Oxidative stress reduction related to the efficacy of n-3 polyunsaturated fatty acids in first episode schizophrenia: Secondary outcome analysis of the OFFER randomized trial.

Intervention studies of n-3 polyunsaturated fatty acids (n-3 PUFA) as add-on therapy in patients with schizophrenia have not examined changes in oxidative stress. A randomized placebo-controlled trial of a 26-week intervention composed of 2.2g/day of n-3 PUFA was found to reduce symptom severity in first-episode schizophrenia patients. The present study is an extension of our previous report, whose secondary aim was to assess the association between the clinical effect of n-3 PUFA and changes in oxidative stress indices. Seventy-one patients aged 16-35 were enrolled in the study and randomly assigned to the study arms. Total plasma antioxidant capacity and 8-epi-isoprostane F2α content were assessed at baseline and at weeks 8 and 26 of the study as secondary outcome measures. Significant changes in oxidative stress indices favouring the intervention group were observed: decreases in 8-isoprostane F2α (p<0.001) and increases in total plasma antioxidant capacity (p<0.001). Significant correlations between changes in clinical scores relevant to symptom severity and changes in oxidative indices were observed. The results of the present study hence suggest that the efficacy of a six-month intervention with n-3 PUFA observed in first-episode schizophrenia may be related to improvement in oxidative stress indices.

The association between polyunsaturated fatty acid consumption and the transition to psychosis in ultra-high risk individuals.

PUFA deficiencies in cellular membranes have been observed in ultra-high risk (HR) individuals and in early schizophrenia. It is uncertain whether dietary PUFA consumption can be associated with the risk of transition to psychosis in HR individuals. The aim of the study was to assess PUFA consumption and confirm whether dietary habits are related to the risk of transition to full-threshold psychosis in HR individuals during a 12-month follow-up. PUFA consumption during the previous year was analyzed in 62 h individuals and 33 healthy controls (HC) at the beginning of the follow-up period using a validated Food-Frequency Questionnaire and the Polish Food Composition Tables. Fifteen HR individuals converted into psychosis (C-HR) during the 12-month follow-up. C-HR individuals reported significantly higher consumption of n-6 fatty acids (linoleic acid, LA and arachidonic acid, AA) in comparison with individuals who did not develop psychosis (NC-HR). The C-HR group reported a significantly higher AA/(EPA+DHA) consumption ratio than the NC-HR group. HC reported significantly higher consumption of most n-3 PUFA and lower consumption of all n-6 PUFA than both groups of HR individuals. The results suggest that dietary patterns of PUFA consumption may play a role in the conversion to psychosis of HR individuals.

Regulation of phospholipase C delta1 by sphingosine.

Sphingosine, which is on the pathway of sphingomyelin degradation, activates phospholipase C (PLC) delta1 moderately. In the liposome assay effect of sphingosine on PLC delta1 activity depends on KCl concentration. Stimulation of PLC delta1 by sphingosine increased as the KCl concentration is increased from 0 to 100 mM, and then diminished with the increasing KCl. In the liposome assay sphingosine diminishes inhibition of PLC delta1 by sphingomyelin. To determine the domain of PLC delta1 which interacts with sphingosine active proteolytic fragments of PLC delta1 were generated by trypsin digestion of the native enzyme. Sphingosine affects the activity of PLC delta1 fragment which lacked the amino-terminal domain (first 60 amino acids) but not the active fragment that has cleaved the domain spanning the X and Y region of PLC delta1. These observations indicate that for interaction of sphingosine with PLC delta1 intact domain that span regions of conservation, designated as X and Y is necessary. When the activity of PLC delta1 was assayed with PIP2 in the erythrocyte membrane as substrate, sphingosine strongly inhibited PLC delta1. The other homolog of sphingosine 4-hydroxysphinganine (phytosphingosine) inhibited PLC delta1 to much lesser extent. The activity of PLC delta1 was inhibited by 68% and 22% in the presence of 20 microM sphingosine and phytosphingosine, respectively. This inhibition was completely abolished by deoxycholate at a concentration of 1.5 mM. These observations suggest that sphingosine may regulate activity of PLC delta1 in the cell.

