Conjugation of the linoleic acid oxidation product, 13-oxooctadeca-9,11-dienoic acid,a bioactive endogenous substrate for mammalian glutathione transferase

The oxidation of linoleic acid leads to the generation of several products with biological activity, including 13-oxooctadeca-9,11-dienoic acid (13-OXO), a bioactive 2,4-dienone that has been linked to cell differentiation. In the current work, the conjugation of 13-OXO by human glutathione transferases (GSTs) of the alpha (A1-1, A4-4), mu (M1-1, M2-2) and pi (the allelic variants P1-1/ile, and P1-1/val) classes, and a rat theta (rT2-2) class enzyme has been evaluated. The kinetics and stereoselectivity of the production of the 13-OXO-glutathione conjugate (13-OXO-SG) have been examined. In contrast to many xenobiotic substrates, the endogenous substrate 13-OXO does not exhibit an appreciable non-enzymatic rate of conjugation under physiological conditions. Therefore, the GST-catalyzed conjugation takes on greater significance as it provides the only realistic means for formation of 13-OXO-SG in most biological systems. Alpha class enzymes are most efficient at catalyzing the formation of 13-OXO-SG with kcat/Km values of 8.9 mM(-1) s(-1) for GST A1-1 and 2.14 mM(-1) s(-1) for GST A4-4. In comparison, enzymes from the mu and pi classes exhibit specificity constants from 0.4 to 0.8 mM(-1) s(-1). Conjugation of 13-OXO with glutathione at C-9 of the substrate can yield a pair of diastereomers that can be resolved by chiral HPLC. GSTs from the mu and pi classes are the most stereoselective enzymes and there is no apparent relationship between catalytic efficiency and stereoselectivity. The role of GST in the metabolic disposition of the bioactive oxidation products of linoleic acid has implications for the regulation of normal cellular functions by these versatile enzymes.     

Conjugation of the linoleic acid oxidation product, 13-oxooctadeca-9,11-dienoic acid,a bioactive endogenous substrate for mammalian glutathione transferase

The oxidation of linoleic acid leads to the generation of several products with biological activity, including 13-oxooctadeca-9,11-dienoic acid (13-OXO), a bioactive 2,4-dienone that has been linked to cell differentiation. In the current work, the conjugation of 13-OXO by human glutathione transferases (GSTs) of the alpha (A1–1, A4–4), mu (M1–1, M2–2) and pi (the allelic variants P1–1/ile, and P1–1/val) classes, and a rat theta (rT2–2) class enzyme has been evaluated. The kinetics and stereoselectivity of the production of the 13-OXO-glutathione conjugate (13-OXO-SG) have been examined. In contrast to many xenobiotic substrates, the endogenous substrate 13-OXO does not exhibit an appreciable non-enzymatic rate of conjugation under physiological conditions. Therefore, the GST-catalyzed conjugation takes on greater significance as it provides the only realistic means for formation of 13-OXO-SG in most biological systems. Alpha class enzymes are most efficient at catalyzing the formation of 13-OXO-SG with k_cat/K_m values of 8.9 mM⁻¹ s⁻¹ for GST A1–1 and 2.14 mM⁻¹ s⁻¹ for GST A4–4. In comparison, enzymes from the mu and pi classes exhibit specificity constants from 0.4 to 0.8 mM⁻¹ s⁻¹. Conjugation of 13-OXO with glutathione at C-9 of the substrate can yield a pair of diastereomers that can be resolved by chiral HPLC. GSTs from the mu and pi classes are the most stereoselective enzymes and there is no apparent relationship between catalytic efficiency and stereoselectivity. The role of GST in the metabolic disposition of the bioactive oxidation products of linoleic acid has implications for the regulation of normal cellular functions by these versatile enzymes.     

