Search PubMed⌕ Search

Biomedical subjects

J A Freedman

Publications and source records attributed to J A Freedman.

29 records · Page 2Linked to original sources
PubMed1988

Effects of antibodies to intact cytochrome-c oxidase and its subunit V on the enzymatic activity.

Antibodies have been raised in rabbits against whole beef heart cytochrome-c oxidase and purified subunit V. Antioxidase recognizes nearly all the enzyme subunits but reacts very strongly with subunits II and IV. Antisubunit V is quite specific against subunit V. Inhibition of enzyme activity by antioxidase is typically biphasic in time, indicating populations of both rapidly and slowly reacting molecules. Variation of cytochrome c concentration shows partially competitive kinetics, but the antibody also affects "internal" enzymatic events, including the catalytic turnover induced by N,N,N',N'-tetramethyl-p-phenylenediamine alone and the spin-state change in cytochrome a3 that follows reduction of cytochrome a. No spectral effects can be seen however. Antioxidase also inhibits proteoliposomal respiration with external cytochrome c, but not that with internally trapped cytochrome c. No functionally significant epitopes are detectable on the N side of the membrane in proteoliposomes, although some small effects can be seen with submitochondrial particles. Antisubunit V inhibits the isolated enzyme by at least 60%. The inhibition at high ionic strength induces a biphasic pattern with respect to cytochrome c concentration. Antisubunit V may thus slow the dissociation of cytochrome c from its complex with the enzyme. Antisubunit V has only small effects on the activities of proteoliposomal and submitochondrial particle oxidase in either orientation. On subunit V, some sites, the binding of which can give rise to inhibition, are thus not accessible to antisubunit V when the enzyme is embedded in a functional membrane system.

Animals↗
PubMed1988

Antibodies as probes of cytochrome oxidase structure and function.

Antibodies have been raised in rabbits against whole beef heart cytochrome oxidase and against purified subunit V. Western blot analysis showed that antioxidase was largely composed of anti-II and anti-IV antibodies but some anti-I and antibodies against small subunits were elicited. Similar analysis of anti-V showed it to be relatively specific against subunit V. Three types of anti-V were identified by ELISA with intact enzyme: (i) weak binding and redox independent, (ii) stronger binding, redox-dependent (binding only to reduced or partially reduced enzyme), and membrane-independent, and (iii) moderate-binding, redox-dependent & membrane-dependent. Inhibition of enzyme activity by anti-oxidase was biphasic in time, indicating populations of rapidly- and slowly- reacting molecules. Variation of cytochrome c concentration showed partially competitive kinetics, but the antibody also affected 'internal' enzymatic events including the turnover rate with TMPD and the spin-state change in cytochrome a3 that follows reduction of cytochrome a. No spectral effects could however be seen. Antioxidase also inhibits proteoliposomal respiration with external cytochrome c but not that with internally-trapped cytochrome c. No functionally significant epitopes occur on the N (matrix) side of the membrane. The more strongly binding anti-V inhibits the isolated enzyme by at least 60%. The inhibition at high ionic strength induces a biphasic pattern with respect to cytochrome c concentration. Anti-V may thus slow the dissociation of cytochrome c from its complex with the enzyme. The redox-dependent, membrane-dependent anti-V (reported previously) gave only about 20% inhibition of the enzyme. The effective anti-V antibody had little if any influence on the activity of proteoliposomal oxidase in either orientation. Some sites on subunit V whose binding can give rise to inhibition are not accessible to anti-V when the enzyme is embedded in a functional membrane system.

Animals↗
PubMed1984

Thermodynamics of reverse electron transfer across site 1: ATP/2e- is greater than one.

ATP-driven, rotenone-sensitive, reverse electron transfer from succinate to acetoacetate was measured in rat liver mitochondria in the presence of cyanide. In the approach to equilibrium, the absolute ratio of the free energy change of electron transfer to that of ATP hydrolysis exceeded 1, tending towards about 1 1/4. The data support an H+/2e- stoichiometry of 5 for Site 1 as predicted by a thirteen-proton model of chemiosmotic coupling.

Acetoacetates↗
PubMed1984

The mechanism of antibody inhibition of proton pumping by cytochrome oxidase vesicles.

Antibodies previously shown to inhibit vectorial proton translocation through cytochrome oxidase vesicles were converted to F(ab')2 and Fab'. Neither fragment inhibited proton pumping, although binding capacity was present. However, when a surrogate Fc was added to F(ab')2, inhibition of proton translocation was restored; indicating that the inhibition is due to steric hindrance. These results provide insight into mechanisms of energy transduction by oxidase.

Animals↗
PubMed1984

Interactions in cytochrome oxidase: functions and structure.

