Nmol Min-1 Mg-1 Research Articles

Isolation of single mammalian proximal tubule cells: effects of hypotonic shocks on cell yield and function.

Nord et al. [Am J Physiol 1986; 250:F539-F550] proposed a method to give a high yield of proximal tubule cells by exposing a suspension of rabbit cortical tubules to a hypotonic shock in calcium-free media. The present study describes the effects of both amplitude and duration of the hypotonic treatment on some transport-related characteristics of individual cells as compared to the starting tubule suspension. The averaged cell yield increased by an order of magnitude when the osmolality of the hypotonic solution was varied in four steps from 200 (C200 cells) to 70 mosm/kg H2O (C70 cells) while the proportion of trypan blue-positive cells progressively decreased from 33% for C200 cells to 9.5% for C70 cells. An increase in duration of the hypotonic shock from 0.5 to 6 min did not change the cell yield of C200 cells while it significantly increased that of C70 cells by 61%. Basal and ouabain-sensitive oxygen consumption (QO2) increased by 57 and 155%, respectively, from C70 to C200 cells but was approximately one order of magnitude smaller than the QO2 measured for tubule suspension. Intracellular ATP content averaged 5.5 +/- 0.8 nmol/mg for the starting tubule suspension, 4.6 +/- 0.8 nmol/mg for C70 cells but only 1.3 +/- 0.1 nmol/mg for C200 cells. The maximal velocity for phloridzin-sensitive alpha-methyl glucose transport averaged 13.7 +/- 1.7 nmol min-1 mg-1 for C70 cells and only 6.3 +/- 1.3 nmol min-1 mg-1 for C200 cells which is approximately one order of magnitude smaller than what can be expected from a tubule presenting a good access to luminal membrane. We conclude from these results that, in the process of isolating individual cells from a polarized epithelium, membrane transport rates have decreased by one order of magnitude and this reduction is intensified by a large hypotonic shock. In comparison with C200 cells, the cells obtained with a large hypotonic shock give a high yield, a larger proportion of trypan blue-negative cells and their lower overall transport rate allows the cells to maintain a better electrochemical gradient for Na and a higher intracellular ATP level.

Purification and characterization of the cytoplasmic domain of human receptor-like protein tyrosine phosphatase RPTP mu.

RPTP mu is a recently described receptor-like protein tyrosine phosphatase (PTP), the ectodomain of which mediates homophilic cell-cell adhesion. The cytoplasmic part contains two homologous PTP-like domains and a juxtamembrane region that is about twice as large as in other receptor-like PTPs. The entire 80-kDa cytoplasmic part of human RPTP mu was expressed in insect Sf9 cells and its enzymatic activity was characterized after purification to electrophoretic homogeneity. In addition, the effects of deletion and point mutations were analyzed following expression in Escherichia coli cells. The purified cytoplasmic part of RPTP mu displays high activity toward tyrosine-phosphorylated, modified lysozyme (Vmax 4500 nmol min-1 mg-1) and myelin basic protein (Vmax 8500 nmol min-1 mg-1) but negligible activity toward tyrosine-phosphorylated angiotensin or the nonapeptide, EDNDpYINASL, that serves as a good substrate for protein tyrosine phosphatase PTP1B. This suggests that RPTP mu and PTP1B have distinct substrate specificities. Catalytic activity is independent of Ca2+ (up to 1 mM) but is strongly inhibited by Zn2+, Mn2+, vanadate, phenylarsenic oxide, and heparin. The first of the two catalytic domains is 5-10 times less active than the expressed catalytic region containing both domains. Mutation of Cys 1095 to Ser in the first catalytic domain abolishes enzymatic activity when analyzed following expression in either E. coli or mammalian COS cells. Deletion of the first 53 amino acids from the juxtamembrane region reduces catalytic activity about 2-fold.

Sphingolipid Long-Chain Base Synthesis in Plants (Characterization of Serine Palmitoyltransferase Activity in Squash Fruit Microsomes).

