Investigation of the Bacillus cereus phosphonoacetaldehyde hydrolase. Evidence for a Schiff base mechanism and sequence analysis of an active-site peptide containing the catalytic lysine residue

Reaction of Bacillus cereus phosphonoacetaldehyde hydrolase (phosphonatase) with phosphonoacetaldehyde or acetaldehyde in the presence of NaBH4 resulted in complete loss of enzymatic activity. Treatment of phosphonatase with NaBH4 in the absence of substrate or product had no effect on catalysis. Inactivation of phosphonatase with [3H]NaBH4 and phosphonoacetaldehyde, NaBH4 and [14C]acetaldehyde, or NaBH4 and [2-3H]phosphonoacetaldehyde produced in each instance radiolabeled enzyme. The nature of the covalent modification was investigated by digesting the radiolabeled enzyme preparations with trypsin and by separating the tryptic peptides with HPLC. Analysis of the peptide fractions revealed that incorporation of the 3H- or 14C-radiolabel into the protein was reasonably selective for an amino acid residue found in a peptide fragment observed in each of the three trypsin digests. Sequence analysis of the 3H-labeled peptide fragment isolated from the digest of the [2-3H]phosphonoacetaldehyde/NaBH4-treated enzyme identified N epsilon-ethyllysine as the radiolabeled amino acid. The ability of the phosphonatase competitive inhibitor (Ki = 230 +/- 20 microM) acetonylphosphonate to protect the enzyme from phosphonoacetaldehyde/NaBH4-induced inactivation suggested that the reactive lysine residue is located in the enzyme active site. Comparison of the relative effectiveness of phosphonoacetaldehyde and acetaldehyde as phosphonatase inactivators showed that the N-ethyllysine imine that is reduced by the NaBH4 is derived from the corresponding N-(phosphonoethyl) imine. On the basis of these findings, a catalytic mechanism for for phosphonatase is proposed in which phosphonoacetaldehyde is activated for P-C bond cleavage by formation of a Schiff base with an active-site lysine. Accordingly, an N-ethyllsysine enamine rather than the high-energy acetaldehyde enolate anion is displaced from the phosphorus.     

An amino acid sequence in the active site of lipoamide dehydrogenase from pig heart

1. The two cysteine residues forming the disulphide bridge that comprises part of the active site of lipoamide dehydrogenase from pig heart were specifically labelled with iodo[2-(14)C]acetic acid. 2. A tryptic peptide containing these carboxymethylcysteine residues was isolated from digests of reduced and S-carboxymethylated lipoamide dehydrogenase and its amino acid sequence of 23 residues was determined. 3. The sequence is highly homologous with a similar sequence containing the active-site disulphide bridge of lipoamide dehydrogenase derived from the 2-oxoglutarate dehydrogenase complex of Escherichia coli (Crookes strain) and it is probable that, as in the bacterial enzyme, the disulphide bridge forms an intrachain loop containing six residues. The results indicate that the bacterial and mammalian proteins have a common genetic origin. 4. Amino acid sequences containing six other unique carboxymethylcysteine residues were also partly determined. 5. The analysis of the primary structure thus far is consistent with the view that the enzyme (mol.wt. approx. 110000) is composed of two identical polypeptide chains.     

Identification of a highly conserved domain in the EcoRII methyltransferase which can be photolabeled with S-adenosyl-L-[methyl-3H]methionine. Evidence for UV-induced transmethylation of cysteine 186

DNA methyltransferases can be photolabeled with S-adenosyl-L-methionine (AdoMet). Specific incorporation of radioactivity has been demonstrated after photolabeling with either [methyl-3H]AdoMet or [35S]AdoMet (Som, S., and Friedman, S. (1990) J. Biol. Chem. 265, 4278-4283). The labeling is believed to occur at the AdoMet binding site. With the purpose of localizing the site responsible for [methyl-3H]AdoMet photolabeling, we cleaved the labeled EcoRII methyltransferase by chemical and enzymatic reactions and isolated the radiolabeled peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography. The labeled peptides were identified by amino-terminal sequencing. A common region was localized which accounted for 65-70% of the total label. This region includes a highly conserved core sequence present in all DNA (cytosine 5)-methyltransferases. One such fragment was digested further with chymotrypsin, and amino acid analysis of the resulting 3H-labeled peptide was consistent with the sequence Ala-Gly-Phe-Pro-(Cys)-Gln-Pro-Phe-Ser-Leu. However, the cysteine residue was not recovered as carboxymethylcysteine. The Pro-Cys bond was found to be protected from cleavage at cysteine residues after cyanylation. These results suggest that the cysteine residue is modified by the labeling reaction. The chymotryptic fragment was hydrolyzed enzymatically to single amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that the cysteine residue is located at or close to the AdoMet binding site of EcoRII methyltransferase.     

