Indication for deletion of two introns in theoxi-3 gene of a respiratory-competentSaccharomyces cerevisiae strain

Physical mapping of the mitochondrial DNA of the wild-typeSaccharomyces cerevisiae strainRXII revealed that most of the restriction sites as well as the location of the apocytochromeb gene were identical in comparison with the known maps of the mitochondrial genome in otherSaccharomyces cerevisiae strains. In the middle of theSalI linearized map of theRXII mitochondrial DNA, a deletion was detected which resulted in the loss of twoEcoRI and oneBamHI restriction sites. The corresponding region, however, exists in most other laboratory strains ofSaccharomyces mapped so far. This region overlaps the introns aI2 and aI3 surrounding exon A3 sequences of the subunit 1 of the cytochrome oxidase gene. The nucleotide sequence of the subunit 1 gene showed that theBamHI site was located close to the aI3-A4 intron-exon junction and the distalEcoRI site close to the aI2-A2 boundary. I therefore conclude that these two introns are deleted in the mitochondrial genome of strainRXII. The exon A3 must have been conserved since this strain was respiratory competent. This result, while being a good example of the morphological diversity of a genome with the same function, may contribute to an understanding of the role of introns in the mitochondrial split genes in yeast.     

Phosphate-Activated Glutaminase in the Crude Mitochondrial Fraction (P2 Fraction) from Human Brain Cortex

The kinetics and other properties of phosphate-activated glutaminase have for the first time been studied in the crude mitochondrial fraction (P2 fraction) from human brain. The enzyme is for unexplained reasons inactivated postmortem. The enzyme activity decreases by storing the tissue or homogenate at 37 degrees C. The inactivation is not caused by formation of a dialysable inhibiting compound. No large proteolytic degradation has occurred, since the phosphate-activated glutaminase-like immunoreactive band did not disappear during the storage. The molecular weight of the subunit of the enzyme as determined by immunoblots of sodium dodecyl sulfate-treated homogenates from human brain is estimated to be approximately 64 K. The enzyme has been shown to have a pH optimum of 8.6; it is activated by phosphate, inhibited by glutamate, and partially inhibited by ammonia. Double-inverse plots of enzyme activity against phosphate are concave-upward, and more so in the presence of an inhibitor. The inhibition by glutamate appears to be noncompetitive with the substrate glutamine, and competitive with the activator phosphate. These kinetic properties are not significantly different from our earlier observations concerning phosphate-activated glutaminase from pig brain and pig kidney.     

var1 Gene on the mitochondrial genome of Torulopsis glabrata

We have cloned and sequenced a region of the Torulopsis glabrata mitochondrial genome homologous to the Saccharomyces cerevisiae var1 gene (var1Sc). An open reading frame that could encode a protein of 339 amino acids was found with 72.7% amino acid and 85.3% nucleotide sequence homology to the S. cerevisiae var1 gene. The T. glabrata gene (var1Tg) is transcribed yielding two stable RNAs, a more abundant 13.5 S RNA and a less abundant 18 S species. We have also identified a candidate for a T. glabrata var1 protein among mitochondrial translation products labeled in isolated mitochondria. The var1Tg gene is even more A + T-rich (93%) than var1Sc (89.6%) and has conserved the strong codon bias of var1Sc. Major differences between the two sequences were found. Significant among these are that no GC clusters are found in var1Tg and the sequences surrounding each of the sites where known polymorphisms exist in var1Sc have deletions at the corresponding sites in var1Tg. These data are discussed with respect to possible origins of these var1 genes and translocation of GC clusters in S. cerevisiae mitochondrial DNA.     

Enzyme kinetics of a highly purified mitochondrial creatine kinase in comparison with cytosolic forms

Summary Mitochondrial creatine kinase (CK) purified from canine myocardium showed a single protein band on SDS-PAGE and was free of MMCK. Its amino acid composition was different than MMCK or BBCK and did not react to antiserum to MMCK or BBCK. Using purified mitochondrial, MM and BBCK, the velocity of reaction (V) was estimated for creatine phosphate (CP), creatine (C), adenosine triphosphate (ATP) and adenosine diphosphate (ADP) over a wide range of concentrations including those at V_max. The values for K_m (mM/L) derived from Lineweaver-Burke plots are shown: The affinity of mitochondrial CK for C is much greater than MMCK which is compatible with the energy shuttle hypothesis, namely ATP is converted by mitochondrial CK to CP, and then diffuses to the myofibril for conversion to ATP for utilization.     

Golgi apparatus activity and membrane flow during scale biogenesis in the green flagellateScherffelia dubia (Prasinophyceae). I: Flagellar regeneration

Summary Cells ofScherffelia dubia regenerate flagella with a complete scale covering after experimental flagellar amputation. Flagellar regeneration was used to study Golgi apparatus (GA) activity during flagellar scale production. By comparing the number of scales present on mature flagella with the flagellar regeneration kinetics, it is calculated that each cell produces ca. 260 scales per minute during flagellar regeneration. Flagellar scales are assembled exclusively in the GA and abstricted from the rims of thetrans-most GA cisternae into vesicles. Exocytosis of scales occurs at the base of the anterior flagellar groove. The central portion of thetrans-most cisterna, containing no scales, detaches from the stack of cisternae and develops a coat to become a coated polygonal vesicle. Scale biogenesis involves continuous turnover of GA cisternae, and scale production rates indicate maturation of four cisternae per minute from each of the cells two dictyosomes. A possible model of membrane flow routes during flagellar regeneration, which involves a membrane recycling loop via the coated polygonal vesicles, is presented.     

The relationship between NADPH-dependent lipid peroxidation and degradation of cytochrome P-450 in adrenal cortex mitochondria

The relationship between NADPH-dependent lipid peroxidation and the degradation of cytochrome P-450 has been studied in bovine adrenal cortex mitochondria. Malondialdehyde formation is accompanied by a corresponding decrease in total cytochrome P-450 content. Inhibitors of lipid peroxidation also prevent the loss of cytochrome P-450, further demonstrating a direct relationship between NADPH-dependent lipid peroxidation and degradation of P-450. To differentiate between cytochrome P-450(11)beta and P-450scc, steroid-induced difference spectra were used to evaluate P-450 degradation. These measurements provide the first evidence that both P-450's are degraded during NADPH-dependent lipid peroxidation with P-450(11)beta being much more susceptible to this process.     

Titration of the binding sites for the oligomycin-sensitivity conferring protein in beef heart submitochondrial particles

The binding parameters of the oligomycin-sensitivity conferring protein (OSCP) in inside-out particles from beef heart mitochondria have been tested by means of two assays, the oligomycin-sensitive ATP-Pi exchange, and the oligomycin-sensitive ATP hydrolysis. The total number of OSCP binding sites in A particles was equal to 220 pmol/mg particle protein. Each mole of ATPase active site was able to bind 1.1 +/- 0.5 mol OSCP with Kd 1.7 nM.