The activity of serine palmitoyltransferase (palmitoyl-coenzyme A [CoA]:L-serine [Ser]-C-palmitoyltransferase [decarboxylating], EC 2.3.1.50), the enzyme catalyzing the first step in the synthesis of the long-chain base required for sphingolipid assembly, has been characterized in a plant system. Enzyme activity in a microsomal membrane fraction from summer squash fruit (Cucurbita pepo L. cv Early Prolific Straightneck) was assayed by monitoring the incorporation of L-[3H]Ser into the chloroform-soluble product, 3-ketosphinganine. Addition of NADPH to the assay system resulted in the conversion of 3-ketosphinganine to sphinganine. The apparent Km for Ser was approximately 1.8 mM. The enzyme exhibited a strong preference for palmitoyl-CoA, with optimal activity at a substrate concentration of 200 [mu]M. Pyridoxal 5[prime]-phosphate was required as a coenzyme. The pH optimum was 7.6, and the temperature optimum was 36 to 40[deg]C. Enzyme activity was greatest in the microsomal fraction obtained by differential centrifugation and was localized to the endoplasmic reticulum using marker enzymes. Two known mechanism-based inhibitors of the mammalian enzyme, L-cycloserine and [beta]-chloro-L-alanine, were effective inhibitors of enzyme activity in squash microsomes. Changes in enzyme activity with size (age) of squash fruit were observed. The results from this study suggest that the properties and catalytic mechanism of Ser palmitoyltransferase from squash are similar to those of the animal, fungal, and bacterial enzyme in most respects. The specific activity of the enzyme in squash microsomes ranged from 0.57 to 0.84 nmol min-1 mg-1 of protein, values 2- to 20-fold higher than those previously reported for preparations from animal tissues.

Expression of rat microsomal epoxide hydrolase in Escherichia coli. Identification of a histidyl residue essential for catalysis

The cDNA containing the complete coding region for rat microsomal epoxide hydrolase (EC 3.3.2.3) was cloned into the expression/secretion vector pIN-III-OmpA3 and expressed in Escherichia coli strain TG1. Recombinant epoxide hydrolase was found to represent 4-9% of total bacterial protein and catalyzed the hydrolysis of styrene oxide and benzo[a]pyrene 4,5-oxide with specific activities of 421 and 734 nmol min-1 mg of epoxide hydrolase-1, respectively. Previous work implicated a histidyl residue at or near the active site of the enzyme (DuBois, G. C., Appella, E., Levin, W., Lu, A. Y. H., and Jerina, D. M. (1978) J. Biol. Chem. 253, 2932-2939). Comparison of the amino acid sequences of rat, human, and rabbit epoxide hydrolases revealed the presence of 14 conserved histidyl residues. To investigate the role of these residues in epoxide hydrolysis, site-specific mutants were generated and expressed in E. coli. Mutants H64L, H82L, H115N, H126N, H129L, H148N, H170L, H176L, H242L, H247L, H301L, H385L, K386M-H387L, delta 385-391, and H407L catalyzed the hydrolysis of benzo[a]pyrene 4,5-oxide with specific activities between 115 and 830 nmol min-1 mg-1. Mutants H431L, H431N, and H431R were all found to have activities of < 5 nmol min-1 mg-1, which is at least 150-fold less than the activity of the wild type enzyme. A Vm versus pH profile for the recombinant wild type epoxide hydrolase revealed a broad pH optimum of 6.5 to 8.5 and the presence of three ionizable groups with pKa values of 5.8 +/- 0.2, 9.2 +/- 0.1, and 9.7 +/- 0.4. The group with a pKa of 5.8 is preferentially unprotonated, while the other two groups are preferentially protonated for catalysis. We propose that histidine 431 corresponds to the group with a pKa of 5.8, while the others, with pKa values of 9.2 and 9.7 likely represent lysyl, cysteinyl, or tyrosyl residues. Thus, the data are consistent with a model where His-431 acts as a general base, abstracting a proton from water, while another residue(s), perhaps lysine, act as a general acid protonating the alkoxide anion that forms upon cleavage of the carbon-oxygen bond.