Prostaglandin dehydrogenase activity of purified rat liver 3 alpha-hydroxysteroid dehydrogenase

Homogeneous 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) from rat liver cytosol displays 9, 11, and 15-hydroxyprostaglandin dehydrogenase activity. Using [14C]-PGF2 alpha as substrate the products of this reaction were separated by TLC and identified by autoradiography as PGE2 and PGB2. The purified enzyme catalyzes this reaction at a rate 200 times faster than cytosol. This corresponds to the rate enhancement observed when the enzyme is purified from cytosol using androsterone (a 3 alpha-hydroxysteroid) as substrate and suggests that it may represent a major 9-hydroxyprostaglandin dehydrogenase in this tissue. Although the 3 alpha-HSD has many properties in common with the 9-hydroxyprostaglandin dehydrogenase of rat kidney, rat kidney contains no protein that is immunodetectable with polyclonal antibody raised against the purified 3 alpha-HSD.     

Serine incorporation into the selenocysteine moiety of glutathione peroxidase

The selenium in mammalian glutathione peroxidase is present as a selenocysteine ([Se]Cys) moiety incorporated into the peptide backbone 41-47 residues from the N-terminal end. To study the origin of the skeleton of the [Se]Cys moiety, we perfused isolated rat liver with 14C- or 3H-labeled amino acids for 4 h, purified the GSH peroxidase, derivatized the [Se]Cys in GSH peroxidase to carboxymethylselenocysteine ([Se]Cys(Cm)), and determined the amino acid specific activity. Perfusion with [14C]cystine resulted in [14C]cystine incorporation into GSH peroxidase without labeling [Se]Cys(Cm), indicating that cysteine is not a direct precursor for [Se]Cys. [14C]Serine perfusion labeled serine, glycine (the serine hydroxymethyltransferase product), and [Se]Cys(Cm) in purified GSH peroxidase, whereas [3-3H]serine perfusion only labeled serine and [Se]Cys(Cm), thus demonstrating that the [Se]Cys in GSH peroxidase is derived from serine. The similar specific activities of serine and [Se]Cys(Cm) strongly suggest that the precursor pool of serine used for [Se] Cys synthesis is the same or similar to the serine pool used for acylation of seryl-tRNAs.     

3-Hydroxy-3-methylglutaryl coenzyme A lyase: affinity labeling of the Pseudomonas mevalonii enzyme and assignment of cysteine-237 to the active site.

Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) lyase is irreversibly inactivated by the reactive substrate analog 2-butynoyl-CoA. Enzyme inactivation, which follows pseudo-first-order kinetics, is saturable with a KI = 65 microM and a limiting k(inact) of 0.073 min-1 at 23 degrees C, pH 7.2. Protection against inactivation is afforded by the competitive inhibitor 3-hydroxyglutaryl-CoA. Labeling of the bacterial enzyme with [1-14C]-2-butynoyl-CoA demonstrates that inactivation coincides with covalent incorporation of inhibitor, with an observed stoichiometry of modification of 0.65 per site. Avian HMG-CoA lyase is also irreversibly inactivated by 2-butynoyl-CoA with a stoichiometry of modification of 0.9 per site. Incubation of 2-butynoyl-CoA with mercaptans such as dithiothreitol results in the formation of a UV absorbance peak at 310 nm. Enzyme inactivation is also accompanied by the development of a UV absorbance peak at 310 nm indicating that 2-butynoyl-CoA modifies a cysteine residue in HMG-CoA lyase. Tryptic digestion and reverse-phase HPLC of the affinity-labeled protein reveal a single radiolabeled peptide. Isolation and sequence analysis of this peptide and a smaller chymotryptic peptide indicate that the radiolabeled residue is contained within the sequence GGXPY. Mapping of this peptide within the cDNA-deduced sequence of P. mevalonii HMG-CoA lyase [Anderson, D. H., & Rodwell, V. W. (1989) J. Bacteriol. 171, 6468-6472] confirms that a cysteine at position 237 is the site of modification. These data represent the first identification of an active-site residue in HMG-CoA lyase.     

