Novel ATP-binding heat-inducible protein of Mr = 37,000 that is sensitive to transformation in BALB/3T3 cells

Using affinity chromatography on ATP-agarose, we have identified a major ATP-binding protein in Nonidet P-40 extracts of avian and mammalian cells labeled with [35S]methionine. After washing ATP-agarose beads with high-ionic-strength buffer (0.4 M NaCl), the 37-kD protein was shown to be one of the major ATP-binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP-agarose. This protein consisted of several spots on two-dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat-shock treatment of BALB/3T3 cells, the major heat-shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP-binding fraction. Besides hsp70, a 37-kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with alpha,alpha'-dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP-binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c-Ha-ras oncogene compared to their normal counterparts. The incorporation of [32P]orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP-binding protein of Mr = 37,000 is a heat-inducible protein and that the synthesis of this protein is regulated by malignant transformation.     

Role of growth hormone in modulating the constitutive and phenobarbital-induced levels of two P-450(6)beta (testosterone 6 beta-hydroxylase) mRNAs in rat livers

The role of growth hormone in the expression of two forms of hepatic cytochrome P-450(P-450), P-450(6)beta-1(6 beta-3), and P-450(6)beta-4, was investigated using RNA blots. The level of P-450(6)beta-1(6 beta-3) mRNA was twenty times higher than that of P-450(6) beta-4 mRNAs in untreated male rat livers. The levels of P-450(6)beta-1(6 beta-3) and P-450(6)beta-4 mRNAs were increased two fold and three fold, respectively, by hypophysectomy of adult male rats. By intermittent injection of human growth hormone (hGH) into hypophysectomized male rats, both mRNAs were decreased to the level of normal rats, and almost disappeared after continuous infusion of hGH. In female rats, these two mRNAs were not detected, but were increased remarkably by hypophysectomy. The increases in these mRNAs were almost abolished after continuous infusion of hGH in hypophysectomized female rats. The effect of hGH on PB-mediated induction of P-450(6)beta-1(6 beta-3) and P-450(6)beta-4 mRNAs was also examined. The PB-mediated increases in P-450(6)beta-1(6 beta-3) and P-450(6)beta-4 mRNAs were higher in hypophysectomized male rats (2.5-fold and 10.9-fold, respectively) than in normal male rats (1.5-fold and 5.2-fold, respectively). Thus, the levels of P-450(6)beta-1(6-beta-3) and P-450(6)beta-4 mRNAs were 4.1-fold and 7.3-fold, respectively, higher in PB-induced hypophysectomized rats than in normal male rats. Concerning the postnatal developmental profiles, P-450(6)beta-1(6 beta-3) mRNA was detectable at neonate and reached a maximal level at around 17 days of age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure of rat brain prostaglandin D synthetase deduced from cDNA sequence

The amino acid sequence of rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi, O. (1985) J. Biol. Chem. 260, 12410-12415) was determined by a combination of cDNA and protein sequencing. cDNA clones specific for this enzyme were isolated from a lambda gt11 rat brain cDNA expression library. Nucleotide sequence analyses of cloned cDNA inserts revealed that this enzyme consisted of a 564- or 549-base pair open reading frame coding for a 188- or 183-amino acid polypeptide with a Mr of 21,232 or 20,749 starting at the first or second ATG. About 60% of the deduced amino acid sequence was confirmed by partial amino acid sequencing of tryptic peptides of the purified enzyme. The recognition sequence for N-glycosylation was seen at two positions of amino acid residues 51-53 (-Asn-Ser-Ser-) and 78-80 (-Asn-Leu-Thr-) counted from the first Met. Both sites were considered to be glycosylated with carbohydrate chains of Mr 3,000, since two smaller proteins with Mr 23,000 and 20,000 were found during deglycosylation of the purified enzyme (Mr 26,000) with N-glycanase. The prostaglandin D synthetase activity was detected in fusion proteins obtained from lysogens with recombinants coding from 34 and 19 nucleotides upstream and 47 and 77 downstream from the first ATG, indicating that the glycosyl chain and about 20 amino acid residues of N terminus were not essential for the enzyme activity. The amino acid composition of the purified enzyme indicated that about 20 residues of hydrophobic amino acids of the N terminus are post-translationally deleted, probably as a signal peptide. These results, together with the immunocytochemical localization of this enzyme to rough-surfaced endoplasmic reticulum and other nuclear membrane of oligodendrocytes (Urade, Y., Fujimoto, N., Kaneko, T., Konishi, A., Mizuno, N., and Hayaishi, O. (1987) J. Biol. Chem. 262, 15132-15136) suggest that this enzyme is a membrane-associated protein.     