Demethylation and degradation of phenylmethylethers by the sulfide-methylating homoacetogenic bacterium strain TMBS 4

Biochemical studies on anaerobic phenylme-thylether cleavage by homoacetogenic bacteria have been hampered so far by the complexity of the reaction chain involving methyl transfer to acetyl-CoA synthase and subsequent methyl group carbonylation to acetyl-CoA. Strain TMBS 4 differs from other demethylating homoacetogenic bacteria in using sulfide as a methyl acceptor, thereby forming methanethiol and dimethylsulfide. Growing and resting cells of strain TMBS 4 used alternatitively CO2 as a precursor of the methyl acceptor CO for homoacetogenic acetate formation. Demethylation was inhibited by propyl iodide and reactivated by light, indicating involvement of a corrinoid-dependent methyltransferase. Strain TMBS 4 contained ca. 750 nmol g dry mass-1 of a corrinoid tentatively identified as 5-hydroxybenzimidazolyl cobamide. A photometric assay for measuring the demethylation activity in cell extracts was developed based on the formation of a yellow complex of Ti3+ with 5-hydroxyvanillate produced from syringate by demethylation. In cell extracts, the methyltransfer reaction from methoxylated aromatic compounds to sulfide or methanethiol depended on reductive activation by Ti3+. ATP and Mg2+ together greatly stimulated this reductive activation without being necessary for the demethylation reaction itself. The specific activity of the transmethylating enzyme system increased proportionally with protein concentration up to 3 mg ml-1 reaching a constant level of 20 nmol min-1 mg-1 at protein concentrations ≥ 10 mg ml-1. The specific rate of activation increased in a non-linear manner with protein concentration. Strain TMBS 4 degraded gallate, the product of sequential demethylations, to 3 acetate through the phloroglucinol pathway as found earlier with Pelobacter acidigallici.

Synthesis of pyrophosphate and ATP by soluble mitochondrial F1

Soluble F1 from beef heart that has been depleted of adenine nucleotides to values of 0.4 mol of ADP and 0.1 mol ATP/mol of enzyme has the capacity to synthetize about 0.1 mol of ATP/mol of enzyme from medium phosphate in the presence of Mg2+ and 30% dimethyl sulfoxide. Under the same conditions, native and adenine nucleotide depleted F1 can also synthesize pyrophosphate to values that range from 0.03 to 0.05 mol/mol of F1. The formation of pyrophosphate requires Mg2+ and dimethyl sulfoxide. The formed pyrophosphate remains bound to F1 during filtration through Sephadex centrifugation columns. In all water media, adenine nucleotide depleted, but not native, F1 can hydrolyze pyrophosphate to values of about 0.2 nmol min-1 mg-1. This activity is inhibited or stimulated by agents (adenylyl imidodiphosphate, aurovertin, and methanol) that produce such effects on the ATPase activity of F1; NaN3 stimulated the activity. Therefore, F1 from bovine heart mitochondria has the capacity to catalyze synthesis and hydrolysis of ATP. Synthesis of pyrophosphate by the soluble F1 appears to follow the same energetic considerations that have been postulated for ATP synthesis by the soluble enzyme [de Meis (1989) Biochim. Biophys. Acta 973, 339-349].

Purification and some properties of 11-hydroxythromboxane B2 dehydrogenase from porcine kidney.

A protein with NAD-dependent 11-hydroxythromboxane B2 dehydrogenase activity was purified to apparent homogeneity from porcine kidney using a relatively simple purification procedure, involving precipitation, anion-exchange chromatography (diethylaminoethyl-cellulose), affinity chromatography (5'-AMP-Sepharose) and gel-filtration chromatography (Protein Pak 125). The dehydrogenase was found to have a molecular mass of 50-55 kDa as determined by comparison with standards on SDS/PAGE. The molecular mass on gel-filtration chromatography was dependent on the ionic strength of the buffer. The apparent Km and Vmax values for thromboxane B2 were also dependent of the ionic strength with a Vmax of 214 nmol min-1 mg-1 using 250 mM Tris/HCl, pH 8.0, and a corresponding Km of 2.9 mM. The enzyme was NAD dependent and was clearly separated from the proteins with 15-hydroxyprostaglandin dehydrogenase activity also present in the kidney. Furthermore, it was found that 11-hydroxythromboxane B2 dehydrogenase did not utilize prostaglandin D2, prostaglandin E2, prostaglandin F2 alpha or cholic acid as substrate, and that the enzyme did not catalyse the reverse reaction, conversion of 11-dehydrothromboxane B2 to thromboxane B2.

