Olanzapine promotes fat accumulation in male rats by decreasing physical activity, repartitioning energy and increasing adipose tissue lipogenesis while impairing lipolysis.

Olanzapine and other atypical antipsychotics cause metabolic side effects leading to obesity and diabetes; although these continue to be an important public health concern, their underlying mechanisms remain elusive. Therefore, an animal model of these side effects was developed in male Sprague-Dawley rats. Chronic administration of olanzapine elevated fasting glucose, impaired glucose and insulin tolerance, increased fat mass but, in contrast to female rats, did not increase body weight or food intake. Acute studies were conducted to delineate the mechanisms responsible for these effects. Olanzapine markedly decreased physical activity without a compensatory decline in food intake. It also acutely elevated fasting glucose and worsened oral glucose and insulin tolerance, suggesting that these effects are adiposity independent. Hyperinsulinemic-euglycemic clamp studies measuring (14)C-2-deoxyglucose uptake revealed tissue-specific insulin resistance. Insulin sensitivity was impaired in skeletal muscle, but either unchanged or increased in adipose tissue depots. Consistent with the olanzapine-induced hyperglycemia, there was a tendency for increased (14)C-2-deoxyglucose uptake into fat depots of fed rats and, surprisingly, free fatty acid (FFA) uptake into fat depots was elevated approximately twofold. The increased glucose and FFA uptake into adipose tissue was coupled with increased adipose tissue lipogenesis. Finally, olanzapine lowered fasting plasma FFA, and as it had no effect on isoproterenol-stimulated rises in plasma glucose, it blunted isoproterenol-stimulated in vivo lipolysis in fed rats. Collectively, these results suggest that olanzapine exerts several metabolic effects that together favor increased accumulation of fuel into adipose tissue, thereby increasing adiposity.

A series of halogenated heterodimeric inhibitors of prostate specific membrane antigen (PSMA) as radiolabeled probes for targeting prostate cancer.

Prostate specific membrane antigen (PSMA) is a validated molecular marker for prostate cancer. A series of glutamate-urea (Glu-urea-X) heterodimeric inhibitors of PSMA were designed and synthesized where X = epsilon-N-(o-I, m-I, p-I, p-Br, o-Cl, m-Cl, p-Cl, p-F, H)-benzyl-Lys and epsilon-(p-I, p-Br, p-Cl, p-F, H)-phenylureido-Lys. The affinities for PSMA were determined by screening in a competitive binding assay. PSMA binding of the benzyllysine series was significantly affected by the nature of the halogen substituent (IC(50) values, Cl < I = Br << F = H) and the ring position of the halogen atom (IC(50) values, p-I < o-I << m-I). The halogen atom had little affect on the binding affinity in the para substituted phenylureido-Lys series. Two lead iodine compounds were radiolabeled with (123)I and (131)I and demonstrated specific PSMA binding on human prostate cancer cells, warranting evaluation as radioligands for the detection, staging, and monitoring of prostate cancer.

Calmodulin enhances the stability of the estrogen receptor.

The estrogen receptor mediates breast cell proliferation and is the principal target for chemotherapy of breast carcinoma. Previous studies have demonstrated that the estrogen receptor binds to calmodulin-Sepharose in vitro. However, the association of endogenous calmodulin with endogenous estrogen receptors in intact cells has not been reported, and the function of the interaction is obscure. Here we demonstrate by co-immunoprecipitation from MCF-7 human breast epithelial cells that endogenous estrogen receptors bind to endogenous calmodulin. Estradiol treatment of the cells had no significant effect on the interaction. However, incubation of the cells with tamoxifen enhanced by 5-10-fold the association of calmodulin with the estrogen receptor and increased the total cellular content of estrogen receptors by 1.5-2-fold. In contrast, the structurally distinct calmodulin antagonists trifluoperazine and CGS9343B attenuated the interaction between calmodulin and the estrogen receptor and dramatically reduced the number of estrogen receptors in the cell. Neither of these agents altered the amount of estrogen receptor mRNA, suggesting that calmodulin stabilizes the protein. This hypothesis is supported by the observation that, in the presence of Ca2+, calmodulin protected estrogen receptors from in vitro proteolysis by trypsin. Furthermore, overexpression of wild type calmodulin, but not a mutant calmodulin incapable of binding Ca2+, increased the concentration of estrogen receptors in MCF-7 cells, whereas transient expression of a calmodulin inhibitor peptide reduced the estrogen receptor concentration. These data demonstrate that calmodulin binds to the estrogen receptor in intact cells in a Ca2+-dependent, but estradiol-independent, manner, thereby modulating the stability and the steady state level of estrogen receptors.

