An extranuclear oligomycin‐resistant mutant of Aspergillus nidulans was isolated and biochemically analyzed. The mutant grows slowly in the absence of oligomycin and contains an excess of cytochrome c. The ATPase complex solubilized from mutant mitochondria with Triton X‐100 is 80 times more resistant to oligomycin than the enzyme of the parental strain. The enzyme complexes of both strains were further purified to electrophoretic homogeneity and resolved by dodecylsulfate gel electrophoresis in the presence of 8 M urea into 16 polypeptide bands of apparent molecular weights between 65000 and 6000. Four proteins of apparent molecular weights 43000, 21000, 11000 and 6000 are synthesized on cycloheximide‐resistant mitochondrial ribosomes. The smallest subunit is selectively extracted from the purified enzyme by neutral chloroform/methanol (2/1). A proteolipid of identical electrophoretic mobility and site of synthesis is the major component of proteins extractable with the same solvent from whole mitochondrial membranes. This protein has been isolated from the mutant and its parental strain, and purified to apparent homogeneity. The proteins from both strains start with N‐formyl‐methionine and end with valine. However, a second internal methionine residue present in the wild‐type protein is absent, together with some other amino acids, from the mutant protein. It is concluded that this small lipophilic protein is coded by a mitochondrial gene determining oligomycin resistance, is synthesized on mitochondrial ribosomes and is associated with the membrane sector of the ATPase complex. The mutational alteration of the protein not only confers oligomycin resistance to the ATPase complex but also causes a structural alteration of the mitochondrial inner membrane.
The dicyclohexylcarbodiimide-binding protein of Aspergillus nidulans has been identified as the smallest subunit of the mitochondrial ATPase complex, and has a molecular weight of approximately 8000. It is extractable from whole mitochondria and from the purified enzyme in neutral chloroform/ methanol, contains 30% polar amino acids, and the N-terminal amino acid has been identified as tyrosine. Using a double-labelling technique in the absence and presence of cycloheximide, followed by immunoprecipitation of the enzyme complex with antiserum against Neurospora crassa F1 ATPase, it has been shown that this subunit is synthesized on cytoplasmic ribosomes.Oligomycin-resistant mutants have been isolated in the filamentous Ascomycete Aspergillus nidulans [l ] and mutations in both nuclear and extranuclear genomes have been shown to affect the oligomycin sensitivity of the mitochondria1 ATPase activity [2,3], suggesting the involvement of at least two subunits of the enzyme complex, one nuclearly coded and one extranuclearly coded, in the expression of oligomycin resistance. Furthermore, nuclear-extranuclear interactions have been observed in strains containing mutations in both genomes [3]. In contrast, only extranuclear oligomycin-resistant mutants in Saccharomyces cerevisiae [4,5] and nuclear oligomycin-resistant mutants in Neurospora crassa [6] have been shown to alter the resistance of the ATPase activity of these organisms in vitro.In order to identify the lesions caused by these mutants, and thus the gene-protein relationships, studies on the ATPase complex are essential. Oligomycin is known to inhibit oxidative phosphorylation by acting on the Fo or membrane components of the complex [7]. Dicyclohexylcarbodiimide acts at a nearby site in a similar manner, and shows irreversible binding to one of the FO components [8-111. This hydrophobic polypeptide, referred to as the dicyclohexylcarbodiimide-binding protein, has now been extracted from a number of sources, including the mitochondria of beef heart [ll, 121, S. cerevisiae [13,14] and N . crassa 11.51, the chloroplasts of lettuce [16] and from membranes of Escherichia coli [17]. In all of these species the protein has a molecular weight of
An equation has been derived correlating the length and content of a loaded zone on a paper chromatogram with the distance travelled by the developing solvent. Use of this equation leads to increased accuracy and avoids the need for plotting calibration graphs.
1. Penicillium chrysogenum and Aspergillus flavus degraded benzylpenicillin in the same way. 2. Degradation of the antibiotic was initiated by the cleavage of phenylacetic acid from 6-aminopenicillanic acid. 3. Phenylacetic acid was left unchanged whereas 6-aminopenicillanic acid was degraded into cysteine and valine. This reaction is probably complex. 4. Cysteine was not utilized but valine was cleaved into acetone and glycine. Catabolism of valine by the preformed mats of the two moulds confirms this result. This step probably involves the formation of propan-2-ol. 5. Cleavage of benzylpenicillin into phenylacetic acid and 6-aminopenicillanic acid was performed through the activity of a cellular-bound enzyme.
The use of paper chromatography for micro-scale quantitative analysis by the zonestrip technique gives accurate and reproducible results within certain concentration limits. Analytical results concerning these limits, obtained under specified experimental conditions, are given. Furthermore, some other conclusions concerning the mode of deviation outside these ranges are given. SHIMI and co-workers1 y2 have described a method for micro-scale quantitative analysis by partitionpaper chromatography. They used narrow strips, and solutions to be assayed were loaded in 2-mm wide zones near the upper end of the strips. They derived the equationwhere I is the length, in cm, of the loaded zone in the developed strip, 1 = Klogclogd c is the concentration of the substance, in pg, that occupies the zone and d is the distance, in cm, travelled by the developing solvent.K is a constant factor that determines the value of I with respect to the two variables, c and d. This constant factor, as well as the concentration limit, are specific for every compound under the experimental conditions adopted.The present study was made to determine the range of concentrations over which application of the zonestrip technique gives accurate and reproducible results for the various compounds, EXPERIMEKTALThe experimental technique used was that described by Shimi and co-workers.1$2 The classes of compounds used in this study were organic, amino-and keto-acids, 2,4-dinitrophenylhydrazones of keto-acids and acetone, sugars and penicillins.
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