A Simple Technique to Determine Glutathione (GSH) Levels and Synthesis in Ocular Tissues as GSH-bimane Adduct: Application to Normal and Galactosemic Guinea-pigs

A fluorimetric technique previously described for other tissues has been applied to determine levels of glutathione and its synthetic rates in ocular tissues of Hartley guinea-pigs. Monochlorobimane forms a stable, fluorescent adduct with glutathione in a reaction catalyzed by glutathione-S-transferase. The fluorescent signal recorded over time is directly proportional to the synthetic rate of glutathione. Lens, cornea and retina were homogenized and cytosolic fractions dialyzed overnight to deplete endogeneous glutathione. Glutathione synthetic rates were determined from a mixture of glutathione precursors and co-factors, viz. cysteine+dithiothreitol, glutamate+glycine, ATP and Mg++ in the presence of monochlorobimane. The mixture was supplemented with glutathione-S-transferase to catalyze the formation of the fluorescent adduct. Glutathione synthetic rates were determined in the absence and presence of buthionine sulfoximine, an inhibitor of γ-glutamyl cysteine synthetase. The difference in fluorescence change over time in the presence and absence of buthionine sulfoximine was used to estimate glutathione synthesis. Basal levels of glutathione in pre-dialyzed cytosolic fractions of the lens, cornea, and retina were 21·8±2·2, 36·5±4·1 and 38·6±2·8 nmol mg-1 protein, respectively. The maximal glutathione synthetic rates in these tissues were 0·52±0·04, 2·25±0·67 and 3·35±0·65 nmol min-1 mg-1 protein, respectively. When γ-glutamyl cysteine is used as a precursor instead of cysteine, the glutathione synthetase activities from lenses and retinas were 0·19 ±0·08 and 1·54± 0·76 nmol-1 min mg-1 protein. The present technique when applied to galactosemic guinea-pigs, showed that galactose feeding for 14 days caused a significant depletion (65 %) of lens glutathione levels, but the rates of glutathione synthesis in those lenses were not altered. The present technique permits measurements of glutathione levels and synthesis in different ocular tissues. It is suggested that the monochlorobimane method may be of value in delineating the role of glutathione in various ocular disorders such as in the galactose-induced cataract.

Human insulin receptor beta-subunit transmembrane/cytoplasmic domain expressed in a baculovirus expression system: purification, characterization, and polylysine effects on the protein tyrosine kinase activity.

We have expressed, purified, and characterized the insulin receptor protein tyrosine kinase (PTK) retaining the transmembrane and downstream domains. The proteins expressed in insect cells using a baculovirus expression system were identified as membrane-bound by immunofluorescence staining and biochemical characterization. One-step purification by immunoaffinity chromatography from Triton X-100 cell extracts resulted in a approximately 360-fold increase in the specific kinase activity with a yield of approximately 50%. An appMr = approximately 60,000 protein was the major component identified by both silver staining of the purified enzyme and immunostaining of the crude extracts after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Using nondenaturing conditions, the molecular weight was estimated to be approximately 250,000 and approximately 500,000 by glycerol gradient centrifugation and gel permeation chromatography, respectively, suggesting that oligomers of the beta-subunit domains such as tetramers and octamers are formed. The basal PTK activity of this enzyme was much higher than those of previously reported soluble-form insulin receptor PTKs expressed in insect cells or the native receptor. Km and Vmax for two substrates, src-related peptide and poly(Glu, Tyr) (4:1), were 2.4 mM and 2.5 mumol min-1 mg-1 and 0.26 mM and 1.2 mumol min-1 mg-1, respectively. Specific activities measured under two previously reported conditions using histone H2B as a substrate were 100 or 135 nmol min-1 mg-1, in contrast to those of soluble PTKs which were reported to be 20 or 70 nmol min-1 mg-1, respectively. The purified enzyme was autophosphorylated at Tyr residues. Autophosphorylation activated the enzyme approximately 3-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Stereoselective inhibition of renal organic cation transport in human kidney.

Amantadine was found to be concentrated by human cortical slices, with apparent Km and Vmax values of 187 +/- 11 microM and 1.37 +/- 0.28 nmol mg-1 min-1 respectively (mean +/- s.e. mean, n = 4). Addition of quinine (8S, 9R-(-)-isomer) or quinidine (8R, 9S-(+)-isomer) competitively inhibited this accumulation (apparent Ki values of 261 +/- 44 (n = 4) and 586 +/- 68 microM (n = 3), respectively). The stereoselectivity of this inhibition is the reverse of that seen with respect to the renal clearances of the diastereoisomers.

Histamine N-methyl transferase: inhibition by drugs.