Glutathione S-transferase-catalyzed conjugation of 9,10-epoxystearic acid with glutathione

The possible role of glutathione S-transferases (GST) in detoxification of fatty acid epoxides generated during lipid peroxidation has been evaluated. Present studies showed that cytosolic human glutathione S-transferases belonging to alpha, mu, and pi classes isolated from human liver and lung catalyzed the conjugation of glutathione and 9,10-epoxystearic acid. The product of enzymatic reaction, i.e., conjugate of GSH and epoxystearic acid, was isolated and characterized. The Michaelis constant (Km) values of the alpha, mu, and pi classes of GSTs for 9,10-epoxystearic acid were found to be 0.47, 0.32 and 0.80 mM, respectively, whereas the maximal velocity (V max) values for the alpha, mu, and pi classes of GSTs were found to be 142, 256, and 52 mol/min/mol, respectively. These results indicate that even though 9,10-epoxystearic acid is a substrate for all the three classes of GSTs, the mu class isozymes have maximum activity toward this substrate and may preferentially metabolize fatty acid epoxides more effectively as compared to the other classes of GSTs.     

Structural determinants of glutathione transferases with azathioprine activity identified by DNA shuffling of alpha class members

A library of alpha class glutathione transferases (GSTs), composed of chimeric enzymes derived from human (A1-1, A2-2 and A3-3), bovine (A1-1) and rat (A2-2 and A3-3) cDNA sequences was constructed by the method of DNA shuffling. The GST variants were screened in bacterial lysates for activity with the immunosuppressive agent azathioprine, a prodrug that is transformed into its active form, 6-mercaptopurine, by reaction with the tripeptide glutathione catalyzed by GSTs. Important structural determinants for activity with azathioprine were recognized by means of primary structure analysis and activities of purified enzymes chosen from the screening. The amino acid sequences could be divided into 23 exchangeable segments on the basis of the primary structures of 45 chosen clones. Segments 2, 20, 21, and 22 were identified as primary determinants of the azathioprine activity representing two of the regions forming the substrate-binding H-site. Segments 21 and 22 are situated in the C-terminal helix characterizing alpha class GSTs, which is instrumental in their catalytic function. The study demonstrates the power of DNA shuffling in identifying segments of primary structure that are important for catalytic activity with a targeted substrate. GSTs in combination with azathioprine have potential as selectable markers for use in gene therapy. Knowledge of activity-determining segments in the structure is valuable in the protein engineering of glutathione transferase for enhanced or suppressed activity.     

Expression of glutathione transferase isoenzymes in the porcine ovary in relationship to follicular maturation and luteinization

The expression of different isoenzymes of glutathione transferase (GST), i.e. the cytosolic subunits GSTA1/A2, A3, A4, A5, M1/2, M2 and P1, T2, and the microsomal GST in follicles of different sizes and in corpora lutea from porcine ovary, was investigated by Western blotting. No immunoreactivity was obtained with anti-rat GSTT2 or anti-rat microsomal GST polyclonal antibodies. In contrast, GSTA1/A2, A3, A4, A5, M1/2, M2 and P1 are all expressed in the cytosol from porcine ovaries. In general, the highest levels of these GST isoenzymes were present in the cytosol from corpora lutea, in agreement with measurements of activity towards 1-chloro-2,4-dinitrobenzene. Immunoreactivity with anti-rat GSTP1 was only obtained with follicles. The cytosolic GSTs from follicles and corpora lutea were affinity purified on glutathione-Sepharose and separated by reversed-phase high-performance liquid chromatography in order to quantitate the different subunits. A peak corresponding to the class pi subunit was present in follicles. This peak was also seen with corpora lutea, although at very low level. There were four peaks containing class mu subunits. The remaining peaks were concluded to contain the class alpha subunits, except for two peaks which are suggested to contain proteins other than GSTs. The levels of the different subunits were quantitated on the basis of the areas under the peaks and the relative amounts in follicles of different sizes and in corpora lutea corresponded well with the Western blot analysis.     

Purification and Characterization of Class Alpha and Mu Glutathione S-Transferases From Porcine Liver

