A mitochondrial protein essential for the formation of the cytochrome c1-c complex. Isolation, purification, and properties

A new mitochondrial protein was isolated to pure form. This protein was indispensable for the formation of the cytochrome c1-c complex; hence, it was provisionally named the hinge protein for formation of the cytochrome c1-c complex, or for simplicity, merely called the hinge protein. The simplest method for the preparation of the pure protein involved essentially pH 5.5 treatment of high purity of "two-band" cytochrome c1 prepared from an improved method. The use of two band cytochrome c1 prepared by an improved method was preferred because the improved method apparently yielded less tight bonding between the heme-containing and colorless protein entities than that from the original methods (King, T. E. (1978) Methods Enzymol. 53, 181-191). The c1-c complex comprised 1 molar equivalent each of the hinge protein, "one-band" cytochrome c1 and cytochrome c. It was demonstrated by gel filtration chromatography that in the absence of the hinge protein, there was no complex formation between cytochromes c and one-band c1. In titration of the complex formed between one-band cytochrome c1 and cytochrome c with the hinge protein present by using the increase of the Soret-Cotton effect as a criterion (Chiang, Y. L., Kaminsky, L. S., and King, T. E. (1976) J. Biol. Chem. 251, 29-36), a sharp break was observed which showed the three species to be present in equivalent amounts. The hinge protein showed low extinction in the 280 nm region and exhibited poor color value and diffuse character of the band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis after staining with Coomassie brilliant blue. The molecular weight was found to be (i) 9,800 from sedimentation equilibrium, (ii) 11,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and (iii) 23,000 with a Stokes radius of 22.4 A from gel filtration chromatography estimated from a standard curve with proteins of known molecular parameters. The disparities in these data from the actual value of 9,175 from calculations based on amino acid sequence, as previously reported (Wakabayashi, S., Takeda, H., Matsubara, H., Kim, C. H., and King, T. E. (1982) J. Biochem. (Tokyo) 91, 2077-2085), have been discussed.     

Ethnic Elders and American Health Care—A Physician's Perspective

The aging process is a fugue composed of innumerable themes; the theme of “ethnicity” is by far one of its more dominant. Due to the increasing incidence of chronic, progressive infirmity and acute, catastrophic illness, the elderly are thrust into direct contact with the health care systems of their society. The experiences of ethnic elders in American health care situations are fraught with conflict and mutual dissatisfaction with the physician-patient relationship. Both providers and consumers of health care services harbor differing culture-bound perceptions of health, illness and the healing process; these cultural beliefs define personal and professional needs and expectations and notions of how those needs are to be met by others. Both physicians and patients can enhance their communication and their compassion for one another by acknowledgment of cultural differences and by increased willingness to interpret motives and behavior within native context.It behooves us in medicine to examine the cultural traditions underlying our own attitudes, beliefs and values about the aged in a universal sense, as well as in a culturally specific sense, that we may gain insight that will be helpful in serving elderly persons more effectively, and in solving some of the problems inherent in the aging process.     

Melting order of successively longer yeast phenylalanine-accepting transfer ribonucleic acid fragments with a common 5' end

Temperature-jump methods were used to study the kinetics of the helix to coil transition in three fragments of yeast tRNAPhe that share a common 5' terminus (the 5' end of the mature tRNA). Correlation of the extrapolated helix dissociation time constants with NMR exchange broadening results allows assignment of the structural basis of the optical melting transition in the fragments. The results confirm nuclear magnetic resonance findings on these fragments: the 5' 1/4 fragment has no helical structure; the 5' 1/2 fragment contains the D stem; and the 5' 3/5 fragment contains the D stem and the anticodon stem. These are the structures expected if sequential folding of the tRNA during biosynthesis were to occur. The D stem is the last helix to melt in the 5' 3/5 fragment. We suggest that structural elements in addition to the four Watson-Crick base pairs of the D-stem helix are responsible for the anomalously high Tm of that hairpin.     

Nonsubstrate induction of a soluble bacterial cytochrome P-450 monooxygenase by phenobarbital and its analogs

A soluble, cytochrome P-450-dependent fatty acid hydroxylase--epoxidase complex from Bacillus megaterium ATCC 14581 can be induced more than 100-fold by the addition of phenobarbital or one of its analogs (hexobarbital) to the growth medium. These barbiturate inducers are apparently not substrates for the enzyme nor do they activate the monooxygenase in the cell-free system. The induction efficiency of both phenobarbital and hexobarbital can be significantly increased with respect to monooxygenase activity by autoclaving the inducer in the growth medium rather than by adding it to the medium after autoclaving. Turnover numbers of about 3 000 nmoles of substrate oxygenated per min per nmole of P-450 were obtained in crude cell-free preparations obtained from maximally induced cultures. Our data indicate that products formed by heating phenobarbital or hexobarbital in the growth medium are significantly better inducers of monooxygenase activity than are the unaltered drugs.     

Enumeration and Identification of Bacillus cereus in Foods: I. 24-Hour Presumptive Test Medium

An egg yolk-polymyxin medium (KG) for rapid enumeration of Bacillus cereus is described. The test is presumptive in that differentiation of B. cereus (and closely related organisms) from other species is based on the formation of turbidity in the agar surrounding the colonies of the cereus group organisms. The medium is formulated to encourage sporulation and release of free spores for serological confirmatory tests within the 24-hr incubation period. The production of turbidity in egg yolk and free-spore production by 25 strains of B. cereus on KG agar were measured. The recovery of food poisoning strains of B. cereus inoculated into nonsterile food slurries was assessed. A comparison of KG agar and mannitol-egg yolk-polymyxin-agar indicated that the two media were comparable in their abilities to recover low levels of B. cereus from naturally contaminated foods. Since KG agar enhances spore formation by B. cereus, thus permitting early serological testing, its use in screening food products is advocated.     

New Class of Streptomycin-Resistant Mutants Incompatible with supX Suppressor Mutations in Salmonella typhimurium

Streptomycin-resistant colonies of Salmonella typhimurium appearing in platings of supX suppressors of strain leu-500 are less variegated in size than are those derived from strain leu-500 counterparts. Several of the streptomycin-resistant leu-500 clones, furthermore, yield suppressors and revertants of the leu-500 auxotrophy at unusually low rates, suggesting that they provide a genetic background inimicable to supX suppression. Two such "suppression-restrictive" leu-500 streptomycin-resistant (str) mutants, designated strains M(1) and M(4), were characterized as to their ability to receive the trp-supX-cysB linkage region by transduction. Coentry of a donor supX deletion mutation with the selected trp(+) marker was not observed even though these sites display more than 10% linkage in control experiments. This was demonstrably the result of nonviability of the combined supX mutant, M(1) or M(4) streptomycin-resistant genotype, rather than the lack of suppression of the leu-500 imparted auxotrophy. Both M(1)- and M(4)-type resistance was accompanied by pleiotropic effects resembling those caused by strB (nonribosomal)- rather than strA (ribosomal)-type resistance, but both restrictive mutants had a high upper limit of resistance corresponding to that of strA-type mutants. Transduction analyses indicated that the str character of neither the M(1) nor the M(4) strain was linked to the strA or the strB gene. These mutations define a previously undescribed locus, which we propose to designate strC, apparently related to streptomycin uptake rather than its intracellular action. Mutation at this locus is evidently incompatible with the inactivation or removal of the supX site, suggesting a functional association between products of the genes.