Mitochondrial cytochrome c oxidase is an exceedingly complex multistructural and multifunctional membranous enzyme. In this review, we will provide an overview of the many interactions of cytochrome oxidase, stressing developments not covered by the excellent monograph of Wikström, Krab, and Saraste (1981), and continuing into early 1983. First we describe its functions (both in the nominal sense, as a transporter of electrons between cytochrome c and oxygen, and in its role in energy transduction). Then we describe its structure, emphasizing the protein (its structure as a whole, the number and stoichiometry of its subunits, their biosynthetic origin, and their interactions with each other, with other components of the enzyme complex, and with the membrane as a whole). Finally, we present a model in which the protein conformation serves as the focus for the dynamic interaction of its two major functions.

Animals↗
PubMed1983

The effect of specific antibodies on oxygen uptake and H+ pumping by cytochrome c oxidase vesicles.

Antibodies to solubilized cytochrome c oxidase and to subunit III were incubated with liposomal oxidase. In oxygen uptake experiments, the inhibiting effects on RCI of anti-oxidase (primarily anti- subunits II and IV) and anti-III were by different mechanisms: the former, by inhibiting the uncoupled rate; the later, by stimulating the coupled rate. In experiments with H+ translocation, anti-oxidase was without effect, while anti-III was a potent inhibitor of proton pumping. These results are conclusive evidence for redox-linked proton extrusion from the vesicles by the oxidase (and its subunit III).

Antibodies↗
PubMed1983

Redox-dependent accessibility of subunit V of cytochrome oxidase. A novel use of ELISA as a probe of intact membranes.

Beef heart cytochrome c oxidase subunit V was isolated by a new one-step procedure. The product was homogeneous to 99% and immunogenically competent. The resulting antibodies inhibited the whole enzyme. A variation of ELISA (enzyme-linked immunosorbent assay) is described which, using a homogeneous antigen, probes the surface of intact membranes to a high degree of specificity. We showed that the subunit is accessible on the surface of inside-out mitochondrial inner membrane particles only when the particles were reduced with ascorbate and N,N,N',N'-tetramethyl-p-phenylene-diamine but is inaccessible on the surface of right-side-out particles regardless of the presence of reductants. These findings have implications with respect to the topography of the enzyme per se and in relation to its neighbors, and with respect to the degree of coupling between electron transport and proton pumping by the enzyme.

Animals↗
PubMed1978

Biosynthesis of mitochondrial membrane proteins: co-ordination with special reference to cytochrome c oxidase.

This paper reviews mechanisms by which the rate of synthesis of subunits of mitochondrial inner membrane protein complexes and the assembly of these subunits are co-ordinated. Current models are evaluated and critically discussed in the light of some recent evidences. The focus is on the incorporation of cytoplasmically-synthesized cytochrome c oxidase subunits in the development of a newer model, which introduces some twists into a combination of several current ideas. A mechanism which governs both organized assembly and the co-ordination of rates of polypeptide synthesis is illustrated and the principles of the model are applied to the elucidation of some odd features of certain mutants. The possibilities that mitochondrial ATPase and cytochrome c reductase may also be synthesized and assembled according to this model are discussed.

Cytoplasm↗
PubMedDate not supplied

An ethnographic, controlled study of the use of a computer-based histology atlas during a laboratory course.

OBJECTIVE: To evaluate the use and effect of a computer-based histology atlas during required laboratory sessions in a medical school histology course. DESIGN: Ethnographic observation of students' interactions in a factorial, controlled setting. MEASUREMENTS: Ethnographer's observations; student and instructor self-report survey after each laboratory session with items rated from 1 (least) to 7 (best); microscope practicum scores at the end of the course. RESULTS: Between groups assigned the atlas and those not, the ethnographer found qualitative differences in the semantic categories used by students in communicating with each other and with the faculty. Differences were also found in the quality of the interactions and in the learning styles used with and without the computer present in the laboratory. The most interactive learning style was achieved when a pair of students shared a computer and a microscope. Practicum grades did not change with respect to historical controls. Students assigned the atlas, compared with those not assigned, reported higher overall satisfaction (a difference in score of 0.1, P = 0.003) and perceived their fellow students to be more helpful (a difference of 0.11, P = 0.035). They rated the usefulness of the microscope lower (a difference of 0.23, P<0.001). CONCLUSION: A computer-based histology atlas induces qualitative changes in the histology laboratory environment. Most students and faculty reacted positively. The authors did not measure the impact on learning, but they found that there are aspects of using the atlas that instructors must manipulate to make learning optimal. Ethnographic techniques can be helpful in delineating the context and defining what the interventions might be.

Analysis of Variance↗