Six cytosolic GSTs from porcine liver were purified by a combination of glutathione affinity chromatography and ion-exchange HPLC. The isoenzymes were characterized by SDS-PAGE, gel filtration, isoelectric focusing, immunoblotting analysis and determination of substrate specificities and inhibition characteristics. The purified GSTs belong to the alpha and mu classes, respectively. No class pi isoenzyme was isolated or detected. The class alpha GST pA1-1* exists as a homodimer (M_r = 25.3 kDa), whereas GST pA2-3* consists of two subunits with different M_r values (27.0 and 25.3 kDa). The estimated pI values were 9.5 and 8.8, respectively. Furthermore, four class mu porcine GSTs, pM1-1*, pM1-2*, pM3-?* and pM4-?*, were isolated. The isoenzyme pM1-1* possesses a relative molecular mass of 27.2 kDa and a pI value of 6.2. Additional pM1 isoenzymes hybridize with the subunit pM2* (M_r = 25.2) to furnish a heterodimer, which shows a pI value of 5.8. The other class mu isoenzymes are heterodimers with pI values of 5.45 and 5.05. Substrate specificities and inhibition characteristics correlate very well with those of the corresponding human isoenzymes. The results are discussed with regard to the usefulness of porcine GSTs as an in vitro testing model.     

Activation of rat glutathione transferases in class mu by active oxygen species

The activities of rat glutathione transferases (GSTs) 3-3, 3-4, 4-4 in Class mu towards 1-chloro-2,4-dinitrobenzene (CDNB) but not 1,2-dichloro-4-nitrobenzene were increased up to 5-fold during preincubation with 0.4 mM xanthine and xanthine oxidase in 50 mM potassium phosphate, pH 7.8, containing 0.1 mM EDTA. The activated GST 3-4, purified by S-hexylglutathione affinity chromatography after the treatment, had a higher specific activity (130 units/mg) than that of the nontreated (35 units/mg), the Km and Vmax values for glutathione or CDNB also were increased. Other rat GSTs in Class alpha and pi were inactivated by the same treatment. In the presence of superoxide dismutase, the activation of GST 3-4 did not occur.     

Metabolism of two Go alpha isoforms in neuronal cells during differentiation

We have previously shown that undifferentiated N1E-115 neuroblastoma cells express only one isoform of Go alpha (pI = 5.8), whereas differentiated neuroblastoma cells expressed, in addition to this isoform, another Go alpha with a more acidic pI (5.55). Moreover, primary cultures of cerebellar granule cells, which are extremely well differentiated cells yielding a high density of synapses, expressed only a single Go alpha isoform with a pI of 5.55 (Brabet, P., Pantaloni, C., Rodriguez Martinez, J., Bockaert, J., and Homburger, V. (1990) J. Neurochem. 54, 1310-1320). In this report, using biosynthetic labeling with [35S]methionine and specific quantitative immunoprecipitation with a polyclonal antibody raised against the purified Go alpha protein, we have determined 1) the degradation rate of total Go alpha (sum of the two isoforms) in differentiated as well as in undifferentiated neuroblastoma cells and in cerebellar granule cells, 2) the degradation rates of each isoform in differentiated neuroblastoma cells. The t 1/2 for total Go alpha protein degradation was very different in the three neuronal cell populations and was 28 +/- 5 h (n = 5), 58 +/- 9 h (n = 5), and 154 +/- 22 h (n = 6) in undifferentiated, differentiated neuroblastoma, and granule cells, respectively. Using two-dimensional gel analysis of immunoprecipitates, we have also determined the individual t 1/2 for degradation of each Go alpha isoform in differentiated neuroblastoma cells, in which the two Go alpha isoforms were expressed. Results indicated that the two Go alpha isoforms exhibit similar t1/2 for degradation (49 +/- 5 h, n = 3). Thus, the t1/2 for degradation of the more basic Go alpha isoform is higher in differentiated neuroblastoma cells (49 +/- 5 h, n = 3) than in undifferentiated neuroblastoma cells (28 +/- 5 h, n = 5) which expressed only the more basic Go alpha isoform. It can be concluded that the degradation rate of the more basic Go alpha isoform is not a characteristic of the protein itself but depends on the state of the cell differentiation. The comparison between the t1/2 for degradation of the more acidic Go alpha isoform is differentiated neuroblastoma cells (51 +/- 6 h, n = 3) with that of cerebellar granule cells (154 +/- 22 h, n = 6) suggests that there is also a decrease in the degradation rate of the more acidic Go alpha isoform during differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and Characterisation of Hepatic Glutathione Transferases from a Herbivorous Marsupial, the Brushtail Possum (Trichosurus vulpecula)

