The acyl-carrier protein in Neurospora crassa mitochondria is a subunit of NADH:ubiquinone reductase (complex I).

We determined the primary structure of a 9.6-kDa subunit of the respiratory chain NADH:ubiquinone reductase (complex I) from Neurospora crassa mitochondria and found a close relationship between this subunit and the bacterial or chloroplast acyl-carrier protein. The degree of sequence identity amounts to 80% in a region of 19 residues around the serine to which the phosphopantetheine is bound. The N-terminal presequence of the subunit has the characteristic features of a mitochondrial import sequence. We cultivated the auxotroph pan-2 mutant of N. crassa in the presence of [14C]pantothenate and recovered all radioactivity incorporated into mitochondrial protein in the 9.6-kDa subunit of complex I. We cultivated N. crassa in the presence of chloramphenicol to accumulate the nuclear-encoded peripheral arm of complex I. This pre-assembled arm also contains the 9.6-kDa subunit. These results demonstrate that an acyl-carrier protein with pantothenate as prosthetic group is a constituent part of complex I in N. crassa.

Primary structures of two subunits of NADH: ubiquinone reductase from Neurospora crassa concerned with NADH-oxidation. Relationship to a soluble NAD-reducing hydrogenase of Alcaligenes eutrophus.

The primary structures of the nuclear-encoded 51 kDa and 78 kDa subunits of the respiratory chain NADH: ubiquinone reductase (complex I) from Neurospora crassa mitochondria were determined by sequencing cDNA and the N-terminus of the mature proteins. Both subunits are related to the soluble NAD-reducing hydrogenase of the bacterium Alcaligenes eutrophus. Sequence comparison between these subunits, the corresponding subunits of the bovine complex I and the bacterial NAD-reducing hydrogenase further confirms the binding sites of NAD(H), FMN and three iron-sulfur clusters.

Primary structure of the nuclear-encoded 18.3 kDa subunit of NADH: ubiquinone reductase (complex I) from Neurospora crassa mitochondria.

The primary structure of the nuclear-encoded 18.3 kDa subunit of the respiratory chain NADH: ubiquinone reductase (complex I) from Neurospora crassa was determined by sequencing cDNA and the N-terminus of the protein. The cDNA contains an open reading frame for a protein of 206 amino acids. The mature protein consists of 173 amino acids and has a molar mass of 18,341 Da. The precursor protein includes a characteristic mitochondrial import sequence with a typical matrix peptidase processing site.

The iron-sulfur clusters in the two related forms of mitochondrial NADH: ubiquinone oxidoreductase made by Neurospora crassa.

Two related forms of the respiratory-chain complex, NADH: ubiquinone oxidoreductase (Complex I) are synthesized in the mitochondria of Neurospora crassa. Normally growing cells make a large, piericidin-A-sensitive form, which consists of some 23 different nuclear- and 6-7 mitochondrially encoded subunits. Cells grown in the presence of chloramphenicol make a small, piericidin-A-insensitive form which consists of only approximately 13 nuclear-encoded subunits. The subunits of the small form are either identical or similar to nuclear-encoded subunits of the large form. The iron-sulfur clusters in these two forms of Complex I are characterized by redox potentiometry and EPR spectroscopy. The large form of Complex I contains four EPR-detectable iron-sulfur clusters, N1, N2, N3 and N4, with the spin concentration of the individual clusters equivalent to the flavin concentration, similar to the mammalian counterparts. The small Complex I contains clusters N1, N3 and N4, but it is devoid of cluster N2. A model of the electron-transfer route through the large form of Complex I has been derived from these findings and an evolutionary pathway which leads to the emergence of large Complex I is discussed.

cDNA and genomic DNA sequence of the 21.3 kDa subunit of NADH:ubiquinone reductase (complex I) from Neurospora crassa.

The primary structure of a nuclear-encoded subunit of the respiratory chain NADH:ubiquinone reductase (complex I) from Neurospora crassa was determined by sequencing cDNA, genomic DNA and the N-terminus of the protein. The sequence correlates to a protein of 200 amino acids and a molecular mass of 21349 Da. The protein is synthesized without a cleavable presequence. It contains two alpha-helices predicted to traverse the bilayer and is a constituent of the membrane part of complex I.

Relationship between a subunit of NADH dehydrogenase (complex I) and a protein family including subunits of cytochrome reductase and processing protease of mitochondria.

The primary structure of a 40 kDa subunit of the respiratory chain NADH:ubiquinone reductase from Neurospora crassa was determined by sequencing cDNA, genomic DNA and the N-terminus of the mature protein. The gene which is interrupted by 7 introns encodes a preprotein consisting of 375 amino acids with a 26 amino acid long presequence typical for a mitochondrial targeting signal. The sequence of the mature subunit shows conspicuous similarities to the recently [(1989) Nature 339, 147-149] discovered protein family which includes subunits I and II of the ubiquinol:cytochrome c reductase, and the processing proteins, matrix processing peptidase and processing enhancing protein, of mitochondria. The possible role of the subunit is discussed.

The 49 K subunit of NADH: ubiquinone reductase (complex I) from Neurospora crassa mitochondria: primary structure of the gene and the protein.