The distribution of enzymes involved in purine metabolism in rat kidney.

Adenosine produced from 5'-AMP has been proposed as a mediator of intrinsic renal regulation. The rates of 5'-AMP and adenosine metabolism are dependent on the activities of enzyme involved in purine metabolism. The activities of adenosine kinase (AK), adenosine deaminase (ADA), 5'-nucleotidase (5'-NT), AMP deaminase, xanthine oxidase and purine nucleoside phosphorylase were measured in cytosolic and membrane fractions from glomeruli, cortical tubules, medullary thick ascending limb of Henle (MTAL) and collecting duct prepared from rat kidney by combinations of sieving and sucrose density gradient centrifugation techniques. In the cytoplasm of glomeruli cells, the activity ratios of ADA/AK and AMP deaminase/5'-NT were 70 and 2.4, respectively. The highest activity of 5'-NT was found in membrane fractions of cortical tubules where it was equally distributed between luminal and antiluminal membranes. Membrane fractions of MTAL did not contain detectable amounts of adenosine deaminase activity. The highest activity of xanthine oxidase and purine nucleoside phosphorylase was in the cytoplasm fraction of glomeruli. These results suggest that deamination of AMP and adenosine may be favored in the cytoplasm of glomeruli cells. In contrast, in the extracellular space of glomeruli and especially in the cortical tubule, AMP can be converted preferentially to adenosine by 5'-NT.

Altered expression of adenosine receptors in heart of diabetic rat.

Diabetes results in functional, biochemical, and morphological abnormalities in the heart. Some of these changes may be attributed to altered adenosine action. This study aimed to examine the expression level of adenosine receptors (AR) in heart of streptozotocin-induced diabetic rat. Performed analyses revealed detectable levels of A1-AR, A2a-AR, A2b-AR, A3-AR mRNA and protein in whole heart and isolated cardiac myocytes. An increase in A1-AR protein content with no changes in mRNA level was observed in isolated cardiac myocytes. Diabetes resulted in an increase of A3-AR mRNA and protein levels in heart and in cardiac myocytes. The level of A2a-AR mRNA was increased in whole diabetic heart, but it decreased in cardiac myocytes with no detectable changes in protein content. We did not observe any changes in expression level of A2b-AR in diabetic heart and isolated cardiac myocytes. Administration of insulin to diabetic rat for four days resulted in returning of the ARs mRNA and protein to the levels observed in heart of normal rat. These changes in ARs genes expression, and receptors protein content correspond to some abnormalities characteristic of the diabetic heart, suggesting involvement in pathogenesis of diabetic cardiomyopathy.

Changes in the structure of pyruvate dehydrogenase complex induced by mono- and divalent ions.

The activity of pyruvate dehydrogenase complex purified from pig kidney cortex was affected by various mono- and di-valent ions and changes in ionic strength. The fluorescence emission spectrum of PDC exposed to 0.04 M ionic strength and excited at 280 nm exhibited a maximum at 334 nm; the fluorescence intensity of PDC appeared to depend upon the ionic strength and the K+ and Na+ content of the incubation buffer. Alteration of ionic strength to which the enzyme complex was exposed produced a change in the absorption of the complex at 230 nm. The presence of HPO4(2-) ions prevented changes in the UV absorption spectrum of PDC induced by the variation in ionic strength. The K+ and Na+ ions alone had no effect on the UV spectrum of PDC. Upon increasing the ionic strength to which the enzyme complex was exposed, dramatic changes in the circular dichroism (CD) pattern were observed. At 0.04 M ionic strength PDC exhibited a CD spectrum with minima at 216, 218 and 222 nm and a cross-over point at 215 nm. At 0.15 M ionic strength the CD spectrum of PDC exhibited minima at 223, 226, 228 nm and a cross-over point at 221 nm. The presence of HPO4(2-) ions prevented alterations in the CD spectrum of PDC induced by variations in ionic strength. The K+ and Na+ ions had no effect on the CD spectrum of PDC.

