Enhancement of glutathione levels in mouse peritoneal macrophages by sodium arsenite, cadmium chloride and glucose/glucose oxidase.

Glutathione content of mouse peritoneal macrophages markedly increased when they were exposed to insulting agents like sodium arsenite, cadmium chloride, and glucose/glucose oxidase which generates hydrogen peroxide. This increase was attributed to the induction of the cystine transport activity by these agents. The transport activity for other amino acids was not induced, but rather diminished by these agents.

Selenium antagonizes the induction of human heme oxygenase by arsenite and cadmium ions.

Effects of selenium compounds on the induction of heme oxygenase in human cells exposed to sodium arsenite or cadmium chloride have been investigated by an immunoblotting technique. Exposure of HeLa cells to arsenite or cadmium ions caused a marked increase in the synthesis of heme oxygenase, and the presence of sodium selenite suppressed the induction. DL-Selenocystine was an effective suppressor, and sodium selenate was less effective.

Molecular cloning and sequence analysis of cDNA encoding human ferrochelatase.

The cDNA encoding human ferrochelatase [EC 4.99.1.

Molecular cloning, sequencing, and expression of mouse ferrochelatase.

The cDNA encoding mouse ferrochelatase (protoheme ferrolyase, EC 4.99.1.

Hemopexin-dependent down-regulation of expression of the human transferrin receptor.

To investigate the regulation mechanism of the uptake of iron and heme iron by the cells and intracellular utilization of iron, we examined the interaction between iron uptake from transferrin and hemopexin-mediated uptake of heme by human leukemic U937 cells or HeLa cells. U937 cells exhibited about 40,000 hemopexin receptors/cell with a dissociation constant (Kd) of 1 nM. Heme bound in hemopexin was taken up by U937 cells or HeLa cells in a receptor-mediated manner.

Induction in mouse peritoneal macrophages of 34 kDa stress protein and heme oxygenase by sulfhydryl-reactive agents.

The synthesis of 34-kDa stress protein was enhanced, with a simultaneous increase in heme oxygenase activity, when mouse macrophages were exposed to diethylmaleate or sodium arsenite. After 7 h of exposure to the sulfhydryl agents, the 34-kDa protein was the most actively synthesized protein. Immunoblot analysis showed that the induced 34-kDa protein reacted with an antibody raised against bovine heme oxygenase.

The 70-kDa peroxisomal membrane protein is a member of the Mdr (P-glycoprotein)-related ATP-binding protein superfamily.

The 70-kDa peroxisomal membrane protein (PMP70) is one of the major integral membrane proteins of rat liver peroxisomes. cDNA clones for PMP70 were isolated and sequenced. The predicted amino acid sequence (659 amino acid residues) revealed that the carboxyl-terminal region of PMP70 has strong sequence similarities to a group of ATP-binding proteins such as MalK and Mdr.

Characterization of ferrochelatase in kidney and erythroleukemia cells.

Ferrochelatase from bovine kidney mitochondria has been purified 1600-fold with a 6.5% yield, exhibiting a specific activity of 490 nmol mesoheme formed/mg of protein per min. The Km values for mesoporphyrin IX and protoporphyrin IX with iron were 12.

Endothelin stimulates accumulations of cellular atrial natriuretic peptide and its messenger RNA in rat cardiocytes.

The effect of endothelin-1 (ET-1) on secretion and synthesis of rat atrial natriuretic peptide (rANP) as well as its mRNA levels was studied in primary cultures of neonatal rat atrial cardiocytes. ET-1 dose-dependently (10(-10)-10(-7) M) increased media and cellular rANP-like immunoreactivity as well as its cytoplasmic mRNA levels in rat cardiocytes during 24 hrs incubation. These results suggest that ET-1 directly stimulates expression of the rANP gene in cardiocytes, thereby leading to enhanced synthesis and secretion of rANP.

Molecular cloning and sequence analysis of the cDNA for rat mitochondrial enoyl-CoA hydratase. Structural and evolutionary relationships linked to the bifunctional enzyme of the peroxisomal beta-oxidation system.

To elucidate structural relationships between the mitochondrial and peroxisomal isozymes of beta-oxidation systems, cDNA of the mitochondrial enoyl-CoA hydratase was cloned and sequenced. The 1454-bp cDNA sequence contained a 870 bp of open reading frame, encoding a polypeptide of 290 amino acid residues. When compared with the amino-terminal sequence of the mature enzyme, the predicted sequence contained a 29-residue presequence at the amino terminus.

The human 32-kDa stress protein induced by exposure to arsenite and cadmium ions is heme oxygenase.

Exposure of HeLa and HL60 cells to sodium arsenite or cadmium chloride led to marked increases in cellular heme oxygenase activity. SDS-polyacrylamide gel electrophoresis of [35S]methionine-labeled cellular proteins indicated that these treatments also resulted in the induction of a 32-kDa protein. Immunoblot analysis further showed that the 32-kDa protein reacted with anti-bovine heme oxygenase antibodies.

The hemopexin receptor on the cell surface of human polymorphonuclear leukocytes.

Promyelocytic leukemia HL-60 cells can be induced to differentiate to granulocytes, under the conditions of cultures in the presence of dimethyl sulfoxide (DMSO). Examination of the binding of 125I-labeled hemopexin to DMSO-induced HL-60 cells showed that the density of hemopexin receptors on the induced-cells was 1.35 times that on the uninduced cells.

Induction of heme oxygenase in rat hepatoma cells by exposure to heavy metals and hyperthermia.