Amino acid sequence of the nucleotide-binding site of D-(-)-beta-hydroxybutyrate dehydrogenase labeled with arylazido-beta-[3-3H]alanylnicotinamide adenine dinucleotide

In the dark, arylazido-beta-alanylnicotinamide adenine dinucleotide (N3-NAD) can replace NAD as cofactor for D-(-)-beta-hydroxybutyrate dehydrogenase (BDH) purified from bovine heart mitochondria. When photoirradiated with visible light, N3-NAD forms a nitrene species that binds covalently to BDH and inhibits the enzyme. NAD(H) protects BDH against photolabeling and inhibition by N3-NAD [Yamaguchi, M., Chen, S., & Hatefi, Y. (1985) Biochemistry 24, 4912-4916]. In the present study, a tryptic peptide of purified BDH photolabeled with arylazido-beta-[3-3H] alanyl-NAD [( 3H]N3-NAD) was isolated and sequenced. The same tryptic peptide was also isolated from BDH not labeled with [3H]N3-NAD and sequenced. Both peptides indicated the sequence Met-Glu-Ser-Tyr-Cys-Thr-Ser-Gly-Ser-Thr-Asp-Thr-Ser-Pro-Val-Ile-Lys. The residue labeled with [3H]N3-NAD was Cys. This heptadecapeptide contains 14 uncharged residues and is marked by having in an undecapeptide segment 8 hydroxy amino acids located symmetrically around a central glycine.     

Location in Muscarinic Acetylcholine Receptors of Sites for [3H]Propylbenzilcholine Mustard Binding and for Phosphorylation with Protein Kinase C

Muscarinic acetylcholine receptors purified from porcine cerebra or atria were covalently labeled with [3H]propylbenzilylcholine mustard ([3H]PrBCM), and then the labeled receptors were subjected to limited hydrolysis with trypsin, V8 protease, and lysyl endopeptidase, followed by analysis involving sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fluorography, autoradiography, or immunostaining. The labeled peptides were located on the basis of their reactivity with antibodies raised against three synthetic peptides with partial sequences of the m1 or m2 receptor, and of their sensitivity to endoglycosidase F, which was taken as evidence that they contain glycosylation sites near the N terminus. The [3H]PrBCM-binding site in both cerebral and atrial receptors was found to be located between the N terminus and the second intracellular loop, because the size of the smallest deglycosylated peptide that contained both the [3H]PrBCM-binding and glycosylation sites was approximately 16 kDa. Cerebral receptors were 32P-phosphorylated with protein kinase C, and the major phosphorylation sites in cerebral muscarinic receptors were found to be located in a C-terminal segment including a part of the third intracellular loop, because a 32P-labeled peptide of 12-14 kDa reacted with anti-(m1 C-terminal peptide) antiserum. The presence of an intramolecular disulfide bond, probably between Cys 98 and Cys 178 in the first and second extracellular loops, respectively, was suggested by the finding that a peptide of approximately 17 kDa containing the [3H]PrBCM-binding site, but not the glycosylation sites, was partly converted to a peptide of approximately 12 kDa on treatment with beta-mercaptoethanol.     

Identification of the reactive cysteines of Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase and their nonessentiality for enzymatic catalysis

Reaction of 5-enolpyruvylshikimate-3-phosphate synthase of Escherichia coli with the thiol reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) leads to a modification of only 2 of the 6 cysteines of the enzyme, with a significant loss of its enzymatic activity. Under denaturing conditions, however, all 6 cysteines of 5-enolpyruvylshikimate-3-phosphate synthase react with DTNB, indicating the absence of disulfide bridges in the native protein. In the presence of shikimate 3-phosphate and glyphosate, only 1 of the 2 cysteines reacts with the reagent, with no loss of activity, suggesting that only 1 of these cysteines is at or near the active site of the enzyme. Cyanolysis of the DTNB-inactivated enzyme with KCN leads to elimination of 5-thio-2-nitrobenzoate, with formation of the thiocyano-enzyme. The thiocyano-enzyme is fully active; it exhibits a small increase in its I50 for glyphosate (6-fold) and apparent Km for phosphoenolpyruvate (4-fold) compared to the unmodified enzyme. Its apparent Km for shikimate 3-phosphate is, however, unaltered. These results clearly establish the nonessentiality of the active site-reactive cysteine of E. coli 5-enolpyruvylshikimate-3-phosphate synthase for either catalysis or substrate binding. Perturbations in the kinetic constants for phosphoenolpyruvate and glyphosate suggest that the cysteine thiol is proximal to the binding site for these ligands. By N-[14C]ethylmaleimide labeling, tryptic mapping, and N-terminal sequencing, the 2 reactive cysteines have been identified as Cys408 and Cys288. The cysteine residue protected by glyphosate and shikimate 3-phosphate from its reaction with DTNB was found to be Cys408.     

