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N Borgese

Publications and source records attributed to N Borgese.

At least 19 recordsLinked to original sources
PubMed1995

The targeting information of the mitochondrial outer membrane isoform of cytochrome b5 is contained within the carboxyl-terminal region.

Two isoforms of mammalian cytochrome b5, which have homologous cytosolic amino-terminal catalytic domains, are located one on endoplasmic reticulum (ER b5) the other on mitochondrial outer membranes (OM b5). A cDNA coding for the previously unknown carboxyl-terminal domain of OM b5 was cloned and a chimera between the catalytic domain of ER b5 and the carboxyl-terminal region of OM b5 was expressed in cultured mammalian cells. The chimera localized to mitochondria, indicating that the carboxyl-terminal 43 amino acids of OM b5 contain sufficient information to target the catalytic domain of ER b5 to the mitochondrial outer membrane.

Amino Acid Sequence↗
PubMed1995

A novel point mutation in a 3' splice site of the NADH-cytochrome b5 reductase gene results in immunologically undetectable enzyme and impaired NADH-dependent ascorbate regeneration in cultured fibroblasts of a patient with type II hereditary methemoglobinemia.

Hereditary methemoglobinemia with generalized deficiency of NADH-cytochrome b5 reductase (b5R) (type II) is a rare disease characterized by severe developmental abnormalities, which often lead to premature death. Although the molecular relationship between the symptoms of this condition and the enzyme deficit are not understood, it is thought that an important cause is the loss of the lipid metabolizing activities of the endoplasmic reticulum-located reductase. However, the functions of the form located on outer mitochondrial membranes have not been considered previously. In this study, we have analyzed the gene of an Italian patient and identified a novel G-->T transversion at the splice-acceptor site of the 9th exon, which results in the complete absence of immunologically detectable b5R in blood cells and skin fibroblasts. In cultured fibroblasts of the patient, NADH-dependent cytochrome c reductase, ferricyanide reductase, and semidehydroascorbate reductase activities were severely reduced. The latter activity is known to be due to b5R located on outer mitochondrial membranes. Thus, our results demonstrate that the reductase in its two membrane locations, endoplasmic reticulum and outer mitochondrial membranes, is the product of the same gene and suggest that a defect in ascorbate regeneration may contribute to the phenotype of hereditary methemoglobinemia of the generalized type.

Amino Acid Sequence↗
PubMed1992

Enzymatic instability of NADH-cytochrome b5 reductase as a cause of hereditary methemoglobinemia type I (red cell type).

Nucleotide substitutions in the gene for NADH-cytochrome b5 reductase were identified in three independent probands of hereditary methemoglobinemia type I. Patients in Kagoshima and Okinawa in Japan were shown to possess the same base change, from guanine to adenine at codon 57, which results in amino acid substitution from Arg to Gln. This nucleotide change was the same as formerly found in a patient in Toyoake, Japan (Katsube, T., Sakamoto, N., Kobayashi, Y., Seki, R., Hirano, M., Tanishima, K., Tomoda, A., Takazakura, E., Yubisui, T., Takeshita, M., Sakaki, Y., and Fukumaki, Y. (1991) Am. J. Hum. Genet. 48, 799-808). A type I patient in Italy was shown to have a base change from guanine to adenine at codon 105 which causes substitution from Val to Met. To characterize the enzymes of type I patients, Arg-57----Gln and Val-105----Met mutant enzymes were overexpressed in Escherichia coli and purified to homogeneity. kcat/Km values (NADH) of these two enzymes were 25% in Arg-57----Gln and 14.5% in Val-105----Met compared with that of the wild type enzyme, while the value of type II (generalized, severe form of the disease) mutant enzyme was 3% of the normal value (Yubisui, T., Shirabe, K., Takeshita, M., Kobayashi, Y., Fukumaki, Y., Sakaki, Y., and Takano, T. (1991) J. Biol. Chem. 266, 66-70). The type I mutant enzymes were less heat-stable and more susceptible to proteinase treatment than the wild type. From these results we conclude that restriction of enzyme deficiency to red cells in hereditary methemoglobinemia type I may be generally derived from instability and increased proteolytic susceptibility of variant NADH-cytochrome b5 reductases due to a point mutation.

Amino Acid Sequence↗
PubMed1992

A single mRNA, transcribed from an alternative, erythroid-specific, promoter, codes for two non-myristylated forms of NADH-cytochrome b5 reductase.