The primary structure of the 49 K subunit of the respiratory chain NADH:ubiquinone reductase (complex I) from Neurospora crassa was determined by sequencing cDNA, genomic DNA and the N-terminus of the mature protein. The sequence lengths correlate to a molecular mass of 54,002 daltons for the preprotein and 49,239 daltons for the mature protein. The presequence consists of 42 amino acids of typical composition for sequences which target nuclear-encoded proteins into mitochondria. The mature protein consists of 436 amino acids and shows 64% similarity to a 49 K subunit of bovine heart NADH:ubiquinone reductase and 33% to a predicted translation product of an open reading frame in the chloroplast DNAs of Marchantia polymorpha and Nicotiana tabacum. Evidence for an iron-sulfur cluster in the subunit is discussed.

Assembly of NADH: ubiquinone reductase (complex I) in Neurospora mitochondria. Independent pathways of nuclear-encoded and mitochondrially encoded subunits.

NADH:ubiquinone reductase, the respiratory chain complex I of mitochondria, consists of some 25 nuclear-encoded and seven mitochondrially encoded subunits, and contains as redox groups one FMN, probably one internal ubiquinone and at least four iron-sulphur clusters. We are studying the assembly of the enzyme in Neurospora crassa. The flux of radioactivity in cells that were pulse-labelled with [35S]methionine was followed through immunoprecipitable assembly intermediates into the holoenzyme. Labelled polypeptides were observed to accumulate transiently in a Mr 350,000 intermediate complex. This complex contains all mitochondrially encoded subunits of the enzyme as well as subunits encoded in the nucleus that have no homologous counterparts in a small, merely nuclear-encoded form of the NADH:ubiquinone reductase made by Neurospora crassa cells poisoned with chloramphenicol. With regard to their subunit compositions, the assembly intermediate and small NADH:ubiquinone reductase complement each other almost perfectly to give the subunit composition of the large complex I. These results suggest that two pathways exist in the assembly of complex I that independently lead to the preassembly of two major parts, which subsequently join to form the complex. One preassembled part is related to the small form of NADH:ubiquinone reductase and contributes most of the nuclear-encoded subunits, FMN, three iron-sulphur clusters and the site for the internal ubiquinone. The other part is the assembly intermediate and contributes all mitochondrially encoded subunits, one iron-sulphur cluster and the catalytic site for the substrate ubiquinone. We discuss the results with regard to the evolution of the electron pathway through complex I.

Detection of ovulation by a radioreceptor assay for human luteinizing hormone.

A specific, sensitive, and rapid radioreceptor assay (RRA), employing membranes from bovine testes as receptor and [125I]hLH as radioligand, has been developed for measurement of human LH in serum. This RRA was used to determine the time of ovulation in seven women. For comparison, four hourly values around midcycle were measured by RIA. The sensitivity of the RRA was 0.78 ng/ml serum and could be increased by prolonged incubation. The coefficient of within and between assay variation at the 50% inhibition level was 7% and 13%, respectively. The mean index of discrimination (RRA/RIA) was 1.02, expressed by the slope of the regression curve. The coefficient of correlation was 0.97. In all women, the LH surge was detected by RRA, and the subsequent ovulation was verified within 30 h by endoscopic examination of the ovaries, as well as serum progesterone concentrations of more than 5 ng/ml on the fifth day after ovulation. As shown, prospective ovulation timing can be done by this simple and accurate method. The RRA can be useful in infertility therapy such as artificial insemination.

PIP: A 2-hour solid-phase radioimmunoassay (RIA) is described for determination of luteinizing hormone (LH) concentrations to detect ovulation. Time of ovulation was determined in 7 women. For comparison, 4 hourly values around midcycle were measured by 2-hour RIA. The 2-hour RIA sensitivity was .78 ng/ml of serum and could be increased by prolonged incubation. The coefficients of within and between assay variation at the 50% inhibition level were 7 and 13%, respectively. The mean index of discrimination between standard RIA and 2-hour RIA was 1.02, expressed by the slope of the regression curve. The coefficient of correlation was .97. In all women, the LH surge was detected by 2-hour RIA, and the subsequent ovulation was verified within 30 hours by endoscopic examination of ovaries as well as serum progesterone concentrations of more than 5 ng/ml on the 5th day after ovulation. Prospective ovulation timing can be done by this simple, accurate method. The 2-hour RIA can be useful in infertility therapy such as artificial insemination.

Ovulation timing by a radioreceptor assay for human luteinizing hormone.

Since predetermination of ovulation would be helpful in treatment of sterility, a quick, sensitive, and specific radioreceptor assay (RRA) for measurement of actual LH concentrations in human serum has been developed. Using partially purified membrane receptors from bovine testes, our assay system enabled precise measurement of LH within 4 hours. The detection limit of the present method is 0.78 ng LER 960/ml serum. The method was used to detect ovulation during four observation cycles each in eleven women who were undergoing treatment for infertility, such as recommended intercourse or artificial insemination because of reduced fertility of their husbands. In all women ovulations could be predicted by LH surge and were verified by laparoscopy within 36 hours. Insemination was carried out at the time of the characteristic increase of LH values around mid-cycle. Pregnancy occurred in three women during the observation period.