1. Histamine N-methyl transferase activity was measured in samples of human liver, brain, kidney, lung and intestinal mucosa. The mean (+/- s.d.) rate (nmol min-1 mg-1 protein) of histamine N-methylation was 1.78 +/- 0.59 (liver, n = 60), 1.15 +/- 0.38 (renal cortex, n = 8), 0.79 +/- 0.14 (renal medulla, n = 8), 0.35 +/- 0.08 (lung, n = 20), 0.47 +/- 0.18 (human intestine, n = 30) and 0.29 +/- 0.14 (brain, n = 13). 2. Inhibition of histamine N-methyl transferase by 15 drugs was investigated in human liver. The IC50 for the various drugs ranged over three orders of magnitude; chloroquine was the most potent inhibitor. 3. The average IC50 values for chloroquine were 12.6, 22.0, 19.0, 21.6 microM in liver, renal cortex, brain and colon, respectively. These values are lower than the Michaelis-Menten constant for histamine N-methyltransferase in liver (43.8 microM) and kidney (45.5 microM). Chloroquine carried a mixed non-competitive inhibition of hepatic histamine N-methyl transferase. Some side-effects of chloroquine may be explained by inhibition of histamine N-methyl transferase.

Pyruvate oxidation by Methanococcus spp.

In the absence of H2, Methanococcus spp. utilized pyruvate as an electron donor for methanogenesis. For Methanococcus voltae A3, Methanococcus maripaludis JJ1, and Methanococcus vannielii, typical rates of pyruvate-dependent methanogenesis were 3.4, 2.8, and 3.9 nmol min-1 mg-1 cell dry wt, respectively. These rates were 1–4% of the rates of H2-dependent methanogenesis. For M. voltae, the concentration of pyruvate required for one-half the maximum rate of methanogenesis was 7 mM, and pyruvate-dependent methanogenesis was linear for 3 days. Radiolabeled acetate was formed from [3-14C]pyruvate, and the stoichiometry of pyruvate consumed per acetate produced was 1.12±0.27. The stoichiometry of pyruvate consumed per CH4 produced was 3.64±0.34. These values are close to the expected values of 1 acetate and 4 CH4. Although 10–30% of total cell carbon could be obtained from exogenous pyruvate during growth with H2, pyruvate did not replace the nutritional requirement for acetate in Methanococcus voltae A3 or two acetate auxotrophs of Methanococcus maripaludis, JJ6 and JJ7. These results suggest that pyruvate was not oxidized in the presence of H2. The inability to oxidize pyruvate during H2-dependent methanogenesis would prevent a futile cycle of pyruvate oxidation and biosynthesis during autotrophic growth.

Large-scale purification and characterisation of a recombinant epidermal growth-factor receptor protein-tyrosine kinase. Modulation of activity by multiple factors.

The human epidermal-growth-factor receptor (EGF-R) is a 170-kDa transmembrane glycoprotein that mediates the mitogenic response of cells to EGF and transforming growth factor alpha. Culture conditions have been developed for the large-scale expression of the cytoplasmic domain of the EGF-R in insect cells using a recombinant baculovirus. From 61 Sf9 cells, grown to high density using a bioreactor, 20 mg of the EGF-R kinase was purified to greater than 95% purity. Purification, which was carried out in the absence of detergents using classical purification methods, yielded an EGF-R protein that was not phosphorylated on tyrosine. This procedure has enabled us to produce high quality enzyme for both structural and biochemical studies on the EGF-R kinase. The in vitro activity of the cytoplasmic domain of the EGF-R kinase was modulated by multiple assay factors which include substrates, divalent cations and conformational modulators. Kinetic analysis in the presence of Mn2+ gave an apparent Vmax value of 20 nmol min-1 mg-1 and Km values of 4.5 microM for ATP and 1.43 mM for angiotensin II. This corresponds to a turnover number of 1.4 mol min-1 mol-1. Ammonium sulfate (1 M) resulted in an eightfold stimulation of kinase activity when assayed using angiotensin II as substrate. The specific activity of the intracellular domain of the EGF-R, when assayed at 20 degrees C in the presence of 1M ammonium sulfate, was 160 nmol min-1 mg-1. Activation of the EGF-R kinase by ammonium sulfate was found to be substrate-specific. No activation was found when assayed using polymeric substrates. Addition of Me(2+)-ATP to the purified enzyme resulted in autophosphorylation and was accompanied by retardation of SDS/PAGE migration. Kinetic constants and metal ion preferences of a number of co-polymers and peptide substrates have been compared. Dramatic differences in kinetic constants were found which were dependent on both the substrate and metal ion used. Activation of EGF-R autophosphorylation was found to be influenced by the use of charged polymers. The random polymer of Glu, Lys, Ala, Tyr (2:5:6:1), which was not phosphorylated by the EGF-R kinase, dramatically activates autophosphorylation of the EGF-R. Thus the intracellular domain of the EGF-R appears to be in a low-activity conformation which, under appropriate assay conditions, can be activated to a similar specific activity to that reported for the purified EGF-R holoenzyme.