Active site labeling of dopamine beta-hydroxylase by two mechanism-based inhibitors: 6-hydroxybenzofuran and phenylhydrazine

6-Hydroxybenzofuran and phenylhydrazine are mechanism-based inhibitors of dopamine beta-hydroxylase (D beta H; EC 1.14.17.1). We report here the isolation and characterization of radiolabeled peptides obtained after inactivation of D beta H with [3H]6-hydroxybenzofuran and [14C]phenylhydrazine followed by digestion with Staphylococcus aureus V8 protease. Inactivation of D beta H with [3H]6-hydroxybenzofuran gave only one labeled peptide, whereas inactivation with [14C]phenylhydrazine gave several labeled peptides. Each inhibitor labeled a unique tyrosine in the enzyme corresponding to Tyr477 in the primary sequence of the bovine enzyme (Robertson, J. G., Desai, P. R., Kumar, A., Farrington, G. K., Fitzpatrick, P. F., and Villafranca, J. J. (1990) J. Biol. Chem. 265, 1029-1035). In addition, [14C]phenylhydrazine also labeled a unique histidine (His249) as well as several other peptides. Examination of the complete peptide profile obtained by high pressure liquid chromatography analysis also revealed the presence of a modified but nonradioactive peptide. This peptide was isolated and sequenced and was identical whether the enzyme was inactivated by 6-hydroxybenzofuran or phenylhydrazine. An arginine at position 503 was missing from the sequence cycle performed by Edman degradation of the modified peptide, but arginine was present in the identical peptide isolated from native dopamine beta-hydroxylase. These data are analyzed based on an inactivation mechanism involving formation of enzyme bound radicals (Fitzpatrick, P. F., and Villafranca, J. J. (1986) J. Biol. Chem. 261, 4510-4518) interacting with active site amino acids that may have a role in substrate binding and binding of the copper ions at the active site.     

Binding site for fungal beta-lactone hymeglusin on cytosolic 3-hydroxy-3-methylglutaryl coenzyme A synthase

We studied the molecular mechanism through which the fungal beta-lactone, hymeglusin, potently and specifically inhibits 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. [(14)C]Hymeglusin covalently bound to purified rat liver and to recombinant hamster cytosolic HMG-CoA synthases. The enzyme activity was completely inhibited at a binding ratio of 1.6-2.0 mol [(14)C]hymeglusin/mol HMG-CoA synthase. Incubating the enzyme with 2 mM iodoacetamide (IAA) or 2 mM N-ethylmaleimide (NEM) but not with 1.0 mM diisopropyl fluorophosphates (DFP) completely inhibited the binding, suggesting that hymeglusin binds to a Cys residue of HMG-CoA synthase. Recombinant hamster HMG-CoA synthase labeled with [(3)H]hymeglusin was digested with V8 protease, and the [(3)H]peptide was purified by high performance liquid chromatography (HPLC). The sequence of the peptide was Ser-Gly-Asn-Thr-Asp-Ile-Glu-Gly-Ile-Asp-Thr-Thr-Asn-Ala-[(3)H]hymeglusyl Cys-Tyr-Gly-Gly-Thr-Ala-Ala-Val-Phe-Asn-Ala-Val-Asn-, which corresponds to the active site sequence (from Ser 115 to Asn 141) of hamster HMG-CoA synthase. These findings showed that hymeglusin inhibits hamster cytosolic HMG-CoA synthase by covalently modifying the active Cys 129 residue of the enzyme.     