Binding of IRS proteins to calmodulin is enhanced in insulin resistance.

The IRS proteins, major endogenous targets of the insulin receptor, bind to calmodulin in a Ca(2+)-dependent manner. Here, we have examined the interaction between these proteins in animal and cultured cell models of insulin resistance. Both IRS-1 and IRS-2 co-immunoprecipitate with calmodulin from insulin target tissues in rats. The interaction between calmodulin and IRS proteins in rat soleus muscle was enhanced when insulin resistance was induced in rats by treatment with dexamethasone for 5 days. Moreover, injection of angiotensin II into the inferior vena cava enhanced the binding in rat cardiac muscle. Similarly, increased binding between calmodulin and IRS-1 was observed in isolated cells incubated with tumor necrosis factor-alpha. Overexpression of calmodulin in Chinese hamster ovary cells reduced the tyrosine phosphorylation of IRS-1 induced by insulin, with a concomitant decrease in insulin-stimulated association of IRS-1 with the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase. Insulin-stimulated phosphatidylinositol 3-kinase activity associated with IRS-1 was also reduced in cells overexpressing calmodulin, while this activity was increased in cells incubated with the cell-permeable calmodulin antagonist trifluoperazine. These data demonstrate an enhanced interaction between calmodulin and IRS proteins in models of insulin resistance and suggest a possible mechanism by which increased intracellular Ca(2+) concentrations may contribute to impaired insulin sensitivity.

PKC-dependent regulation of transepithelial resistance: roles of MLC and MLC kinase.

The mechanisms by which protein kinase C (PKC) activation results in increased transepithelial resistance (TER) are unknown [G. Hecht, B. Robinson, and A. Koutsouris. Am. J. Physiol. 266 (Gastrointest. Liver Physiol. 29): G214-G221, 1994]. We have previously shown that phosphorylation of the regulatory light chain of myosin II (MLC) is associated with decreases in TER and have suggested that contraction of the perijunctional actomyosin ring (PAMR) increases tight junction (TJ) permeability [J. R. Turner, B. K. Rill, S. L. Carlson, D. Carnes, R. Kerner, R. J. Mrsny, and J. L. Madara. Am. J. Physiol. 273 (Cell Physiol. 42): C1378-C1385, 1997]. We therefore hypothesized that PKC activation alters TER via relaxation of the PAMR. Activation of PKC by the phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in a progressive dose-dependent increase in TER that was apparent within 15 min (111% of controls) and maximal within 2 h (142% of controls). Similar increases were induced by a diacylglycerol analog, and the effects of both PMA and the diacylglycerol analog were prevented by the PKC inhibitor bisindolylmaleimide I. PMA treatment caused progressive decreases in MLC phosphorylation, by 12% at 15 min and 41% at 2 h. Phosphorylation of MLC kinase (MLCK) increased by 64% within 15 min of PMA treatment and was stable over 2 h (51% greater than that of controls). Thus increases in MLCK phosphorylation preceded decreases in MLC phosphorylation. These data suggest that PKC regulates TER via decreased phosphorylation of MLC, possibly due to inhibitory phosphorylation of MLCK. The decreased phosphorylation of MLC likely reduces PAMR tension, leading to decreased TJ permeability.

IQGAP1 integrates Ca2+/calmodulin and Cdc42 signaling.

Calmodulin regulates diverse Ca2+-dependent cellular processes, including cell cycle progression and cytoskeletal rearrangement. A recently identified calmodulin-binding protein, IQGAP1, interacts with both actin and Cdc42. In this study, evidence is presented that, in the absence of Ca2+, IQGAP1 bound to Cdc42, which maintained Cdc42 in the active GTP-bound state. Addition of Ca2+ both directly abrogated the effect of IQGAP1 on the intrinsic GTPase activity of Cdc42 and, in the presence of calmodulin, dissociated Cdc42 from IQGAP1. In addition, in vitro binding assays revealed that calmodulin associated with both the calponin homology domain and the IQ motifs of IQGAP1. Moreover, F-actin competed with Ca2+/calmodulin for binding to the calponin homology domain, but not the IQ motifs, of IQGAP1. Analysis of cell lysates revealed that calmodulin bound to IQGAP1 in a ternary complex with Cdc42. Increasing the Ca2+ concentration enhanced the interaction between calmodulin and IQGAP1, with a concomitant decrease in the association of IQGAP1 with Cdc42. Our data suggest that IQGAP1 functions as a scaffolding protein, providing a molecular link between Ca2+/calmodulin and Cdc42 signaling.