Effect of sphingomyelin and its metabolites on the activity of human recombinant PLC delta 1.

In an attempt to obtain sufficient quantities of pure phospholipase C delta 1 (PLC delta 1) necessary for structural and kinetic studies, human fibroblast PLC delta 1 was cloned in the pPROEX-1 vector, expressed in E. coli cells as a (6xHis) fusion protein and purified to homogeneity. From 11 of E. coli culture 21 mg of pure PLC delta 1 was obtained by a two-step purification procedure, which includes Ni(2+)-NAT agarose and Mono S cation exchange chromatography. Catalytic properties of recombinant PLC delta 1 with respect to activation by spermine and calcium ions and inhibition by sphingomyelin were similar to or identical to PLC delta 1 purified from rat liver. Calcium activation of PLC delta 1 was dependent on the presence of spermine. Half-maximal activity was attained at 250 and 170 nM of free Ca2+ in the presence and absence of spermine, respectively. Sphingomyelin and lysosphingomyelin were mixed type inhibitors with respect to PIP2. Ceramide inhibits PLC delta 1 very weakly. GM1, which is a ceramide bound glucosidically to the oligosaccharide moiety, was a strong non-competitive inhibitor of PLC delta 1. In the absence of spermine, sphingosine and phytosphingosine weakly activated PLC delta 1. The results indicate that the effect of sphingomyelin and its metabolites on PLC delta 1 activity depends on the presence of spermine. It is postulated that, among other factors, in vivo, activity of PLC delta 1 may depend on the turnover of sphingomyelin.

Recombinant protein kinase C-gamma phorbol binding domain upon microinjection blocked insulin-induced maturation of Xenopus laevis oocytes.

The second cysteine-rich (Cys-2) domain of rat brain PKC-gamma regulatory region C1 (92-173) was expressed in Escherichia coli cells and purified. NMR studies of Cys-2 protein identified the phorbol and other phospholipid binding sites within this molecule (Xu, R.X., Pawelczyk, T., Xia, T-H. and Brown, S.T. (1997) Biochemistry 37, 10709-10717). Here, we tested the ability of this domain to bind other proteins. Using an overlay assay we show that the Cys-2 domain binds other proteins in Xenopus oocyte soluble fraction. Unlike the kinase activity, binding of Cys-2 to other proteins was detected in the absence of added phospholipids. Microinjection of Cys-2 protein into Xenopus leavis oocytes inhibited insulin-induced but not progesterone-induced maturation. The smallest dose that enhanced insulin-induced maturation was 0.45 x 10(-12) mol injected Cys-2. These results demonstrate that the PKC-gamma Cys-2 domain beside being the binding site for phorbol ester/DAG and phosphatidylserine binds also other proteins. The proteins that interact with Cys-2 domain of PKC are essential for insulin-induced maturation program in oocytes.

Effect of ionic strength on the regulatory properties of 2-oxoglutarate dehydrogenase complex.

