Mitochondrial proteinases of the yeast Saccharomyces cerevisiae: electrophoretic detection, substrate specificity and sensitivity to inhibitors

The proteins of submitochondrial particles solubilized with 0.1% Triton X-100 were separated by polyacrylamide gel electrophoresis. Hydrolysis of several proteinase substrates was registered directly in the gel after completion of electrophoresis. According to the data obtained the inner mitochondrial membrane contains one or two enzymes which catalyze hydrolysis of cytochrome c as well as one or two enzymes splitting synthetic substrate of trypsin-like proteinases, e. g. N-alpha-benzoyl-L-arginine-p-nitroanilide (BAPA) and N-alpha-benzoyl-L-arginine-beta-naphthylamide (BANA). Submitochondrial particles were shown to catalyze hydrolysis of 3H-labelled cytochrome c. This activity is suppressed by the same inhibitors as the hydrolysis of mitochondrial translation products, i. e. phenyl-methylsulfonylfluoride, p-chloromercuribenzosulfonate, leupeptin and antipain. Presumably these two processes are catalyzed by the same enzyme localized in the inner mitochondrial membrane. Physiological functions of BAPA- and BANA-hydrolyzing enzyme(s) are still unclear.     

Mechanism of stabilization of synaptosomes with alpha-tocopherol during exposure to phospholipase A2

Changes in potential-dependent fluorescence were studied, using fluorescent probe di-S-C3-(5), in synaptosome suspensions exposed to phospholipase A2, alpha-tocopherol and its derivatives. Phospholipase A2 increased potential-dependent fluorescence, i.e. depolarization of synaptosome membranes. The damaging phospholipase A2 effect was prevented and/or abolished by alpha-tocopherol added to synaptosome suspensions before and after phospholipase A2. Alpha-tocopherol derivatives (2,2,5,7,8-pentamethyl-6-hydroxychromane and alpha-tocopheryl-acetate as well as 4-methyl-2,6-di-tert-butylphenol) failed to exert a protective effect on synaptosome membranes modified by phospholipase A2. It is suggested that alpha-tocopherol effect is determined by its interaction with fatty acids, with 6-hydroxy groups of chromanol nucleus and phytol chain being essential for the complex formation.     

Presence of single-stranded DNA in PCR products of slow electrophoretic mobility.

PCR products were characterized by electrophoresis, blotting and hybridization. In addition to the bands of expected size, bands of slower electrophoretic mobility were often detected. The slower bands completely disappeared when the PCR products were subjected to slow cooling, treated with S1 nuclease or run on an alkaline gel, whereas the bands of expected size were unaffected. The slower bands are therefore likely to contain single-stranded DNA.     

Morphological pathway of flagellar assembly in Salmonella typhimurium.

The process of flagellar assembly was investigated in Salmonella typhimurium. Seven types of flagellar precursors produced by various flagellar mutants were purified by CsCl density gradient protocol. They were characterized morphologically by electron microscopy, and biochemically by two-dimensional gel electrophoresis. The MS ring is formed in the absence of any other flagellar components, including the switch complex and the putative export apparatus. Four proteins previously identified as rod components, FlgB, FlgC, FlgF, FlgG, and another protein, FliE, assemble co-operatively into a stable structure. The hook is formed in two distinct steps; formation of its proximal part and elongation. Proximal part formation occurs, but elongation does not occur, in the absence of the LP ring. FlgD is necessary for hook formation, but not for LP-ring formation. A revised pathway of flagellar assembly is proposed based on these and other results.     

Mapping and characterization of a novel human myc-like (MYCLK1) sequence.

The myc family of proto-oncogenes consists of several members that possess regions of sequence homology and some have known similarities in structure and function. We have isolated an 8.8-kb EcoRI fragment from a human genomic library by hybridization to a 28-base oligonucleotide probe derived from a region of the second exon of MYC, which is highly conserved in the myc gene family. Sequence analysis of this myc-like (MYCLK1) DNA fragment has revealed the existence of a region with 85% homology to the 28-base oligonucleotide probe. An open reading frame of 207 nucleotides containing the region of homology was found. We have mapped MYCLK1 to human chromosome 7 at band p15 by chromosome in situ hybridization; this site is distinct from the map location of previously characterized myc genes. Whether MYCLK1 represents a new functional member of the myc family of proto-oncogenes remains to be determined.     

NMR studies of the conformational change in human N-p21ras produced by replacement of bound GDP with the GTP analog GTP gamma S.

1H-Detected 15N-edited NMR in solution was used to study the conformational differences between the GDP- and GTP gamma S-bound forms of human N-p21ras. The amide protons of 15N-labeled glycine and isoleucine were observed. Resonances were assigned to residues of particular interest, glycines-60 and -75 and isoleucines-21 and -36, by incorporating various 13C-labeled amino acids in addition to [15N]glycine and [15N]iosleucine and by replacing Mg2+ by Co2+. When GTP gamma S replaced GDP in the active site of p21ras, only 5 of the 14 glycine amide resonances show major shifts, indicating that the conformational effects are fairly localized. Responsive glycines-10, -12, -13, and -15 are in the active site. Gly-75, located at the far end of a conformationally-active loop and helix, also responds to substitution of GTP gamma S for GDP, while Gly-77 does not, supporting a role for Gly-75 as a swivel point for the conformational change. The amide proton resonances of isoleucines-36 and -21 and a third unidentified isoleucine also undergo major shifts upon replacement of GDP by GTP gamma S. Thus, the effector-binding loop containing Ile-36 is confirmed to be involved in the conformational change, and the alpha-helix containing Ile-21 is also shown to be affected.