cDNA library, we isolated a rat homolog of GATA‐1. Rat GATA‐1 expressed as a GST‐fusion protein in E. coli bound to the GAT(A/T) motif in the serine dehydratase gene. Northern analysis show that GATA‐1 and GATA‐4 mRNAs are expressed in fetal hepatocytes.

Transaminase of branched chain amino acids X. High activity in stomach and pancreas

The activity of branched chain amino acid transaminase (EC 2.6. 1.6) was found to be 8 to 10 times higher in rat stomach and pancreas than in heart and kidney, which were previously thought to be the tissues with the highest activity. For comparison, the activities of two other transaminases, aspartate transaminase (EC 2.6.1.1) and alanine transaminase (EC 2.6.1.2) in different parts of the digestive tract were measured. However, their activities were not especially high in the stomach and pancreas, and in the pancreas the activity of branched chain amino acid transaminase was higher than those of the two other transaminases. The isozyme of branched chain amino acid transaminase in the stomach and pancreas was identified as enzyme I by DEAE cellulose chromatography and immunochemistry. The rates of oxidation of [U-14C]-L-leucine by slices of stomach and pancreas were also higher than by slices of other tissues.

Molecular cloning of two types of cDNA encoding subunit RC6-I of rat proteasomes

A new subunit, named RC6-I, of the rat 20 S proteasome was purified and the partial amino acid sequences of several peptide fragments obtained by digestion with lysyl-endopeptidase were determined by Edman degradation. Amplification of cDNAs encoding RC6-I by the polymerase chain reaction (PCR) technique revealed two types of cDNA, tentatively designated as RC6-IL and RC6-IS in order of size. The nucleotide sequences of the two cDNAs are identical except that RC6-IL contains an insertion of 18 nucleotides in the coding region compared with RC6-IS. The polypeptide predicted from the open reading frame of RC6-IS cDNA consists of 248 amino acid residues with a calculated molecular weight of 27,783. These values are consistent with those obtained by protein chemical analyses. Computer-assisted homology analysis showed that RC6-I belongs to the alpha-type subfamily of the proteasome gene family, which shows similarity to the alpha-subunit of the archaebacterium Thermoplasma acidophilum proteasome, and that the 18 nucleotide insert, encoding six amino acid residues, VVASVS, appears to be unique to RC6-IL, because this motif has not been conserved in any other alpha-type subunit. By reverse transcription (RT)-PCR analysis, the mRNAs for both RC6-IL and RC6-IS were found in all the rat tissues examined. These results suggest that proteasomes are present as a heterogeneous population, possibly for acquisition of diversity of functions.