The effects of changing ionic strength on the activity of the 2-oxoglutarate dehydrogenase complex from pig kidney cortex were explored. This enzyme complex is found to be influenced in many ways by the ionic strength of the reaction medium. The enzyme shows an optimum activity at 0.1 M ionic strength. Increase in ionic strength from 0.1 M to 0.2 M resulted in a decrease of S0.5 for 2-oxoglutarate, and in an increase of S0.5 for NAD. Changes in ionic strength over the range of 0.05-0.2 M have little, if any, effect on S0.5 for CoA. The Hill coefficient for 2-oxoglutarate and NAD at 0.2 M ionic strength was 1.0, whereas at 0.05 M ionic strength it was 0.85 and 1.2 for 2-oxoglutarate and NAD, respectively. At 0.05 M ionic strength the pH optimum of the enzyme ranges between 7.4-7.6, but at 0.15 M ionic strength the pH optimum shifts to 7.8. The magnitude of inhibition of enzyme activity by ATP is not influenced by changes in ionic strength in the absence of calcium. However, in the presence of Ca2+, increases in ionic strength lower the inhibitory effects of ATP. The Si0.5 for ATP in both presence and absence of Ca2+ was not affected by changes in ionic strength in the range of 0.1-0.2 M. In contrast, the Sa0.5 for ADP in the absence of Ca2+ decreases as ionic strength increases. In the presence of calcium and 0.2 M ionic strength ADP has no effect on 2-oxoglutarate dehydrogenase complex activity.

The effect of different molecular species of sphingomyelin on phospholipase C delta 1 activity.

Bovine brain sphingomyelin was separated into different molecular species using a reverse phase column. PLC delta 1 was inhibited by all molecular species of sphingomyelin. The extent of this inhibition was dependent on the hydrophobicity. Based on fatty acid analysis, we conclude that the inhibition of PLC delta 1 depends on the chain length and degree of unsaturation of the fatty acid moiety of SM. N-palmitoyl-D-sphingomyelin and N-stearoyl-D-sphingomyelin inhibited PLC delta 1 less then N-oleoyl-D-sphingomyelin. In the absence of Ca2+ (1 mM EGTA) all tested molecular species of SM inhibited weakly the enzyme. The sensitivity of PLC delta 1 to inhibition by SM increased with increasing Ca2+ concentration. The shape of calcium curve differed for molecular species with saturated and unsaturated fatty acids. Inhibition of PLC delta 1 by N-palmitoyl-D-sphingomyelin and N-stearoyl-D-sphingomyelin reached a maximum at 0.2 microM Ca2+, while inhibition by N-oleoyl-D-sphingomyelin reached maximum at 2 microM Ca2+. PLC delta 1 is more sensitive to inhibition by SM when it is maximally activated by spermine and calcium and the extent of this inhibition depends on the length and degree of fatty acid unsaturation of the molecular species.

Effects of TRH, prolactin and TSH on cell proliferation in the intermediate lobe of the rat pituitary gland.

The effect of TRH on cell proliferation in the anterior lobe of the pituitary is well known and documented. On the other hand, there are no data on the effects of TRH on the intermediate lobe of the pituitary gland. The aim of this study was to investigate the effect of TRH and its analogues (pGlu-HIs-Gly, pGlu-His-Gly-NH2) on cell proliferation in the intermediate pituitary lobe. The bromodeoxyuridine technique was used to detect the proliferating cells. It was found that TRH stimulated cell proliferation 24 h after a single injection at a dose of 100 micrograms/kg body weight. The TRH analogues did not exert any significant stimulatory effect either 12 h or 24 h after the injection. The second experiment was carried out to distinguish the probable mechanism of the action of TRH. The effects of TSH and prolactin (PRL) on intermediate lobe cell proliferation were examined. It was found that both PRL and TSH exerted a significant stimulatory effect 24 h after a single s.c. injection of PRL at a dose of 150 IU/kg body weight or TSH at a dose 20 IU/kg body weight. It therefore appears that the stimulatory effect of TRH on intermediate pituitary lobe cell proliferation is mediated by PRL and TSH.

Inhibition of phospholipase C delta by hexadecylphosphorylcholine and lysophospholipids with antitumor activity.