Identification of an isoprenylated cysteine methyl ester hydrolase activity in bovine rod outer segment membranes.

Proteins from eucaryotic cells which have a carboxyl-terminal CAAX motif are posttranslationally modified by isoprenylation. The pathway involves the linkage of an all-trans-farnesyl (C15) or an all-trans-geranylgeranyl (C20) moiety to the cysteine residue followed by proteolysis which generates the modified cysteine as the carboxyl-terminal residue. Carboxylmethylation of the modified cysteine residue completes the pathway. This latter methylation reaction is the only potentially reversible reaction in the pathway and thus of possible regulatory significance. A specific esterase is required to reverse the methylation. It is demonstrated here that simple isoprenylated cysteine derivatives, such as N-acetyl-S-farnesyl-L-cysteine methyl ester (L-AFCM) and N-acetyl-S-geranylgeranyl-L-cysteine methyl ester (L-AGGCM), are substrates for a rod outer segment (ROS) membrane esterase activity. The KM and Vmax values for L-AFCM and L-AGGCM are 186 microM and 2.2 nmol mg-1 min-1 and 435 microM and 4.8 nmol mg-1 min-1, respectively. The enzyme(s) is stereoselective rather than stereospecific because D-AFCM is enzymatically hydrolyzed with KM and Vmax values of 157 microM and 0.46 nmol mg-1 min-1, respectively. The enzyme(s) does not process N-acetyl-L-cysteine methyl ester, demonstrating that the isoprenyl moiety is required for substrate activity. Ebelactone B is a potent mechanism-based inactivator of the enzyme with a KI = 42 microM and a kinh = 3.7 x 10(-3) s-1. Importantly, L-AFCM, L-AGGCM, and ebelactone B all inhibit the demethylation of the endogenous ROS substrates, showing that the same enzymatic activity is involved in the processing of the synthetic and physiological substrates.

Anaerobic degradation of 2-aminobenzoic acid (anthranilic acid) via benzoyl-coenzyme A (CoA) and cyclohex-1-enecarboxyl-CoA in a denitrifying bacterium.

The enzymes catalyzing the initial reactions in the anaerobic degradation of 2-aminobenzoic acid (anthranilic acid) were studied with a denitrifying Pseudomonas sp. anaerobically grown with 2-aminobenzoate and nitrate as the sole carbon and energy sources. Cells grown on 2-aminobenzoate are simultaneously adapted to growth with benzoate, whereas cells grown on benzoate degrade 2-aminobenzoate several times less efficiently than benzoate. Evidence for a new reductive pathway of aromatic metabolism and for four enzymes catalyzing the initial steps is presented. The organism contains 2-aminobenzoate-coenzyme A ligase (2-aminobenzoate-CoA ligase), which forms 2-aminobenzoyl-CoA. 2-Aminobenzoyl-CoA is then reductively deaminated to benzoyl-CoA by an oxygen-sensitive enzyme, 2-aminobenzoyl-CoA reductase (deaminating), which requires a low potential reductant [Ti(III)]. The specific activity is 15 nmol of 2-aminobenzoyl-CoA reduced min-1 mg-1 of protein at an optimal pH of 7. The two enzymes are induced by the substrate under anaerobic conditions only. Benzoyl-CoA is further converted in vitro by reduction with Ti(III) to six products; the same products are formed when benzoyl-CoA or 2-aminobenzoyl-CoA is incubated under reducing conditions. Two of them were identified preliminarily. One product is cyclohex-1-enecarboxyl-CoA, the other is trans-2-hydroxycyclohexane-carboxyl-CoA. The complex transformation of benzoyl-CoA is ascribed to at least two enzymes, benzoyl-CoA reductase (aromatic ring reducing) and cyclohex-1-enecarboxyl-CoA hydratase. The reduction of benzoyl-CoA to alicyclic compounds is catalyzed by extracts from cells grown anaerobically on either 2-aminobenzoate or benzoate at almost the same rate (10 to 15 nmol min-1 mg-1 of protein). In contrast, extracts from cells grown anaerobically on acetate or grown aerobically on benzoate or 2-aminobenzoate are inactive. This suggests a sequential induction of the enzymes.