Amino acid replacements at the H2-activating site of the NAD-reducing hydrogenase from Alcaligenes eutrophus

The role of amino acid residues in the H(2)-activating subunit (HoxH) of the NAD-reducing hydrogenase (SH) from Alcaligenes eutrophus has been investigated by site-directed mutagenesis. Conserved residues in the N-terminal L1 (RGxE) and L2 (RxCGxCx(3)H) and the C-terminal L5 (DPCx(2)Cx(2)H/R) motifs of the active site-harboring subunit were chosen as targets. Crystal structure analysis of the [NiFe] hydrogenase from Desulfovibrio gigas uncovered two pairs of cysteines (motifs L2 and L5) as coordinating ligands of Ni and Fe. Glutamate (L1) and histidine residues (L2 and L5) were proposed as being involved in proton transfer [Volbeda, A., Charon, M.-H., Piras, C., Hatchikian, E. C., Frey, M., and Fontecilla Camps, J. C. (1995) Nature 373, 580-587]. The A. eutrophus mutant proteins fell into three classes. (i) Replacement of the putative four metal-binding cysteines with serine led to the loss of H(2) reactivity and blocked the assembly of the holoenzyme. Exchange of Cys62, Cys65, or Cys458 was accompanied by the failure of the HoxH subunit to incorporate nickel, supporting the essential function of these residues in the formation of the active site. Although the fourth mutant of this class (HoxH[C461S]) exhibited nickel binding, the modified protein was catalytically inactive and unable to oligomerize. (ii) Mutations in residues possibly involved in proton transfer (HoxH[E43V], HoxH[H69L], and HoxH[H464L]) yielded Ni-containing proteins with residual low levels of hydrogenase activity. (iii) The most promising mutant protein (HoxH[R40L]), which was identified as a metal-containing tetrametric enzyme, was completely devoid of H(2)-dependent oxidoreductase activity but exhibited a remarkably high level of D(2)-H(+) exchange activity. These characteristics are compatible with the interpretation of a functional proton transfer uncoupled from the flow of electrons.     

Site specificity of histone H4 methylation by wheat germ protein-arginine N-methyltransferase

CNBr treatment of calf thymus [methyl-14C]histone H4, methylated in vitro with S-adenosyl-L-[methyl-14C]methionine by a highly histone-specific wheat germ protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23), produced two peptide fragments corresponding to residues 1-83 and 84-102, with the former being radioactive. Two-dimensional peptide mapping of the chymotryptic and tryptic digest of [methyl-14C]histone H4 and analysis of the chymotryptic digest on HPLC have shown that only a single peptide is radiolabeled. In order to define the exact site of methylation (arginine residue), the radioactive peptide from the chymotryptic digest of [methyl-14C]histone H4 was further purified on HPLC by linear and then isocratic elution. The purified chymotryptic peptide was then digested with trypsin and purified on HPLC, and its amino acid composition was determined on HPLC. These results indicate that the peptide corresponding to residues 24-35 of histone H4 is radiolabeled. Since this peptide contains a single arginine residue at position 35, we have concluded that the enzyme is specific not only to the protein substrate but also to the methylation site.     

Reaction of oxidized dopamine with endogenous cysteine residues in the human dopamine transporter

There is evidence to suggest that dopamine (DA) oxidizes to form dopamine ortho-quinone (DAQ), which binds covalently to nucleophilic sulfhydryl groups on protein cysteinyl residues. This reaction has been shown to inhibit dopamine uptake, as well as other biological processes. We have identified specific cysteine residues in the human dopamine transporter (hDAT) that are modified by this electron-deficient substrate analog. DAQ reactivity was inferred from its effects on the binding of [(3)H]2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane (beta-CFT) to hDAT cysteine mutant constructs. One construct, X5C, had four cysteines mutated to alanine and one to phenylalanine (Cys(90)A, Cys(135)A, C306A, C319F and Cys(342)A). In membrane preparations 1 mM DAQ did not affect [(3)H]beta-CFT binding to X5C hDAT, in contrast to its effect in wild-type hDAT in which it reduced the B:(max) value by more than half. Wild-type cysteines were substituted back into X5C, one at a time, and the ability of DAQ to inhibit [(3)H]beta-CFT binding was assessed. Reactivity of DAQ with Cys(90) increased the affinity of [(3)H]beta-CFT for the transporter, whereas reactivity with Cys(135) decreased the affinity of [(3)H]beta-CFT. DAQ did not change the K:(D) for [(3)H]beta-CFT binding to wild-type. The reactivity of DAQ at Cys(342) decreased B:(max) to the same degree as wild-type. The latter result suggests that Cys(342) is the wild-type residue most responsible for DAQ-induced inhibition of [(3)H]beta-CFT binding.     