The antineoplastic compound hexadecylphosphorylcholine (HPC) was shown to be a highly effective inhibitor of phospholipase C delta (PLC delta 1), with an I50 of about 30 nmol/mL (30 microM) in the presence and absence of 200 microM spermine. A number of lysophospholipids, of which HPC can be considered to be a structural analog, also inhibited PLC. Lysosphingomyelin, lysophosphatidylserine, and lysophosphatidylcholine exhibited I50 values of 15, 10, and 7 nmol/mL, respectively, in the presence of 200 microM spermine. The I50 values were increased to 21-53 nmol/mL in the absence of spermine. N,N-Dimethylsphingosine and N,N,N-trimethylsphingosine, which inhibit the metastatic potential of human and murine tumor cells, were weak activators of PLC delta 1. It is postulated that HPC is more effective as an antineoplastic agent than lysophospholipids because HPC is metabolized slowly, while the lysophospholipids are metabolized rapidly in vivo.

Amplification of c-myc gene and overexpression of c-Myc protein in breast cancer and adjacent non-neoplastic tissue.

BACKGROUND: Deregulated c-Myc expression and alterations of c-myc oncogene have been reported to play an important role in breast cancer tumorigenesis. We examined the relationship between c-Myc protein level, amplification of c-myc oncogene and commonly used clinical and pathologic factors. METHODS: The studies were conducted on 94 ductal and lobular cancers. Amplification of c-Myc was assessed by the semiquantitative multiplex PCR assay. The amount of c-Myc protein was estimated by the densitometry analysis of Western blots. RESULTS: Amplification of c-Myc was found in 21% of examined cancers. There was no association of c-myc amplification with established risk factors. Overexpression of c-Myc protein without c-myc amplification was associated with negative status of axillary lymph node. The size of lobular carcinoma displaying overexpression of c-Myc and the normal copy number of c-myc gene was significantly smaller than the size of tumor with elevated c-Myc and amplification of c-myc gene (p < 0.01). Within tumors displaying overexpression of c-Myc protein and c-myc gene amplification the size of ductal carcinoma was smaller than the size of lobular carcinoma (p < 0.007). CONCLUSION: Data presented in this study suggest that alterations of c-myc gene and c-Myc protein level might be related to breast cancer progression. The prognostic utility of elevated level of c-Myc protein associated with normal status of c-myc gene for patients with lobular carcinoma requires further studies.

Isozymes delta of phosphoinositide-specific phospholipase C.

Phospholipase C (PLC, EC 3.1.4.11) is the major starting point in the phosphatidylinositol pathway, which generates intracellular signals that regulate protein kinase C and intracellular calcium concentration. To date, three major types of phosphoinositide-specific PLC species named beta, gamma and delta, have been characterized. This article reviews recent studies on isozymes delta of PLC. Four such isozymes have been cloned and termed delta1-4. Their structural organization, regulation of activity and the interaction with membrane lipid are considered. The intracellular localization of delta isozymes and distribution in various tissues are presented. Attention is given to the pathological conditions in which an abnormal protein level of PLC delta or its activity have been observed.

Expression, purification and kinetic properties of human recombinant phospholipase C delta 3.

To obtain sufficient quantities of pure phospholipase C delta 3 (PLC delta 3) necessary for structural and kinetic studies, cDNA of human fibroblast PLC delta 3 was cloned in the pPROEX-1 vector, expressed in E. coli cells as a (6 x His) fusion protein and purified to homogeneity. From 1 L of E. coli culture 8 mg of pure PLC delta 3 was obtained by a two step purification procedure, which includes phosphocellulose and Mono S cation exchange chromatography. The presence of His tag did not affect the catalytic and regulatory properties of PLC delta 3. The K(app) for PIP2 was 142 +/- 11 and 156 +/- 12 microM for His.PLC delta 3 and PLC delta 3, respectively. Recombinant PLC delta 3 showed an absolute requirement for Ca2+. Increasing the free Ca2+ concentration from 0.2 to 0.5 microM resulted in a sharp increase in enzyme activity. In comparison with human recombinant PLC delta 1 the delta 3 isoenzyme was more sensitive to low Ca2+ concentration. The Ca2+ concentration yielding maximal activation of PLC delta 1 and PLC delta 3 was 10 and 1 microM, respectively. The activity of PLC delta 3 was stimulated by polyamines and by basic proteins such as protamine, histone and mellitin. PLC delta 3 was activated most effectively by spermine and histone but the extent of this activation was lower than for PLC delta 1. The data presented indicate that the expression of PLC delta 3 in E. coli cells permits to obtain active enzyme. The catalytic and regulatory properties of PLC delta 3 are similar to those of PLC delta 1.