A single glutathione transferase isoenzyme was purified from hepatic cytosol of the brushtail possum and shown to represent 3.6 ± 0.3% of the total cytosolic protein. Characterisation of the enzyme, termed Possum GST 1–1, indicated that it possessed similar catalytic activity and structural homology with isoenzymes belonging to the alpha class of glutathione transferases. This homodimeric GST exhibited a single band with an apparent molecular mass of 25.4 kDa on sodium dodecyl sulphate-polyacrylamide gels and an apparent pI of 9.8. Inhibition studies demonstrated that Possum GST 1–1 displays binding affinity for a range of inhibitors similar to that shown by alpha class GSTs purified from other mammals. Immunoblot analysis demonstrated immuno-cross reactivity between Possum GST 1–1 and antisera raised against human alpha GST, while this GST did not cross-react with antisera raised against human mu and pi GST. N-terminal sequencing of purified Possum GST 1–1 revealed that the N-terminus of the protein is chemically blocked. Sequence analysis of three internal peptide sequences demonstrated homology with mammalian alpha GSTs. Of particular interest is the significant substrate specificity that Possum GST 1–1 displays with both organic and inorganic hydroperoxides. It is proposed that this substrate specificity is an evolutionary adaptation to a diet high in potentially toxic plant allelochemicals.     

Characterisation of aminopeptidase activity in scab mites (Psoroptes spp.)

Soluble and membrane-bound aminopeptidase activities were demonstrated in extracts of P. cuniculi (Delafond). Leucine aminopeptidase (LAP) activity in the soluble fraction of P. cuniculi extracts displayed substrate preference for amino acid derivatives with terminal leucine and methionine over those with acidic, basic or heterocyclic groups. P. cuniculi LAP was inhibited by leucinethiol (IC(50) = 1.4 +/- 0.4 nM), bestatin (IC(50) = 3.9 +/- 1.7 microM), Arphamenine A (IC(50) = 0.37 +/- 0.03 mM) the chelating agent 1,10-phenanthroline (IC(50) = 2.3 +/- 0.5 mM), Zn(2+), Cu(2+) Ni(2+), and Co(2+), and activated by Mn(2+) and Mg(2+). The LAP activity was visualised as a single major band after electrophoresis on native gels and eluted from a size exclusion column as a single major peak representing a molecular mass range of 85-116 kDa. Degenerate oligonucleotide primers were used to amplify short fragments of genomic DNA containing nucleotide sequence coding for the cation-binding motifs of the co-catalytic Zn(2+) binding domains of dizinc leucine aminopeptidases in both P. cuniculi and P.ovis (Hering). The major soluble aminopeptidase from these mites therefore displays most of the characteristics associated with typical cytosolic leucine aminopeptidases belonging to the M17 family of metalloproteinases.     

Purification and characterization of human muscle glutathione S-transferases: evidence that glutathione S-transferase zeta corresponds to a locus distinct from GST1, GST2, and GST3.

Human muscle glutathione S-transferase isozyme, GST zeta (pI 5.2) has been purified by three different methods using immunoaffinity chromatography, DEAE cellulose chromatography, and isoelectric focusing. GST zeta prepared by any of the three methods does not recognize antibodies raised against the alpha, mu, or pi class glutathione S-transferases of human tissues. GST zeta has a blocked N-terminus and its peptide fingerprints also indicate it to be distinct from the alpha, mu, or pi class isozymes. As compared to GSTs of alpha, mu, and pi classes, GST zeta displays higher activities toward t-stilbene oxide and Leukotriene A4 methyl ester. GST zeta also expresses GSH-peroxidase activity toward hydrogen peroxide. The Kms of GST zeta for CDNB and GSH were comparable to those reported for other human GSTs but its Vmax for CDNB, 7620 mol/mol/min, was found to be considerably higher than that reported for other human GSTs. The kinetics of inhibition of GST zeta by hematin, bile acids, and other inhibitors also indicate that it was distinct from the three classes of GST isozymes. These studies suggest that GST zeta corresponds to a locus distinct from GST1, GST2, and GST3 and probably corresponds to the GST4 locus as suggested previously by Laisney et al. (1984, Human Genet. 68, 221-227). The results of peptide fingerprints and kinetic analysis indicate that as compared to the pi and alpha class isozymes, GST zeta has more structural and functional similarities with the mu class isozymes. Besides GST zeta several other GST isozymes belonging to pi and mu class have also been characterized in muscle. The pi class GST isozymes of muscle have considerable charge heterogeneity among them despite identical N-terminal sequences.     