Carboxylation of phenylphosphate by phenol carboxylase, an enzyme system of anaerobic phenol metabolism.

Several lines of evidence indicate that the first step in the anaerobic metabolism of phenol is phenol carboxylation to 4-hydroxybenzoate; this reaction is considered a biological Kolbe-Schmitt carboxylation. A phenol carboxylase system was characterized by using a denitrifying Pseudomonas strain, K 172, which catalyzes an isotope exchange between 14CO2 and the carboxyl group of 4-hydroxybenzoate. The enzymatic isotope exchange activity (100 nmol min-1 mg-1 of protein) requires Mn2+ and K+. We show that this system also catalyzes the carboxylation of phenylphosphate (the phosphoric acid monophenyl ester) to 4-hydroxybenzoate and phosphate. The specific activity of phenylphosphate carboxylation at the optimal pH of 6.5 is 12 nmol of CO2 fixed min-1 mg-1 of protein. Phenylphosphate cannot be replaced by Mg(2+)-ATP and phenol. The carboxylase activity requires Mn2+ but, in contrast to the isotope exchange activity, does not require K+. The apparent Km values are 1.5 mM dissolved CO2 and 0.2 mM phenylphosphate. Several convenient assays for phenylophosphate carboxylation are described. The isotope exchange reaction and the net carboxylation reaction are catalyzed by the same oxygen-sensitive enzyme, which has a half-life in an air-saturated solution of less than 1 min. Both activities cochromatographed with a protein with a Mr of 280,000, and both activities were induced only after anaerobic growth on phenol. The carboxylation of phenylphosphate suggests that phenylphosphate itself is the physiological CO2 acceptor molecular of this novel CO2 fixation reaction. Alternatively, phenylphosphate could simulate the unknown natural precursor. It is suggested that the formation of an enzyme-bound phenolate anion from the activated phenolic compound is the rate-determining step in the carboxylation reaction.

Heterogeneity of glutathione S-transferase enzyme and gene expression in ovarian carcinoma.

Expression of the three major cytosolic classes of glutathione S-transferases (GST; Pi, Alpha and Mu) was examined by 2D gel analysis and Western blotting of biopsies from 26 patients diagnosed with ovarian carcinoma. In contrast to other tissues, at least one 'constitutive' subunit from each of the three major cytosolic GST classes was expressed. In most cases, pi appeared to be the major form present, although levels of alpha and mu subunit expression were approximately equal to pi in some patients. There was no detectable effect of prior chemotherapy on enzyme activity. Mean transferase activity for primary carcinoma was 79.9 +/- 11.9 (mean +/- SEM; nmol min-1 mg-1), with three pair-matched normal tissues showing minor decreases in transferase activity. One sample, in which a 32% increase in tumour enzyme activity was noted, was from a patient with primary disease and was associated with marked overexpression of a relatively basic form of alpha which was absent from the matching normal tissue, but present in 20% of all tumours examined. RFLP analysis of genomic tumour DNA using a human mu class cDNA probe indicated that at least two of the three mu forms (the 'constitutive' form and one other) observed in ovarian tissue were allelic variants, as a one-to-one correlation was observed between the presence of two Hind III fragments at 13.1 and 2.2 kb and expression of a second, more basic, variable form. This latter form was positively identified as the mu class subunit mu based on Southern analysis and was seen to be present in 40% of the samples examined. However, in the absence of mu expression, at least one other mu class subunit probably corresponding to GST psi, was seen to be present. Thus, at least in ovarian tissues, absence of the mu subunit does not necessarily imply a lack of ability to metabolize mu substrates, as psi has similar catalytic activity. A third mu subunit, probably corresponding to GST phi based on its relatively acidic pI, was also noted in 72% of samples examined, but has unknown substrate specificity. Increased expression of both alpha and mu forms may be of relevance to disease diagnosis and drug response.