Calmodulin activates phosphatidylinositol 3-kinase.

Calmodulin and phosphatidylinositol 3-kinase are vital components of a number of common intracellular events. Calmodulin, a ubiquitous Ca2+-dependent effector protein, regulates multiple processes in eukaryotic cells, including cytoskeletal organization, vesicular trafficking, and mitogenesis. Phosphatidylinositol 3-kinase participates in events downstream of the receptors for insulin and other growth factors. Here we demonstrate by coimmunoprecipitation and affinity chromatography that Ca2+/calmodulin associates with Src homology 2 domains in the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase, thereby significantly enhancing phosphatidylinositol 3-kinase activity in vitro and in intact cells. Furthermore, CGS9343B, a calmodulin antagonist, inhibited basal and Ca2+-stimulated phosphorylation of phosphatidylinositol in intact cells. These data demonstrate a novel mechanism for modulating phosphatidylinositol 3-kinase and provide a direct link between components of two fundamental signaling pathways.

Calmodulin modulates the interaction between IQGAP1 and Cdc42. Identification of IQGAP1 by nanoelectrospray tandem mass spectrometry.

Calmodulin regulates numerous fundamental metabolic pathways by binding to and modulating diverse target proteins. In this study, calmodulin-binding proteins were isolated from normal (Hs578Bst) and malignant (MCF-7) human breast cell lines with calmodulin-Sepharose and analyzed by SDS-polyacrylamide gel electrophoresis. A protein that migrated at approximately 190 kDa bound to calmodulin in the presence of Ca2+ and was the only calmodulin-binding protein detected in the absence of Ca2+. This 190-kDa protein was identified as IQGAP1 by nanoelectrospray mass spectrometry and collision-induced dissociation tandem mass spectrometry. IQGAP1 coimmunoprecipitated with calmodulin from lysates of MCF-7 cells. Moreover, overlay with 125I-calmodulin confirmed that IQGAP1 binds directly to calmodulin. Analysis of the functional effects of the interaction revealed that Ca2+/calmodulin disrupted the binding of purified IQGAP1 to the Ras-related protein Cdc42 in a concentration-dependent manner. These data clearly identify IQGAP1 as the predominant calmodulin-binding protein in Ca2+-free breast cell lysates and reveal that calmodulin modulates the interaction between IQGAP1 and Cdc42.

Ca2+ regulates calmodulin binding to IQ motifs in IRS-1.

IRS-proteins couple the receptors for insulin and various cytokines to signalling proteins containing Src homology 2 (SH2) domains. Here we demonstrate that calmodulin, a mediator of Ca(2+)-dependent physiological processes, associates with IRS-1 in a phosphotyrosine-independent manner. IRS-1 coimmunoprecipitated with calmodulin from lysates of Chinese hamster ovary cells expressing IRS-1. The interaction was modulated by Ca2+, and calmodulin binding to IRS-1 was enhanced by increasing intracellular Ca2+ with A23187. In contrast, trifluoperazine, a cell-permeable calmodulin antagonist, decreased binding of calmodulin to IRS-1. Insulin stimulated tyrosine phosphorylation of IRS-1, but did not significantly alter the interaction between calmodulin and IRS-1. IQ-like motifs occur between residues 106-126 and 839-859 of IRS-1. Synthetic peptides based on the these sequences inhibited the association between IRS-1 and calmodulin. These data demonstrate that calmodulin binds to IRS-1 in intact cells in a Ca(2+)-regulated manner, providing a molecular link between the signalling pathways.

Identification of insulin-stimulated phosphorylation sites on calmodulin.