5'-AMP-activated protein kinase activity and subunit expression in exercise-trained human skeletal muscle.

5'-AMP-activated protein kinase (AMPK) has been proposed to be a pivotal factor in cellular responses to both acute exercise and exercise training. To investigate whether protein levels and gene expression of catalytic (alpha(1), alpha(2)) and regulatory (beta(1), beta(2), gamma(1), gamma(2), gamma(3)) AMPK subunits and exercise-induced AMPK activity are influenced by exercise training status, muscle biopsies were obtained from seven endurance exercise-trained and seven sedentary young healthy men. The alpha(1)- and alpha(2)-AMPK mRNA contents in trained subjects were both 117 +/- 2% of that in sedentary subjects (not significant), whereas mRNA for gamma(3) was 61 +/- 1% of that in sedentary subjects (not significant). The level of alpha(1)-AMPK protein in trained subjects was 185 +/- 34% of that in sedentary subjects (P < 0.05), whereas the levels of the remaining subunits (alpha(2), beta(1), beta(2), gamma(1), gamma(2), gamma(3)) were similar in trained and sedentary subjects. At the end of 20 min of cycle exercise at 80% of peak O(2) uptake, the increase in phosphorylation of alpha-AMPK (Thr(172)) was blunted in the trained group (138 +/- 38% above rest) compared with the sedentary group (353 +/- 63% above rest) (P < 0.05). Acetyl CoA-carboxylase beta-phosphorylation (Ser(221)), which is a marker for in vivo AMPK activity, was increased by exercise in both groups but to a lower level in trained subjects (32 +/- 5 arbitrary units) than in sedentary controls (45 +/- 1 arbitrary units) (P < 0.01). In conclusion, trained human skeletal muscle has increased alpha(1)-AMPK protein levels and blunted AMPK activation during exercise.     

Effects of imidazole and indomethacin on fluid balance in isolated sheep lungs

We determined the effects of extracorporeal perfusion with a constant flow (75 ml . min-1 . kg-1) of autologous blood on hemodynamics and fluid balance in sheep lungs isolated in situ. After 5 min, perfusate leukocyte and platelet counts fell by two-thirds. Pulmonary arterial pressure (Ppa) increased to a maximum of 32.0 +/- 3.4 Torr at 30 min and thereafter fell. Lung lymph flow (QL), measured from the superior thoracic duct, and perfusate thromboxane B2 (TXB2) concentrations followed similar time courses but lagged behind Ppa, reaching maxima of 4.1 +/- 1.2 ml/h and 2.22 +/- 0.02 ng/ml at 60 min. Lung weight gain, measured as the opposite of the weight change of the extracorporeal reservoir, and perfusate 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) concentration increased rapidly during the first 60 min and then more gradually. After 210 min, weight gain was 224 +/- 40 g and 6-keto-PGF1 alpha concentration, 4.99 +/- 0.01 ng/ml. The ratio of lymph to plasma oncotic pressure (pi L/pi P) at 30 min was 0.61 +/- 0.06 and did not change significantly. Imidazole (5 mM) reduced the changes in TXB2, Ppa, QL, and weight and platelet count but did not alter 6-keto-PGF1 alpha, pi L/pi P, or leukocyte count. Indomethacin (0.056 mM) reduced TXB2, 6-keto-PGF1 alpha, and the early increases in weight, Ppa, and QL but did not alter the time courses of leukocyte or platelet counts. Late in perfusion, however, Ppa and QL were greater than in either untreated or imidazole-treated lungs.(ABSTRACT TRUNCATED AT 250 WORDS)     

3-Methyleneoxindole: an affinity label of glutathione S-transferase pi which targets tryptophan 38.