Treatment of rat hepatoma dRLh-84 cells with sodium arsenite, cadmium chloride and cobalt chloride resulted in marked induction of protein with a molecular mass of 32 kDa. To examine the possibility that the induced 32 kDa protein may be heme oxygenase, the enzyme activity was measured, and then the activity in the cells increased by these metals, and heat shock treatments. Immunoblot analysis showed that the induction of 32 kDa protein reacted with anti-heme oxygenase antibody occurred by the treatments.

Mechanisms involved in the cellular uptake of hematoporphyrin by rat hepatoma cells.

To clarify the mechanisms involved in the specific uptake of hematoporphyrin by cancer cells, we investigated the interaction of the heme- and/or hematoporphyrin-hemopexin complexes with rat hepatoma dRLh-84 cells. Hemopexin bound to the cells in a saturable, time- and temperature-dependent manner. The cells exhibited 0.

A new immunoblotting assay for thymidylate synthetase and its application to the regulation of enzyme activity in regenerating rat liver.

A highly sensitive and specific immunoblot assay has been developed to quantitate the content of rat liver thymidylate synthetase (EC 2.1.1.

Characterization of photoreceptor cell differentiation in the rat retinal cell culture.

Photoreceptor cell differentiation in the rat retina was studied in vivo and in vitro, using an immunohistochemical method to demonstrate opsin-like immunoreactivity. Cells in a dissociated monolayer culture expressed some properties characteristic of rat rod cells developing in vivo, including a ciliary structure and opsin-like immunoreactivity. Immunoblot analysis revealed that cultured retinal cells synthesize a polypeptide with the same molecular weight as that synthesized by the intact retina.

Solubilization, purification, and characterization of (H+, K+)ATPase from hog gastric microsomes.

The (H+,K+)ATPase-enriched microsomal fraction prepared from hog gastric mucosa by sucrose density gradient centrifugation was effectively solubilized with Emulgen, with apparent preservation of the enzyme activity, and then the ATPase was highly purified by polyethylene glycol fractionation, and Blue Sepharose CL-6B and amino-hexyl Sepharose chromatographies. The purified enzyme showed a single band, with an apparent molecular mass of approximately 94 kDa, on SDS-PAGE, and exhibited both K+-ATPase and K+-stimulated-p-nitrophenyl phosphatase (pNPPase) activities. The optimum pH for the ATPase activity was 7.

Isolation of the hemopexin receptor from human placenta.

A hemopexin receptor detected in detergent-solubilized placental membranes was purified from the human placenta, using hemopexin-Sepharose affinity chromatography. The solubilized membranes exhibited binding sites of 2.77 pmol of hemopexin/mg of protein with a dissociation constant (Kd) of 6.

Cell surface receptor for hemopexin in human leukemia HL60 cells. Specific binding, affinity labeling, and fate of heme.

The binding of 125I-labeled human hemopexin to human leukemia HL60 cell at 4 degrees C was saturable with time and with increasing concentrations of 125I-hemopexin. Scatchard analysis of the binding data revealed the presence of approximately 42,000 binding sites/cell with an apparent dissociation constant (Kd) of 1.0 X 10(-9) M.

Association of ferrochelatase with Complex I in bovine heart mitochondria.

The location of ferrochelatase in bovine heart mitochondria has been studied. When the mitochondria were fractionated into Complexes I, II and III, ferrochelatase activity was only found in Complex I. Complex I also showed heme synthesis from ferric ion in the presence of NADH as an electron donor.

The (Na+, K+)ATPase of rat kidney: purification, biosynthesis, and processing.

(Na+, K+)ATPase was purified from rat renal outer medulla by concanavalin A- and wheat germ agglutinin-lectin Sepharose affinity chromatographies. The antibody, which was raised in rabbits, markedly inhibited ATPase activity. The monospecificity of this antibody was assayed by the Ouchterlony double immunodiffusion and Western blotting tests.

Receptor-mediated heme uptake from hemopexin by human erythroleukemia K562 cells.

Using human erythroleukemia K562 cells, existence of receptors for hemopexin has been investigated. Hemopexin was bound to the cells in saturable, time- and temperature-dependent manner. The cells exhibited approximately 8,400 binding sites/cell for hemopexin and apohemopexin.

Phorbol ester-induced regulation of transferrin receptors in human leukemia K562 cells.

The treatment of human leukemia K562 cells with 12-0-tetradecanoyl phorbol-13-acetate (TPA) caused a decrease of transferrin receptors. The mechanism of the decrease of the receptors with TPA has been investigated. In cells incubated with TPA, the rate of biosynthesis of transferrin receptors was reduced to 10-20% of that in untreated cells.

Expression and phosphorylation of transferrin receptors in mitogen-activated peripheral blood lymphocytes.

Expression of transferrin receptors of cultured human lymphocytes has been investigated by using monoclonal antibody (5E9) specific for human transferrin receptors. When isolated lymphocytes were cultured in a medium containing fetal calf serum, the biosynthesis of transferrin receptor was barely detectable. The addition of concanavalin A or human serum to the medium caused a slight stimulation of the biosynthesis.

Reconstitution of heme-synthesizing activity from ferric ion and porphyrins, and the effect of lead on the activity.

We examined the activity of heme synthesis when ferrochelatase purified from rat liver mitochondria was incubated with ferric chloride and mesoporphyrin IX as substrates in the absence of reducing reagents. In the presence of the NADH dehydrogenase-rich fraction and NAD(P)H, mesoheme was synthesized; the addition of FMN or FAD markedly enhanced the activity. These results indicate that the NAD(P) H-oxidizing system reduces ferric ion to ferrous ion.