Insulin enhances calmodulin phosphorylation in vivo. To determine the insulin-sensitive phosphorylation sites, phosphocalmodulin was immunoprecipitated from Chinese hamster ovary cells expressing human insulin receptors (CHO/IR). Calmodulin was constitutively phosphorylated on serine, threonine, and tyrosine residues, and insulin enhanced phosphate incorporation on serine and tyrosine residues. Phosphocalmodulin immunoprecipitated from control and insulin-treated CHO/IR cells, and calmodulin phosphorylated in vitro by the insulin receptor kinase and casein kinase II were resolved by two-dimensional phosphopeptide mapping. Several common phosphopeptides were detected. The phosphopeptides from the in vitro maps were eluted and phosphoamino acid analysis, manual sequencing, strong cation exchange chromatography, and additional proteolysis were performed. This strategy demonstrated that Tyr-99 and Tyr-138 were phosphorylated in vitro by the insulin receptor kinase and Thr-79, Ser-81, Ser-101 and Thr-117 were phosphorylated by casein kinase II. In vivo phosphorylation sites were identified by comigration of phosphopeptides on two-dimensional maps with phosphopeptides derived from calmodulin phosphorylated in vitro and by phosphoamino acid analysis. This approach revealed that Tyr-99 and Tyr-138 of calmodulin were phosphorylated in CHO/IR cells in response to insulin. Additional sites remain to be identified. The identification of the insulin-stimulated in vivo tyrosine phosphorylation sites should facilitate the elucidation of the physiological role of phosphocal-modulin.

Phosphorylation of calmodulin by plasma-membrane-associated protein kinase(s).

Plasma-membrane-associated protein kinase(s) from normal rat liver phosphorylates exogenous bovine brain calmodulin in the absence of Ca2+ and in the presence of histone or poly(L-lysine). Maximum levels of calmodulin phosphorylation are obtained at a poly(L-lysine)/calmodulin molar ratio of 0.4. Phosphoamino acid analysis revealed that calmodulin is phosphorylated on serine, threonine and tyrosine residues. Endogenous plasma-membrane-associated calmodulin was also phosphorylated by plasma-membrane-associated protein kinase(s) in the absence of added cationic protein or polypeptide. The identity of endogenous phosphocalmodulin was confirmed by immunoprecipitation with a specific anti-calmodulin monoclonal antibody. Ehrlich ascites tumor cell plasma membranes do not contain endogenous calmodulin. However, membrane-associated protein kinase(s) from these tumor cells phosphorylates bovine brain calmodulin in the presence of poly(L-lysine). These data demonstrate that phosphocalmodulin is present in liver plasma membranes and suggest that this post-translational modification could have a physiological role in this location.

The activity of calmodulin is altered by phosphorylation: modulation of calmodulin function by the site of phosphate incorporation.

Calmodulin transduces Ca2+ signals by binding to and activating essential regulatory enzymes. The large number of intracellular targets for calmodulin raises the possibility that mechanisms in addition to Ca2+ may modulate calmodulin activity. Phosphocalmodulin is found in cells and tissues, and calmodulin phosphorylation is enhanced by several mitogens. Phosphorylation of calmodulin on serine/threonine residues by casein kinase II decreased its ability to activate Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II). The major effect was a 2.5-fold increase in the concentration at which half-maximal velocity (K0.5) was attained, with no apparent alteration in the Vmax, or the K0.5 for Ca2+. In contrast, calmodulin phosphorylated on tyrosine residues by the insulin receptor kinase produced an increase in the Vmax, with no alteration in the affinity for CaM-kinase II or the K0.5 for Ca2+. Direct determination by surface plasmon resonance of the dissociation constants with a synthetic peptide corresponding to the calmodulin-binding domain of CaM-kinase II revealed that phosphorylation on serine/threonine residues of calmodulin significantly decreased its affinity for the peptide, while tyrosine phosphorylation had no effect on binding. In contrast to CaM-kinase II, neither serine/threonine nor tyrosine phosphorylation of calmodulin altered its ability to activate calcineurin. These data indicate that phosphorylation of calmodulin differentially modifies its interaction with individual target enzymes. Moreover, the amino acid residues phosphorylated provide an additional level of control. These results demonstrate that phosphorylation is an in vitro regulatory mechanism in the targeting of calmodulin responses and, coupled with the stoichiometric phosphorylation of calmodulin in rat hepatocytes, suggest that it may be relevant in intact cells.

Intramitochondrial protein synthesis is regulated by matrix adenine nucleotide content and requires calcium.