The compound 3-methyleneoxindole (MOI), a photooxidation product of the plant auxin indole-3-acetic acid, functions as an affinity label of the dimeric pi class glutathione S-transferase (GST) isolated from pig lung. MOI inactivates the enzyme to a limit of 14% activity. The k for inactivation by MOI is decreased 20-fold by S-hexylglutathione but only 2-fold by S-methylglutathione, suggesting that MOI does not react entirely within the glutathione site. The striking protection against inactivation provided by S-(hydroxyethyl)ethacrynic acid indicates that MOI reacts in the active site region involving both the glutathione and the xenobiotic substrate sites. Incorporation of [(3)H]MOI up to approximately 1 mol/mol of enzyme dimer concomitant with maximum inactivation suggests that there are interactions between subunits. Fractionation of the proteolytic digest of [(3)H]MOI-modified GST pi yielded Trp38 as the only labeled amino acid. The crystal structure of the human GST pi-ethacrynic acid complex (2GSS) shows that the indole of Trp38 is less than 4 A from ethacrynic acid. Similarly, MOI may bind in this substrate site. In contrast to its effect on the pi class GST, MOI inactivates much less rapidly and extensively alpha and mu class GSTs isolated from the rat. These results show that MOI reacts preferentially with GST pi. Such a compound may be useful in novel combination chemotherapy to enhance the efficacy of alkylating cancer drugs while minimizing toxic side effects.     

Characterization of a novel alpha-class anionic glutathione S-transferase isozyme from human liver

A novel, alpha-class glutathione S-transferase (GST) isozyme has been isolated from human liver using glutathione (GSH) affinity chromatography, DEAE-cellulose ion-exchange chromatography, and immunoaffinity chromatography. The isozyme is a dimer of approximately 25,000 Mr with blocked N termini. Structural, kinetic, and immunological properties of this enzyme indicate that it belongs to the alpha class of GSTs. Noticeable differences between the properties of this enzyme and the other alpha-class GSTs of human liver are its anionic nature (pI 5.0), GSH peroxidase activity toward hydrogen peroxide, and relatively higher GSH conjugating activities toward CDNB and epoxide substrates as compared to other alpha-class GSTs. Results of these studies indicate that anionic GST omega characterized previously (Y. C. Awasthi, D. D. Dao, and R. P. Saneto, 1980, Biochem. J. 191, 1-10) from human liver is a mixture of GST pi and a novel alpha-class GST. We have, therefore, reassigned the name GST omega to this new alpha-class anionic GST of human liver.     

Cellular localization and substrate specificity of isoelectric forms of human liver neuraminidase activity.

The four major isoelectric forms of human liver neuraminidase (with pI values between 3.4 and 4.8) have been isolated by preparative isoelectric focusing and characterized with regard to their substrate specificity using glycoprotein, glycopeptide, oligosaccharide and ganglioside natural substrates. All forms exhibited a rather broad linkage specificity and were capable of hydrolyzing sialic acid glycosidically linked alpha 2-3, alpha 2-6 and alpha 2-8, although differential rates of hydrolysis of the substrates were found for each form. The most acidic form 1 (pI 3.4) was most active on sialyl-lactose, whereas form 2 (pI 3.9) and 3 (pI 4.4) were most active on the more hydrophobic ganglioside substrates. Form 4 (pI 4.8) was most active on the low-Mr hydrophilic substrates (fetuin glycopeptide, sialyl-lactose). Each form was less active on the glycoprotein fetuin than on a glycopeptide derived from fetuin. Organelle-enriched fractions were prepared from fresh human liver tissue and neuraminidase activity on 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid was recovered in plasma membrane, microsomal, lysosomal and cytosolic preparations. Isoelectric focusing of the neuraminidase activity recovered in each of these preparations resulted in significantly different isoelectric profiles (number, relative amounts and pI values of forms) for each preparation. The differential substrate specificity of the isoelectric forms and the different isoelectric focusing profiles of neuraminidase activity recovered in subcellular-enriched fractions suggest that specific isoelectric forms with broad but defined substrate specificity are enriched at separate sites within the cell.     