Cooperation of Ca2+ and pH in regulation of the activity of the 2-oxoglutarate dehydrogenase complex and its components from bovine kidney cortex.

A modified procedure for preparation of the 2-oxoglutarate dehydrogenase complex from bovine kidney cortex is presented. The enzymatic preparation obtained showed a specific activity of 18.5 mumol X min-1 X mg-1. This activity was dependent on Ca2+ (1-40 microM) and hydrogen ion concentration. At pH 7.6 in the absence of Ca2+ (less than 10(-9) M), S0.5 for 2-oxoglutarate was 2.5 mM, and in the presence of Ca2+ it was decreased to 0.3 mM. The maximum reaction rate at this pH was increased by Ca2+ by 33%. The increase of pH from 7.0 to 8.4 resulted in a 150-fold increase of S0.5. The activity of 2-oxoglutarate decarboxylase, a subunit of the dehydrogenase complex, was also dependent on Ca2+ and pH. The activity of 2-oxoglutarate decarboxylase, determined in the presence of ferrocyanide as electron acceptor, showed three different partial Michaelis constants for 2-oxoglutarate, low (K1m), medium (K2m) and high (K3m). At pH 6.9, K3m was 0.11 mM, and 0.005 mM in the absence and presence of Ca2+, respectively. The maximum reaction rate at pH 6.9 in the presence of Ca2+ was by 72% higher than in its absence. A change of pH from 6.9 to 7.6 led to an increase in K1m from 0.005 to 0.01 mM, and K3m from 0.11 to 0.60 mM. Ca2+ had no effect on the activity of lipoamide dehydrogenase or lipoamide succinyltransferase. These results indicate that, over the pH range 6.5 - 7.2, calcium ions affect the activity of the whole complex by regulating the activity of 2-oxoglutarate decarboxylase, whereas over the pH range 7.2 - 8.4 they affect the activity of the 2-oxoglutarate dehydrogenase complex by acting on the structure of the whole complex rather than by changing the activity of 2-oxoglutarate decarboxylase.

Comparison of the kinetic properties of the pyruvate dehydrogenase complex from pig kidney cortex and medulla.

The activity of the pyruvate dehydrogenase complex (PDC) purified from pig kidney medulla was affected by K+, Na+, Cl-, HCO3-, HPO4(2-) and changes in ionic strength. Increased ionic strength influenced the activity of PDC from medulla by decreasing the Vmax and S0.5 for pyruvate and increasing the Hill coefficient. The magnitude of these changes was smaller than the corresponding changes for PDC purified from the cortex. In the presence of K+ (80 mM), Na+ (20 mM), Cl- (20 mM), HCO3- (20 mM), HPO4(2-) (10 mM) and at ionic strength of 0.15 M the S0.5 for pyruvate of PDC from medulla was 117 microM and the enzyme complex was saturated by 1.1 mM pyruvate. Under these conditions the S0.5 for pyruvate of PDC derived from cortex was 159 microM and the enzyme was saturated at 4.5 mM pyruvate. Based on the results presented in this report it is suggested that PDC in kidney medulla may be regulated not only by a phosphorylation/dephosphorylation system and end-product inhibition but also via changes in ionic strength.

Expression level of adenosine kinase in rat tissues. Lack of phosphate effect on the enzyme activity.