The hypothesis that fluctuations in matrix adenine nucleotide content (ATP + ADP + AMP) may regulate intramitochondrial protein synthesis was investigated in newborn and adult rat liver mitochondria. Protein synthesis in mitochondria from 0-h-old newborns, which contain 3.4 +/- 0.3 nmol adenine nucleotide/mg protein, was > 90% lower than protein synthesis in mitochondria from 4-h-old newborns, which contain 9.1 +/- 0.2 nmol adenine nucleotide/mg protein. If 0-h newborn mitochondria were preincubated to accumulate adenine nucleotides to 16.8 nmol/mg protein in vitro, the protein synthesis rate increased 25-fold compared to control. Adult rat liver mitochondria normally contain 12-14 nmol adenine nucleotide/mg protein and exhibit a brisk rate of protein synthesis. Following a preincubation to deplete adenine nucleotides in vitro down to 3 nmol/mg protein, protein synthesis in adult liver mitochondria was nearly abolished. Conversely, when adult mitochondria were preincubated to superload adenine nucleotides (to 29 nmol/mg protein), the rate of protein synthesis was doubled. Protein synthesis was also inhibited when the matrix ATP/ADP ratio was lowered by adding FCCP or by omitting phosphate. In adult mitochondria, protein synthesis was inhibited by 0.5 mM EGTA and was increased in proportion to buffered free calcium between 0 and 20 microM. The rate of intramitochondrial RNA synthesis was not inhibited by EGTA nor affected by variations in matrix adenine nucleotide content. The results show that intramitochondrial translation requires matrix calcium and is regulated by changes in the matrix adenine nucleotide content that affect the matrix ATP concentration. The matrix adenine nucleotide content is controlled by the ATP-Mg/Pi carrier. In newborns, the matrix adenine nucleotide content increases 3-fold within 2-4 h after birth, stimulating mitochondrial translation 10-fold and probably contributing to the onset of postnatal mitochondrial biogenesis and adaptation to aerobic metabolism.

Insulin-dependent phosphorylation of calmodulin in rat hepatocytes.

Insulin-stimulated phosphorylation of calmodulin in vivo was examined using a highly specific anti-calmodulin monoclonal antibody combined with high resolution two-dimensional gel electrophoresis. The two major isoforms of calmodulin immunoprecipitated from insulin-treated hepatocytes migrated on two-dimensional gel electrophoresis to the same position as nonphosphorylated calmodulin and calmodulin phosphorylated in vitro. Immunoblotting verified the identity of calmodulin. Insulin enhanced the phosphorylation of calmodulin 3.1 +/- 0.4-fold (mean +/- S.E., n = 10), with a stoichiometry in insulin-treated hepatocytes of 0.47 +/- 0.06 (mean +/- S.E., n = 3) mol of phosphate/mol of calmodulin. Two-dimensional phosphopeptide mapping of calmodulin immunoprecipitated from rat hepatocytes and calmodulin phosphorylated in vitro by the insulin receptor kinase or casein kinase II revealed several common phosphopeptides. The common phosphopeptides that appeared insulin-sensitive in intact cells comprised 61 and 40% of casein kinase II- and insulin receptor-catalyzed 32P incorporation into calmodulin in vitro, respectively. This suggests that casein kinase II and the insulin receptor kinase are, at least in part, responsible for insulin-stimulated phosphorylation of calmodulin in rat hepatocytes. These data indicate that phosphorylation of calmodulin in intact hepatocytes is significantly enhanced by insulin, supporting a critical role for calmodulin in insulin signal transduction.

Net adenine nucleotide transport in rat kidney mitochondria.