Dissociation between changes in muscle Na+-K+-ATPase isoform abundance and activity with consecutive days of exercise and recovery

The early plasticity of vastus lateralis Na(+)-K(+)-ATPase to the abrupt onset of prolonged submaximal cycling was studied in 12 untrained participants (Vo(2 peak) 44.8 +/- 2.0 ml x kg(-1) x min(-1), mean +/- SE) using a 6-day protocol (3 days of exercise plus 3 days of recovery). Tissue samples were extracted prior to (Pre) and following exercise (Post) on day 1 (E1) and day 3 (E3) and on each day of recovery (R1, R2, R3) and analyzed for changes in maximal protein (beta(max)) (vanadate-facilitated [(3)H]ouabain binding), alpha- and beta-isoform concentration (quantitative immunoblotting) and maximal Na(+)-K(+)-ATPase activity (V(max)) (3-O-methylfluorescein K(+)-stimulated phosphatase assay). For beta(max) (pmol/g wet wt), an increase (P < 0.05) of 11.8% was observed at R1 compared with E1-Pre (340 +/- 14 vs 304 +/- 17). For the alpha-isoforms alpha(1), alpha(2), and alpha(3), increases (P < 0.05) of 46, 42, and 31% were observed at R1, respectively. For the beta-isoform, beta(1) and beta(2) increased (P < 0.05) by 19 and 28% at R1, whereas beta(3) increased (P < 0.05) by 18% at R2. With the exception of alpha(2) and alpha(3), the increases in the isoforms persisted at R3. Exercise resulted in an average decrease (P < 0.05) in V(max) by 14.3%. No differences were observed in V(max) at E1 - Pre and E3 - Pre or between R1, R2, and R3. It is concluded that 3 days of prolonged exercise is a powerful stimulus for the rapid upregulation of the Na(+)-K(+)-ATPase subunit isoforms. Contrary to our hypothesis, the increase in subunit expression is not accompanied by increases in the maximal catalytic activity.     

The tyrosine kinase activity of the epidermal-growth-factor receptor is necessary for phospholipase A2 activation.

Cytosolic glutathione transferases (GSTs) were purified from the rat spleen by S-hexyl-GSH-Sepharose chromatography, and two major forms were identified as GSTs 2-2 and 7-7 (GST P). Besides these forms an acidic form (pI 5.8) was purified by chromatofocusing at pH 7-4 and it accounted for about 1% of the total GST activity bound to S-hexyl-GSH-Sepharose. Two-dimensional gel electrophoresis revealed that it is a homodimer (subunit Mr 26,000 with pI 5.8). Immunoblot analysis demonstrated that it was immunologically related to GSTs 2-2 and 1-1, and its N-terminal amino acid was apparently blocked, similarly to other forms of the class Alpha. This form had a low activity towards cumene hydroperoxide or 4-hydroxynon-2-enal, indicating that this form differed from GSTs 10-10 and 8-8 as well as from GSTs 1-1 and 2-2. These results suggest that it is a new form of GST belonging to the class Alpha.     

Muscle Na+-K+-ATPase response during 16 h of heavy intermittent cycle exercise

This study investigated the effects of a 16-h protocol of heavy intermittent exercise on the intrinsic activity and protein and isoform content of skeletal muscle Na(+)-K(+)-ATPase. The protocol consisted of 6 min of exercise performed once per hour at approximately 91% peak aerobic power (Vo(2 peak)) with tissue sampling from vastus lateralis before (B) and immediately after repetitions 1 (R1), 2 (R2), 9 (R9), and 16 (R16). Eleven untrained volunteers with a Vo(2 peak) of 44.3 +/- 2.3 ml x kg(-1) x min(-1) participated in the study. Maximal Na(+)-K(+)-ATPase activity (V(max), in nmol x mg protein(-1) x h(-1)) as measured by the 3-O-methylfluorescein K(+)-stimulated phosphatase assay was reduced (P < 0.05) by approximately 15% with exercise regardless of the number of repetitions performed. In addition, V(max) at R9 and R16 was lower (P < 0.05) than at R1 and R2. Vanadate-facilitated [(3)H]ouabain determination of Na(+)-K(+)-ATPase content (maximum binding capacity, pmol/g wet wt), although unaltered by exercise, increased (P < 0.05) 8.3% by R9 with no further increase observed at R16. Assessment of relative changes in isoform abundance measured at B as determined by quantitative immunoblotting showed a 26% increase (P < 0.05) in the alpha(2)-isoform by R2 and a 29% increase in alpha(3) by R9. At R16, beta(3) was lower (P < 0.05) than at R2 and R9. No changes were observed in alpha(1), beta(1), or beta(2). It is concluded that repeated sessions of heavy exercise, although resulting in increases in the alpha(2)- and alpha(3)-isoforms and decreases in beta(3)-isoform, also result in depression in maximal catalytic activity.