The beta subunit of tryptophan synthase. Clarification of the roles of histidine 86, lysine 87, arginine 148, cysteine 170, and cysteine 230

Our studies, which are aimed at understanding the catalytic mechanism of the beta subunit of tryptophan synthase from Salmonella typhimurium, use site-directed mutagenesis to clarify the functional roles of several putative active site residues. Although previous chemical modification studies have suggested that histidine 86, arginine 148, and cysteine 230 are essential residues in the beta subunit, our present findings that beta subunits with single amino acid replacements at these positions have partial activity show that these 3 residues are not essential for catalysis or substrate binding. These conclusions are consistent with the recently determined three-dimensional structure of the tryptophan synthase alpha 2 beta 2 complex. Amino acid substitution of lysine 87, which forms a Schiff base with pyridoxal phosphate in the wild type beta subunit, yields an inactive form of the beta subunit which binds alpha subunit, pyridoxal phosphate, and L-serine. We also report a rapid and efficient method for purifying wild type and mutant forms of the alpha 2 beta 2 complex from S. typhimurium from an improved enzyme source. The enzyme, which is produced by a multicopy plasmid encoding the trpA and trpB genes of S. typhimurium expressed in Escherichia coli, is crystallized from crude extracts by the addition of 6% poly(ethylene glycol) 8000 and 5 mM spermine. This new method is also used in the accompanying paper to purify nine alpha 2 beta 2 complexes containing mutant forms of the alpha subunit.     

Sequence requirements for cytochrome P-450IIB1 catalytic activity. Alteration of the stereospecificity and regioselectivity of steroid hydroxylation by a simultaneous change of two hydrophobic amino acid residues to phenylalanine

The phenobarbital-inducible P-450 forms IIB1 and IIB2 are identical in sequence except for 14 amino acid differences within the carboxyl-terminal half of the molecule. IIB1 has about a 5-10-fold higher turnover number for most monooxygenase substrates examined although the substrate specificities of both enzymes are virtually identical. Both P-450s oxygenate testosterone to yield the 16 alpha-hydroxy, 16 beta-hydroxy, 17-keto, and 16 beta-hydroxy, 17-keto metabolites as major products. A variant IIB2 cDNA, isolated from an uninduced rat liver lambda gt11 library, and when expressed in Hep G2 cells using a vaccinia virus vector, was found to code for a protein that produced the 16 alpha-hydroxy and 17-keto metabolites of testosterone but no 16 beta-hydroxylated products. Although the published sequences of IIB1 and IIB2 are identical within the N-terminal halves of the proteins, sequence analysis of the variant cDNA revealed two amino acid substitutions in this region; Leu58----Phe and I1e114----Phe. When these two amino acid changes were incorporated into IIB1, via construction of a chimeric cDNA, the resultant expressed enzyme did not catalyze the 16 beta-hydroxylation of testosterone or androstenedione. Formation of the 16 alpha-hydroxy and 17-keto metabolites, however, was only slightly reduced compared with the parent IIB1. A IIB1 protein that possessed only the I1e114----Phe replacement catalyzed the production of all four testosterone metabolites with only slightly different product ratios compared with the parent enzyme. The substrate specificity of a IIB1 variant containing only the Leu58----Phe replacement could not be determined, since that protein did not accumulate in cells infected with the corresponding recombinant vaccinia virus. These data suggest that two distinct amino acid residues located within the amino-terminal fourth of IIB1 and IIB2 can affect substrate orientation at the active site.     