Two forms of NADH-cytochrome b5 reductase are produced from one gene: a myristylated membrane-bound enzyme, expressed in all tissues, and a soluble, erythrocyte-specific, isoform. The two forms are identical in a large cytoplasmic domain (Mr approximately 30,000) and differ at the NH2-terminus, which, in the membrane form, is responsible for binding to the bilayer, and which contains the myristylation consensus sequence and an additional 14 uncharged amino acids. To investigate how the two differently targeted forms of the reductase are produced, we cloned a reductase transcript from reticulocytes, and studied its relationship to the previously cloned liver cDNA. The reticulocyte transcript differs from the liver transcript in the 5' non-coding portion and at the beginning of the coding portion, where the seven codons specifying the myristoylation consensus are replaced by a reticulocyte-specific sequence which codes for 13 non-charged amino acids. Analysis of genomic reductase clones indicated that the ubiquitous transcript is generated from an upstream "housekeeping" type promoter, while the reticulocyte transcript originates from a downstream, erythroid-specific, promoter. In vitro translation of the reticulocyte-specific mRNA generated two products: a minor one originating from the first AUG, and a major one starting from a downstream AUG, as indicated by mutational analysis. Both the AUGs used as initiation codons were in an unfavorable sequence context. The major, lower relative molecular mass product behaved as a soluble protein, while the NH2-terminally extended minor product interacted with microsomes in vitro. The generation of soluble reductase from a downstream AUG was confirmed in vivo, in Xenopus oocytes. Thus, differently localized products, with respect both to tissues and to subcellular compartments, are generated from the same gene by a combination of transcriptional and translational mechanisms.

Amino Acid Sequence↗
PubMed1990

Both the outer mitochondrial membrane and the microsomal forms of cytochrome b5 reductase contain covalently bound myristic acid. Quantitative analysis on the polyvinylidene difluoride-immobilized proteins.

NADH-cytochrome b5 reductase is known to be located on two distinct membranes, i.e. endoplasmic reticulum and outer mitochondrial membranes. The endoplasmic-reticulum-associated form of the enzyme contains myristic acid in an amide linkage to its N-terminal glycine [Ozols, Carr & Strittmatter (1984) J. Biol. Chem. 259, 13349-13354]. To investigate whether the dual subcellular localization of the reductase corresponds to a difference in fatty acylation, the enzyme was purified from well-characterized rat liver microsomal and mitochondrial fractions and analysed by a new quantitative analytical procedure. The purified reductases were run on SDS/polyacrylamide gels and blotted on to polyvinylidene difluoride membranes. The reductase-containing bands were treated with hydroxylamine, and amide-linked fatty acids were then detached by acid hydrolysis. The detached fatty acids were extracted, derivatized and analysed as phenylacyl esters by reverse-phase h.p.l.c., and the protein content of the samples was determined by amino acid analysis of the acid hydrolysates. Myristic acid was found in both the microsomal and mitochondrial reductases in a molar ratio of 1:1 with protein. These results demonstrate for the first time the presence of a myristylated protein on outer mitochondrial membranes, and show that the microsomal and mitochondrial reductases are also identical in their fatty acylation.

Amino Acids↗
PubMed1988

Two transcripts encode rat cytochrome b5 reductase.

A cDNA expression library in lambda gt11 was screened with affinity-purified polyclonal anti-rat cytochrome b5 reductase antibodies. One positive clone out of 450,000 clones was isolated and found to be incomplete. This clone was used to rescreen the library, and a second, overlapping clone that contained the entire coding sequence was isolated. RNA gel blots showed that the two overlapping clones contained approximately 90% of the reductase mRNA sequence. Sequencing data showed (i) that rat reductase has a 93% sequence similarity with bovine and human reductase and (ii) that reductase is not synthesized as a high molecular weight precursor. Results of Southern blot analysis were consistent with the hypothesis that a single gene codes for the soluble and membrane-bound (microsomal and mitochondrial) forms of the reductase, present in erythrocytes and liver, respectively. The cloned cDNA was used to study reductase transcripts in liver and reticulocytes. Two antisense RNA probes that together covered the entire coding region and part of the noncoding region of reductase mRNA were used in RNase A protection experiments. These probes detected only one transcript in liver, suggesting that endoplasmic reticulum and mitochondrial reductase are translated from the same mRNA. In contrast, two transcripts were detected in reticulocytes, one of which mismatched the liver probe approximately 30 nucleotides downstream from the initiation codon. Since the soluble and membrane form of the reductase are known to differ at the N terminus, we suggest that this second transcript encodes soluble reductase.

