524 Session 7: Oxidative Mechanisms of Cell “Death”

7:33 OXIDATIVE STRESS TRANSFERS PROTEIN INTO

“MOLTEN GLOBULE”-LIKE STATE

Lev Weiner and 1 Israel Silman

Departments of Organic Chemistry and lNeurobiology, The

Weizmann Institute of Science, Rehovot 76100, Israel

Oxygen radicals oxidize amino acids of proteins and, thus,

cause secondary and tertiary structural changes (K. Davies et

al. 1987). We have studied the effect of hydroxyl radicals on

the structure of acetylcholinesterase (AChE) from Torpedo

Californica. When affected by OH radicals, the enzyme was

inactivated. This transformation was accompanied by a

considerable decrease of ellipticity of circular dichroism (CD)

spectra in the near-UV and by a much smaller decrease in the

far-UV CD spectra of AChE. The radical-treated enzyme can

bind the amphiphilic probe, 1-anilino-8-naphthalene-

sulfonate and is characterized by enhanced susceptibility to

trypsinolysis. After radical treatment, maximum intrinsic

protein fluorescent shifts to the red side demonstrating

partial exposure of tryptophans to the solvent. These changes

are characteristic of the protein in a partly unfolded state, so-

called “molten-globule” (Dolginova et al. Biochemistry 31,

12248, 1992). We postulate that oxidative stress in cells

transforms proteins to “molten-globules”. This in turn may

stimulate synthesis of molecular chaperons, thus explaining

known phenonena that enhanced synthesis of certain shock

proteins is evoked by oxidative stress.

7:35 OXYGEN RADICAL PRODUCTION BY HUMAN CYT. P450 J. Rashba-Step, Y. Dai, and A. I. Cederbaum. Department of Biochemistry, Mount Sinai School of Medicine, New York, New York, 10029 USA.

Reactive oxygen species (ROS) have been implicated in cellular damage and pathogenesis of certain diseases. Many studies characterizing the generation of ROS have utilized rat liver microsomes; there are few reports concerning the activity of human liver microsomes in generating ROS. Ethanol-inducible cytochrome P4502El produces elevated levels of 4’ and l-&4. To study the generation of ROS by human liver P450 and by P4502El two systems were used: human liver microsomes; HepG2 cells infected with the human P4502El cDNA to produce a cell line which stably expressed this isoform. With human liver microsomes, NADPH-dependent superoxide radical production, measured by ESR and 44 generation was 3 times higher than the NADH-dependent reactions. Redox cycling agents caused huge increases in 4’ generation. In the presence of appropriate ferric complexes human liver microsomes generated -OH, promoted cleavage of vicinal diols and underwent lipid peroxidation. NADPH- and NADH-dependent rates were similar for these reactions as were rates of ferric reduction. These results suggest that human liver microsomes may be an important source of ROS. Microsomes from HepG2 infected with virus lacking (MV clone) or containing the P4502El cDNA (2El clones) were assayed for the production of ROS. Western and Northern blots, and catalytic activities validated expression of P4502El. ESR spectroscopy showed that 2El -clone microsomes produced OPT at an order of magnitude higher than on control clones. Production of l+Oz was also higher in 2El clones as was lipid peroxidation. The rates of 4’ and @Oz production and of lipid peroxidation were 7-20-fold higher on a per nanomole of P450 basis with PEl-clone microsomes compared to human liver microsomes, indicating that the human P4502El is an isozyme especially reactive in production of ROS.

EFFECT OF INHIBITORS AND SUBSTRATES ON OXYGEN 7:34 RADICAL PRODUCTION BY CYTOCHROME P450 J. Rashba-Step, L. Clejan, and A. I. Cederbaum. Department of Biochemistry, Mount Sinai School of Medicine, New York, New York, 10029 USA.

Oxygen radical production has been suggested to play a significant role in the development of alcohol liver injury. Elevated rates of superoxide and 44 production found in microsomes after chronic ethanol treatment correlates with the content of the cytochrome P4502El isoform. The goal of this study was to evaluate and compare the effect of some commonly used inhibitors and substrates of cytochrome P450 on its mixed function oxidase activity and its catalytic activity in producing reactive oxygen species (ROS). In rat liver microsomes and reconstituted system containing NADPH- reductase. cytochrome P4502E1, cytochrome b, certain inhibitors such as tryptamine or miconazole decreased both P450 catalytic activity and generation of ROS, however this was not specific for P4502El since inhibition was also found with P4502Bl. Other inhibitors, relatively specific for P4502El such as diethyldithiocarbamate (DDC) or diallyl sulfide (DAS), decreased catalytic activity but had no effect on production of ROS. Antibodies against P4502El inhibited catalytic activity and &Oz production but was more effective against the former. In general, substrates for P4502Ei inhibited each other’s metabolism but had no effect on production of ROS. Substrates and inhibitors had no effect on reduction of non-heme iron by P4502El. Differences in effects on oxygen radical production compared to substrate oxidation may reflect the more stringent requirements for a nonperturbed protein conformation to be capable of maximum catalytic activity, as compared to the process of decomposition of P450 Oz complex to product 4 4. These data show that although complex, inhibitors are valuable tools to probe the role of P4502El in production of ROS.

COMPARISON OF THE EFFICACY OF A 21-AMINO- 7:36 STEROID DELIVERED TO CELLS USING TWO METHODS IN PROTECTION OF CARDIAC MYOCYTES FROM H,O, Douglas Decker’, Stephen Buxsef, Juliette Wallnei, and Lawrence Horwiti yell Biology, Upjohn Laboratories, Kalamazoo, MI 49001; ‘Department of Cardiology, University of Colorado Health Sciences Center, Denver, CO 80262 USA

Primary cultures of cardiac myocytes isolated from chicken embryos were used tn evaluate the effects of H,O, and protection by U74500. The process of H,O, toxicity, as measured by the release of the intracellular enzyme lactate dehydrogenase (LDH), consists of two sequential steps. The first step appears as a lag period preceding LDH release into the culture medium. Following the lag period, the kinetics of release of LDH into the culture medium was measured directly and used to calculate a cell injury index (CII). Minimal protection from H,O,-induced cell injury was observed when U74500 was added to the culture medium by dilution from an organic solvent, conditions that lead to precipitation of U74500. However, significant protection from an H,O, insult was observed when U74500 incorporated in emulsions was added to the extracellular medium. Furthermore, the degree of protection was proportional to the amount of emulsion- incorporated U74500 added. Analysis of the dose-response curve showed that the toxicity and protection are consistent with straight-forward assumptions regarding the chemistry of radical generation from H,O, and quenching with U74500. Additionally, the results indicate that U74500 functions better to quench reactive oxygen species when present in emulsion form than when present as a microprecipitate. Thus, it is compelling to explore the implications for improving efficacy of compounds including the Sl-aminosteroids by improving the form in which they are delivered to cells.