Spontaneous inflammatory disease in transgenic rats expressing HLA-B27 and human beta 2m: an animal model of HLA-B27-associated human disorders

Humans who have inherited the human class I major histocompatibility allele HLA-B27 have a markedly increased risk of developing the multi-organ system diseases termed spondyloarthropathies. To investigate the role of B27 in these disorders, we introduced the B27 and human beta 2-microglobulin genes into rats, a species known to be quite susceptible to experimentally induced inflammatory disease. Rats from one transgenic line spontaneously developed inflammatory disease involving the gastrointestinal tract, peripheral and vertebral joints, male genital tract, skin, nails, and heart. This pattern of organ system involvement showed a striking resemblance to the B27-associated human disorders. These results establish that B27 plays a central role in the pathogenesis of the multi-organ system processes of the spondyloarthropathies. Elucidation of the role of B27 should be facilitated by this transgenic model.     

Clonal dental pulp cells (RDP4-1, RPC-C2A) synthesize and secrete osteopontin (SPP1, 2ar).

Dental pulp cells play an important role in maintaining dental mineralized tissue throughout life. Supplementary mineralization such as reparative dentin and pulp stone frequently occurs after primary dentin formation. Dental pulp cells are thought to be closely associated with such mineralization. We found that clonal rat dental pulp cells, RDP4-1 and RPC-C2A, produce and secrete osteopontin, but do not synthesize phosphophoryn which is a major noncollagenous protein found in dentin. The dental pulp osteopontin was highly phosphorylated and identified by thrombin susceptibility and immunoprecipitation with osteopontin/2ar antibody. Osteopontin synthesis markedly increased by 12-O-tetradecanoylphorbol-13-acetate (TPA) as observed in many osteoblastic cells. This study indicates that these cells can produce osteopontin as a major phosphoprotein and suggests that the synthesis of osteopontin could be used as a characteristic marker of dental pulp cells.     

Electron microscopic radioautographic study of the localization of an anti-allergic agent, Tranilast, in rat mast cells.

We have previously reported that Tranilast, an anti-allergic agent, was rapidly taken into the cytoplasm of rat mast cells in vitro by means of light microscopic radioautography. The present study was performed at the electron microscopic level to elucidate the fine localization of this agent in the mast cells. The results revealed that the number of radioautographic silver grains in the cells increased by the incubation with 3H-labelled Tranilast for 0 to 60 min. and that many silver grains were localized on the specific granules, especially on the perigranular membranes. These results suggest that the mode of inhibitory action of mast cell degranulation by Tranilast is related to the specific localization of this agent on the perigranular membranes.     

A multiple-stimuli-responsive as-1-related element of parA gene confers responsiveness to cadmium but not to copper.

The expression of parA, an auxin-regulated gene expressed during the culture of tobacco (Nicotiana tabacum L.) mesophyll protoplasts, is induced by cadmium. To identify the cadmium-responsive element, we examined the parA promoter using the GUS reporter gene. Cadmium responsiveness was retained in a 5' deletion of the parA promoter to -78 bp, but it was nullified by further deletion to -49bp, which implies that the region -49 to -78 bp contained a cadmium-responsive element. This region contains a sequence similar to as-1, an enhancer sequence from the cauliflower mosaic virus 35S RNA promoter that binds the nuclear factor ASF-1. We named the sequence in the parA promoter pas. Gel-shift assays revealed that pas and as-1 compete for the same DNA-binding nuclear protein(s). Since pentamers of either pas and as-1 were able to confer cadmium responsiveness on a minimal promoter but mutant as-1 was not, we propose that pas and as-1 are involved in cadmium-responsive gene expression. Neither pas nor as-1 conferred responsiveness to copper. The specificity of this response, involving the function of as-1-related elements including pas, is discussed.