Amino Acid Sequence↗
PubMed1987

Three translationally regulated mRNAs are stored in the cytoplasm of clam oocytes.

In situ hybridization was used to examine the spatial distributions of three translationally controlled maternal RNAs in oocytes and two-cell embryos of the clam Spisula. 3H-labeled single-stranded RNA probes were generated from SP6 recombinant clones containing DNA inserts encoding portions of histone H3 (the DNA sequence which is presented here), cyclin A, and the small subunit of ribonucleotide reductase. Hybridization of these probes to oocytes, in which the mRNAs are translationally inactive, shows that these mRNAs are stored in the cytoplasm. There is no evidence for sequestration of any of the RNAs within the nucleus or any other discrete structure. Instead they appear to be evenly distributed throughout the cytoplasm.

Amino Acid Sequence↗
PubMed1987

Concentration of NADH-cytochrome b5 reductase in erythrocytes of normal and methemoglobinemic individuals measured with a quantitative radioimmunoblotting assay.

The activity of NADH-cytochrome b5 reductase (NADH-methemoglobin reductase) is generally reduced in red cells of patients with recessive hereditary methemoglobinemia. To determine whether this lower activity is due to reduced concentration of an enzyme with normal catalytic properties or to reduced activity of an enzyme present at normal concentration, we measured erythrocyte reductase concentrations with a quantitative radioimmunoblotting method, using affinity-purified polyclonal antibodies against rat liver microsomal reductase as probe. In five patients with the "mild" form of recessive hereditary methemoglobinemia, in which the activity of erythrocyte reductase was 4-13% of controls, concentrations of the enzyme, measured as antigen, were also reduced to 7-20% of the control values. The concentration of membrane-bound reductase antigen, measured in the ghost fraction, was similarly reduced. Thus, in these patients, the reductase deficit is caused mainly by a reduction in NADH-cytochrome b5 reductase concentration, although altered catalytic properties of the enzyme may also contribute to the reduced enzyme activity.

Animals↗
PubMed1986

Distribution of the integral membrane protein NADH-cytochrome b5 reductase in rat liver cells, studied with a quantitative radioimmunoblotting assay.

The intracellular localization of the post-translationally inserted integral membrane protein, NADH-cytochrome b5 reductase, was investigated, using a quantitative radioimmunoblotting method to determine its concentration in rat liver subcellular fractions. Subcellular fractions enriched in rough or smooth microsomes, Golgi, lysosomes, plasma membrane and mitochondrial inner or outer membranes were characterized by marker enzyme analysis and electron microscopy. Reductase levels were determined both with the NADH-cytochrome c reductase activity assay, and by radioimmunoblotting, and the results of the two methods were compared. When measured as antigen, the reductase was relatively less concentrated in microsomal subfractions, and more concentrated in fractions containing outer mitochondrial membranes, lysosomes and plasma membrane than when measured as enzyme activity. Rough and smooth microsomes had 4-5-fold lower concentrations, on a phospholipid basis than did mitochondrial outer membranes. Fractions containing Golgi, lysosomes and plasma membrane had approximately 14-, approximately 16, and approximately 9-fold lower concentrations of antigen than did mitochondrial outer membranes, respectively, and much of the antigen in these fractions could be accounted for by cross-contamination. No enzyme activity or antigen was detected in mitochondrial inner membranes. Our results indicate that the enzyme activity data do not precisely reflect the true enzyme localization, and show an extremely uneven distribution of reductase among different cellular membranes.

Animals↗
PubMed1986

Purification and characterization of two plasma membrane domains from ejaculated bull spermatozoa.

