Role of cysteine residues in human NADH-cytochrome b5 reductase studied by site-directed mutagenesis. Cys-273 and Cys-283 are located close to the NADH-binding site but are not catalytically essential.

Two forms of NADH-cytochrome b5 reductase (b5R), an erythrocyte-restricted soluble form, active in methemoglobin reduction, and a ubiquitous membrane-associated form involved in lipid metabolism, are produced from one gene. In the rat, the two forms are generated from alternative transcripts differing in the first exon, however, biogenesis of human b5R was less understood. Recently, two different transcripts (M and S), differing in the first exon were also described in humans. Here, we have investigated the tissue-specificity and the role of the S-transcript in the generation of soluble b5R. By RNase protection assays designed to simultaneously detect alternative b5R transcripts in the same sample, the S transcript was undetectable in nonerythroid and in erythroleukemic K562 cells induced to differentiate, but was present in terminal erythroblast cultures, and represented a major b5R transcript in reticulocytes. Analysis of the translation products of the M- and S-transcripts in HeLa cells transfected with the corresponding cDNAs demonstrated that the S-transcript generates soluble b5R, presumably from an internal initiation codon. Our results indicate that the S-transcript is expressed at late stages of erythroid maturation to generate soluble b5R.

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Mechanistic Studies on CDP-6-deoxy-.DELTA.3,4-glucoseen Reductase: The Role of Cysteine Residues in Catalysis As Probed by Chemical Modification and Site-Directed Mutagenesis

Biochemistry ◽

10.1021/bi00013a003 ◽

1995 ◽

Vol 34 (13) ◽

pp. 4159-4168 ◽

Cited By ~ 15

Author(s):

Olivier Ploux ◽

Yenyoung Lei ◽

Kirsi Vatanen ◽

Hung-wen Liu

Keyword(s):

Chemical Modification ◽

Site Directed Mutagenesis ◽

Cysteine Residues ◽

Directed Mutagenesis ◽

Mechanistic Studies

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Cysteine residues in the Na+/dicarboxylate co-transporter, NaDC-1

Biochemical Journal ◽

10.1042/bj3440205 ◽

1999 ◽

Vol 344 (1) ◽

pp. 205-209 ◽

Cited By ~ 9

Author(s):

Ana M. PAJOR ◽

Sally J. KRAJEWSKI ◽

Nina SUN ◽

Rama GANGULA

Keyword(s):

Protein Trafficking ◽

Direct Relationship ◽

Transmembrane Domain ◽

Cysteine Residue ◽

Site Directed Mutagenesis ◽

Cysteine Residues ◽

Directed Mutagenesis ◽

Transport Activity ◽

Specific Reagent

The role of cysteine residues in the Na+/dicarboxylate co-transporter (NaDC-1) was tested using site-directed mutagenesis. The transport activity of NaDC-1 was not affected by mutagenesis of any of the 11 cysteine residues, indicating that no individual cysteine residue is necessary for function. NaDC-1 is sensitive to inhibition by the impermeant cysteine-specific reagent, p-chloromercuribenzenesulphonate (pCMBS). The pCMBS-sensitive residues in NaDC-1 are Cys-227, found in transmembrane domain 5, and Cys-476, located in transmembrane domain 9. Although cysteine residues are not required for function in NaDC-1, their presence appears to be important for protein stability or trafficking to the plasma membrane. There was a direct relationship between the number of cysteine residues, regardless of location, and the transport activity and expression of NaDC-1. The results indicate that mutagenesis of multiple cysteine residues in NaDC-1 may alter the shape or configuration of the protein, leading to alterations in protein trafficking or stability.

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The role of cysteine residues 129 and 329 in Escherichia coli K1 CMP-NeuAc synthase

Biochemical Journal ◽

10.1042/bj2950485 ◽

1993 ◽

Vol 295 (2) ◽

pp. 485-491 ◽

Cited By ~ 4

Author(s):

G Zapata ◽

P P Roller ◽

J Crowley ◽

W F Vann

Keyword(s):

Escherichia Coli ◽

Specific Activity ◽

Site Directed Mutagenesis ◽

Cysteine Residues ◽

Directed Mutagenesis ◽

Wild Type ◽

Acetylneuraminic Acid ◽

Denatured States ◽

Escherichia Coli K1

N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Escherichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catalytic activity and structure of the protein has been investigated by site-directed mutagenesis and chemical modification. The enzyme is inactivated by the thiol-specific reagent dithiodipyridine. Inactivation by this reagent is decreased in the presence of the nucleotide substrate CTP, suggesting that a thiol residue is at or near the active site. Site-directed mutagenesis of either residue Cys-129 to serine or Cys-329 to selected amino acids has minor effects on the specific activity of the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. The limited reactivity of the enzyme to other thiol-blocking reagents suggests that its cysteine residues are partially exposed. The accessibility and role of the cysteine residues in enzyme structure were investigated by fluorescence, c.d. and denaturation studies of wild-type and mutant enzymes. The mutation of Cys-129 to serine makes the enzyme more sensitive to heat and chemical denaturation, but does not cause gross changes in the protein structure as judged by the c.d. spectrum. The mutant containing Ser-129 instead of Cys-129 had a complex denaturation pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthase consisting of several partially denatured states. Cys-329 reacts more readily with N-[14C]ethylmaleimide when the enzyme is in a heat-induced relaxed state. Cys-129 is less reactive and is probably a buried residue.

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