Purification to homogeneity of an NAD dependent 11-hydroxythromboxane B2 dehydrogenase from porcine kidney.

A protein with NAD dependent 11-hydroxythromboxane B2 dehydrogenase activity was purified to homogeneity from porcine kidney using a simple purification procedure, involving precipitation, anion exchange chromatography (DE-52), affinity chromatography (5'-AMP-Sepharose) and gel filtration chromatography (Protein Pak 300SW). The dehydrogenase was found to have a molecular mass of 50 kDa and 42 kDa as determined by comparison with standards on SDS/PAGE and gel filtration chromatography respectively. The apparent Km and Vmax values for thromboxane B2 were 220-250 microM and 15-30 nmol min-1 mg-1 respectively. The enzyme was clearly separated from the proteins with 15-hydroxyprostaglandin dehydrogenase activity also present in the kidney. Furthermore it was found that 11-hydroxythromboxane B2 dehydrogenase did not utilize prostaglandin D2 prostaglandin E2 or prostaglandin F2 alpha as substrate, and that the enzyme did not catalyze the reverse reaction, conversion of 11-dehydrothromboxane B2 to thromboxane B2.

Purification and characterization of the chaperonin 10 and chaperonin 60 proteins from Rhodobacter sphaeroides

Two heat-shock proteins that show high identity with the Escherichia coli chaperonin 60 (groEL) and chaperonin 10 (groES) chaperonin proteins were purified and characterized from photolithoautotrophically grown Rhodobacter sphaeroides. The proteins were purified by using sucrose density gradient centrifugation and Mono-Q anion-exchange chromatography. In the presence of 1 mM ATP, the chaperonin 10 and chaperonin 60 proteins bound to each other and comigrated as a large complex during sucrose density gradient centrifugation. The native molecular weights of each protein as determined by gel filtration chromatography were 889,200 for chaperonin 60 and 60,000 for chaperonin 10. Chaperonin 60 is comprised of monomers with a molecular weight of 61,000 and chaperonin 10 is comprised of monomers with a molecular weight of 12,700 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chaperonin 60 was 9.3% of the total soluble cell protein during photolithoautotrophic growth which increased to 28.5% following heat-shock treatment. When cells were grown photoheterotrophically or chemoheterotrophically, chaperonin 60 was reduced to 6.7% and 3.5%, respectively, of the total soluble protein. The N-terminal amino acid sequence of each protein was determined; chaperonin 60 of R. sphaeroides showed 72% identity to E. coli chaperonin 60 protein, and R. sphaeroides chaperonin 10 showed 45% identity with E. coli chaperonin 10. R. sphaeroides chaperonin 60 catalyzed ATP hydrolysis with a specific activity of 134 nmol min-1 mg-1 (kcat = 0.13 s-1) and was inhibited by R. sphaeroides chaperonin 10, but not E. coli chaperonin 10. The E. coli chaperonin 60 ATPase activity was inhibited by chaperonin 10 from both R. sphaeroides and E. coli.

Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters

Forty-five individually transformed clonal tobacco callus lines were simultaneously assayed for both chloramphenicol acetyltransferase (CAT) and beta-glucuronidase (GUS) activity resulting from expression of introduced reporter genes driven by the adjacent and divergent mannopine (mas) promoters. Excluding lines in which one or both of the enzyme activities was essentially zero, the activities of the reporter genes varied by as much as a factor of 136 (CAT) and 175 (GUS) between individual transformants. Superimposed upon the high degree of inter-clonal expression variability was an intra-clonal variability of 3-4-fold. The observed degree of intra-clonal reporter gene activity may be more extreme because of the regulatory characteristics of the mannopine promoters, but must still be addressed when considering the limitations of reporter gene-based analysis of transgene function and structure. There was no consistent correlation between the expression levels of the introduced CAT and GUS genes since the ratio of GUS to CAT activities (nmol min-1 mg-1) within individual lines varied from 0.05 to 49. Even divergent transcription from two directly adjacent promoter regions (both contained within a 479 bp TR-DNA fragment) is insufficient to guarantee concurrent expression of two linked transgenes. Our quantitative data were compared to published data of transgene expression variability to examine the overall distribution of expression levels in individual transformants. The resulting frequency distribution indicates that most transformants express introduced transgenes at relatively low levels, suggesting that a potentially large number of Agrobacterium-mediated transformation events may result in silent transgenes.