This study investigated the hypothesis that changes in the adenine nucleotide (ATP + ADP + AMP) content of kidney mitochondria can occur by a transport mechanism that catalyzes net transfer of adenine nucleotides across the inner mitochondrial membrane. The adenine nucleotide content of isolated kidney mitochondria was 8.23 +/- 0.85 nmol/mg mitochondrial protein. This amount increased or decreased as a function of the external [ATP-Mg] when mitochondria were incubated in phosphate-containing medium. The increases and decreases were inhibited to different extents by 100 microM EGTA (ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid) or 5 microM carboxyatractyloside (CAT), suggesting two transport mechanisms. The unidirectional components (influx and efflux) of net flux were examined separately for the CAT-insensitive (EGTA-sensitive) and CAT-sensitive (EGTA-insensitive) mechanisms. CAT-insensitive adenine nucleotide influx and efflux were stimulated by [Ca2+]free up to 2 microM; for ATP influx, Km was 1.7 mM, Vmax was 3.5 nmol/min/mg protein, and Mg2+ was required. Efflux varied as a function of both the external and matrix [ATP] and was completely inhibited by mersalyl. ATP was a better substrate than ADP, and ADP transport did not require Mg2+. The CAT-sensitive mechanism was characterized by studying phosphate-induced adenine nucleotide efflux. Efflux varied with external [Pi] and with matrix [ATP] and was not inhibited by cyclosporin. The amount of CAT required for maximal inhibition was 800 pmol/mg protein. In contrast to CAT-insensitive efflux, this pathway was only partially inhibited by mersalyl and showed no preference for ATP vs ADP. In conclusion, two distinct mechanisms for net adenine nucleotide transport were demonstrated. Both exchange adenine nucleotides (ATP-Mg or ADP) for Pi. One mechanism is identical to the CAT-insensitive ATP-Mg/Pi carrier known in liver mitochondria; the other is a CAT-sensitive mechanism that is not present in liver and may represent a novel function of the ADP/ATP translocase or another CAT-sensitive carrier.

The ATP-Mg/Pi carrier of rat liver mitochondria catalyzes a divalent electroneutral exchange.

Net transport of ATP-Mg or ADP in exchange for phosphate in isolated rat liver mitochondria has been shown to be an electroneutral process mediated by the ATP-Mg/Pi carrier. We compared the steady state distribution ratios of phosphate, ATP-Mg, and ADP at a pH of 7.4 to determine whether the divalent or monovalent form of these anions is the transported substrate. The log of the divalent ATP-Mg distribution ratio (in/out) approached the log of the divalent phosphate distribution ratio (approximately 0.85), which was approximately twice the value of the delta pH (approximately 0.40) across the inner mitochondrial membrane. This steady state relationship held under several different conditions, e.g. when the medium ATP concentration was varied or if the phosphate gradient was modified by partial uncoupling with the proton ionophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Unidirectional ADP efflux in exchange for external ADP or ATP-Mg was stimulated by an increase in matrix H+. The log of the trivalent ADP distribution ratio (approximately 1.20) approached 3 times the value of delta pH. All these data are consistent with the model of an electroneutral exchange of divalent phosphate (HPO2-4) for divalent ATP-Mg (ATP-Mg2-) or for divalent protonated ADP (HADP2-). We conclude that this transport mechanism accounts for the adenine nucleotide concentration gradient that normally exists between the matrix and external medium.

Mitochondrial toxicity of cationic photosensitizers for photochemotherapy.

The triarylmethane derivative Victoria Blue-BO (VB-BO) and the chalcogenapyrylium (CP) dyes have potential for use in photochemotherapy, because they are taken up by the mitochondria of malignant cells and cause cell death. To clarify the mechanism of cell killing we examined the phototoxic effects of VB-BO and a series of three CP dyes on bioenergetic function in isolated rat liver mitochondria. Without photoirradiation, and irrespective of the respiratory substrate used, each of the compounds tested induced some uncoupling of oxidative phosphorylation. Visible irradiation of VB-BO produced an inhibition of mitochondrial respiration when glutamate plus malate, but not succinate, was used as the respiratory substrate. With photoirradiation VB-BO was also shown to inhibit rotenone-sensitive NADH-cytochrome c reductase activity, but it had no effect on succinate-cytochrome c reductase activity. These data indicate that photoactivation of VB-BO produces selective inhibition of mitochondrial respiratory complex I. Photoirradiation of the CP dyes inhibited both complex I and complex II initiated respiratory activity. With photoirradiation, the CP dyes also inhibited both NADH- and succinate-cytochrome c reductase activities, as well as other membrane-bound enzymes, cytochrome c oxidase and succinate dehydrogenase, but not the mitochondrial matrix enzyme, citrate synthetase, or the cytosolic enzyme, lactate dehydrogenase. alpha-Tocopherol protected bioenergetic activities against CP dye photodamage. These results suggest that mitochondrial photosensitization by CP compounds is mediated by the production of membrane-damaging singlet oxygen which causes nonspecific damage to membranes and membrane-bound enzymes.