Plasma membranes were detached from ejaculated bull spermatozoa by a brief sonication in a moderately hypotonic medium, and the released plasma membranes were partially purified by differential centrifugation. The resulting fraction was enriched 8- and 15-fold in alkaline phosphatase and 5' nucleotidase activities, respectively, compared with the starting sonicated spermatozoa. This total plasma membrane fraction was separated into two distinct fractions by equilibrium density centrifugation on a continuous linear sucrose gradient. Two peaks of light scattering material were formed at densities of 1.117 and 1.148 g/ml. The denser peak contained most of the protein of the plasma membrane fraction, whereas nearly all the concanavalin A binding activity was found in the lighter peak. The two bands had distinctly different polypeptide compositions when analyzed by SDS PAGE. Polyclonal antibodies were raised in rabbits against a major integral membrane glycoprotein of each fraction (Mr of 92,000 in the light peak and 98,000 in the dense peak). The two antigens were detected on the surface of intact spermatozoa by indirect immunofluorescence microscopy. The 92-kD protein (present in the lighter band) was detected only on the plasma membrane of the acrosomal and anterior postacrosomal regions of the head. The 98-kD antigen, present in the heavier band, was localized to the surface of the postacrosomal region of the head, to the principal piece of the tail, and to the connecting piece between the head and tail. The exclusive localization of the 92-kD polypeptide to the surface of the anterior portion of the head was confirmed by immunoelectron microscopy. These data show that the two fractions isolated on the sucrose gradient originate from different regions of the sperm cell plasma membrane.

Animals↗
PubMed1983

The effects of Bothrops jararacussu venom and its components on frog nerve-muscle preparation.

The effect of Bothrops jararacussu venom was studied in cutaneous pectoris nerve muscle preparations and in the desheathed sciatic nerve of the frog. The venom rapidly inhibited muscle twitch--tension, evoked either directly or indirectly through the motor nerve and abolished the compound action potential of the muscle and of the sciatic nerve. After fractionation of the venom by Sephadex G-50 column chromatography, all the activity was recovered in a fraction containing 30% of the total venom protein and highly enriched in two polypeptides with apparent Mr of 13-15,000, as revealed by two-dimensional polyacrylamide gel electrophoresis. The concentration of active subfraction required to obtain 50% paralysis in 1 h was 8 micrograms protein/ml. The active subfraction contained low levels of phospholipase A activity, whereas no proteolytic activity was detected. The paralyzing activity of the active subfraction on nerve-muscle preparations was not dependent on the presence of Ca2+, suggesting that phospholipase A activity is not required for the toxic effect. The active subfraction was found to cause an initial spontaneous contracture and fasciculation of the nerve-muscle preparation, and a rapid depolarization of the muscle membrane. The frequency of miniature endplate potentials was normal throughout the period of exposure to the active subfraction, although occasionally initial transient bursts were observed. At the end of the incubation, nerve endings still responded to high [K+] and to black widow spider venom. The exposure (1-2 h) to blocking concentrations of venom active subfraction provoked different degrees of morphological alteration of the muscle fibers. In contrast, no ultrastructural alterations were observed in nerve terminals, giving further support to the idea that terminals are not a prime site of the venom action. In addition to its effect on the nerve muscle-preparation, the active subfraction at higher concentrations, showed a Ca2+-dependent hemolytic activity. In the light of these results, the properties of the active subfraction of B. jararacussu venom are compared with those of other known membrane-active toxins.

Animals↗
PubMed1982

Rat erythrocyte NADH-cytochrome b5 reductase. Quantitation and comparison between the membrane-bound and soluble forms using an antibody against the rat liver enzyme.

The subcellular distribution of rat erythrocyte NADH-cytochrome b5 reductase was determined by radioimmunoassay, using a rabbit antibody against the cathepsin D cleaved water-soluble fragment of rat liver microsomal reductase (I-reductase), which is known to be immunologically similar to the red cell enzyme. Erythrocytes contained approximately 30 ng of reductase/mg of protein, of which 90% were recovered in the hemolysate supernatant and 2.3% in the ghost fraction. After concentration by precipitation with 70% saturated (NH4)2SO4, the NADH-cytochrome c reductase activity of the soluble enzyme could be assayed in the presence of cytochrome b5, and was found to be inhibited by anti 1-reductase antibodies. The sodium dodecyl sulfate-polyacrylamide gel electrophoretic mobilities of erythrocyte membrane-associated and soluble reductase of the liver microsomal enzyme and its cathepsin D cleaved hydrophilic fragment (I-reductase) were examined in crude fractions by blotting followed by specific and highly sensitive immunostaining. The intact microsomal enzyme and the two erythrocyte reductases all had similar mobilities and migrated behind 1-reductase. However, the ghost-associated reductase, which was not attributable to contaminating leukocyte or reticulocyte membranes, was distinguishable from the soluble form by two criteria: (i) a lower dependence on exogenous cytochrome b5 in the NADH-cytochrome c reductase assay; and (ii) a larger apparent Mr upon gel filtration in the presence of Triton X-100, presumably because of detergent binding. Considering these results, possible biogenetic relations between membrane-bound and soluble erythrocyte reductase are discussed.

Animals↗