In this report we describe cloning and expression of rat adenosine kinase (AK) in Esccherichaia coli cells as a fusion protein with 6xHis. The recombinant protein was purified and polyclonal antibodies to AK were generated in rabbits. Immunoblot analysis of extracts obtained from various rat tissues revealed two protein bands reactive with anti-AK IgG. The apparent molecular mass of these bands was 48 and 38 kDa in rat kidney, liver, spleen, brain, and lung. In heart and muscle the proteins that react with AK antibodies have the molecular masses of 48 and 40.5 kDa. In order to assess the relative AK mRNA level in rat tissues we used the multiplex PCR technique with beta-actin mRNA as a reference. We found the highest level of AK mRNA in the liver, which decreased in the order kidney > spleen > lung > heart > brain > muscle. Measurement of AK activity in cytosolic fractions of rat tissues showed the highest activity in the liver (0.58 U/g), which decreased in the order kidney > spleen > lung > brain > heart > skeletal muscle. Kinetic studies on recombinant AK as well as on AK in the cytosolic fraction of various rat tissues showed that this enzyme is not affected by phosphate ions. The data presented indicate that in the rat tissues investigated at least two isoforms of adenosine kinase are expressed, and that the expression of the AK gene appears to have some degree of tissue specificity.

The dependence of electrophoretic and spectroscopic properties of the pyruvate dehydrogenase complex on mono- and divalent ions.

The effects of various mono- and divalent ions on the pyruvate dehydrogenase complex (PDC) were investigated. To determine the radius of PDC under various conditions a two-dimensional agarose gel electrophoresis technique was used. The radius of PDC cross-linked with glutaraldehyde at ionic strength 0.04 M was calculated to be 22.0 +/- 0.1 nm. The presence of K+, Na+ or HPO4(2-) prevented changes in electromobility and of the calculated radius of PDC induced by alteration in ionic strength. The fluorescence emission spectra of PDC depended on the ionic strength and monovalent cations. The fluorescence intensity of PDC increased in the presence of 80 mM K+, and decreased in the presence of 80 mM Na+ with no shift in the emission maximum wavelength. Changes in the ionic strength to which PDC was exposed resulted in alteration of the UV absorption spectra in the 230 nm region. These alterations were prevented by HPO4(2-), whereas Na+ or K+ ions had no effect on the UV absorption spectrum of PDC.

The ultraviolet studies on protein-lipid interaction of a protein kinase C-gamma phorbol-binding domain.

Family of protein kinase C (PKC) isozymes play a key role in transducing a vast number of signals into the cells. The members of classical PKC family are activated by binding of various lipid ligands to one of the several cysteine-rich domains of the enzyme. Second cysteine-rich (Cys2) domain of PKC-gamma was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST) using the cDNA sequence from rat brain. The Cys2 protein after cleavage from GST was purified to homogeneity using glutathione-agarose and Mono-S cation exchanger column. In order to investigate the interaction of lipids and calcium with Cys2 protein we used UW spectroscopy. The UV spectrum of Cys2 protein exhibited a maximum at 205 nm. Exposition of Cys2 protein to phosphatidylserine (PS) vesicles resulted in significant decrease in the absorbance in the 210 nm region. Changes in UW spectrum of Cys2 protein induced by phorbol 12,13-dibutyrate (PDB) were smaller than those induced by PS, and addition of PDB with PS had no effect on the PS induced changes in UV spectrum of Cys2. Neither phosphatidylcholine (PC) nor phosphatidylethanolamine (PE) affected UV spectrum of Cys2 but in the presence of phosphatidylinositol 4,5 bisphosphate (PIP2) or phosphatidyliinositol 4-phosphate (PIP) vesicles some changes were observed. Calcium ions alone or in the presence of PS had no effect on the UV spectrum of Cys2 protein. These data indicate that PS comparing to PDB, interacts with a larger area of Cys2 protein, and that the binding sites for these two molecules are at least overlapping. The site of PIP and PIP2 interaction with PKC-gamma is distinct from that of phorbol ester binding site.