Amino acid sequences of two tryptic peptides from D(-)-beta-hydroxybutyrate dehydrogenase radiolabeled at essential carboxyl and sulfhydryl groups

D(-)beta-hydroxybutyrate dehydrogenase (BDH) purified from bovine heart mitochondria contains essential thiol and carboxyl groups. A tryptic BDH peptide labeled at an essential thiol with [3H]N-ethylmaleimide (NEM), and another tryptic peptide labeled at an essential carboxyl with N,N'-dicyclohexyl [14C]carbodiimide (DCCD), were isolated and sequenced. The peptide labeled with [3H]NEM had the sequence Met.Glu.Ser.Tyr.Cys*.Thr.Ser. Gly.Ser.Thr.Asp.Thr.Ser.Pro.Val.Ile.Lys. The label was at Cys. The same peptide was isolated from tryptic digests of BDH labeled at its nucleotide-binding site with the photoaffinity labeling reagent, arylazido- -[3-3H] alanyl-NAD. These results suggest that the essential thiol of BDH is located at its nucleotide-binding site, and agree with our previous observation that NAD and NADH protect BDH against inhibition by thiol modifiers. The [14C]DCCD-labeled peptide had the sequence Glu.Val.Ala.Glu*.Val. Asn. Leu.Trp.Gly.Thr.Val.Arg. DCCD appeared to modify the glutamic acid residue marked by an asterisk. Sequence analogies between these peptides and other proteins have been discussed.     

Characterization of SH groups in porin of bovine heart mitochondria. Porin cysteines are localized in the channel walls.

Porin from bovine heart mitochondria contains probably two cysteines (Cys126 and Cys230 in human porin, Kayser, H., Kratzin, H. D., Thinnes, F. P., G?tz, H., Schmidt, W. E., Eckart, K. & Hilschmann, N. (1989) Biol. Chem. Hoppe-Seyler 370, 1265-1278). Reduced and oxidized forms of these cysteines were investigated in purified protein and in intact mitochondria using the agents dithioerythritol, cuprous(II) phenantroline, diamide and performic acid. Furthermore, intact mitochondria were labelled with the sulfhydryl-alkylating agents N-[14C]ethylmaleimide, eosin-5-maleimide and N-(1-pyrenyl)-maleimide. Affinity chromatography of bovine heart porin was performed with cysteine-specific material. The results can be summarized as follows: (1) Porin has one reduced and two oxidized forms of apparent molecular masses between 30 and 35 kDa. The native form of porin is the reduced 33 kDa form. The oxidized forms only appear after denaturation with SDS. (2) The 35-kDa reduced and the 33.5-kDa oxidized forms of porin show the same pore-forming properties after reconstitution of the protein into lipid bilayer membranes. (3) Labelling of cysteines by eosin-5-maleimide and N-(1-pyrenyl)-maleimide suggested their location at a boundary between the water-phase and the lipid-phase. Incubation of intact mitochondria with N-ethylmaleimide prior to eosin-5-maleimide and N-(1-pyrenyl)maleimide treatment resulted in the inhibition of the fluorescent labelling. Among the cysteines present in the primary structure, Cys126 is the most sensitive to N-ethylmaleimide binding. (4) Bovine heart mitochondrial porin covalently bound to Affi-Gel 501 (with a 1.75 nm long spacer), but not to Thiopropyl-Sepharose 6B (with a 0.51 nm spacer). This suggests that at least one of the cysteines is localized between 0.51 nm and 1.75 nm deep in the protein micelle.     

Characterization of a cysteine-containing peptide after affinity labelling of Ca2+-ATPase of sarcoplasmic reticulum with the disulfide of 3'(2')-O-biotinyl-thioinosine triphosphate

3'(2')-O-Biotinyl-thioinosine triphosphate is a substrate of the Ca2+ pump of sarcoplasmic reticulum. Its disulfide inactivates the Ca2+-ATPase with two different velocities. The rapidly inactivated sulfhydryl group cannot be protected by ATP and is therefore considered to be outside the ATP binding site. The slowly reacting sulfhydryl group interacts with the disulfide of 3'(2')-O-biotinyl-thioinosine triphosphate with a dissociation constant of Kd = 137 microM and an inactivation velocity constant of 1.7 X 10(-3) s-1. It is protected by ATP with two different dissociation constants of the enzyme-ATP complex of Kd = 221 microM and 1130 microM. The slowly reacting sulfhydryl group is therefore considered to be part of the ATP binding site. Since it was impossible to isolate a tryptic peptide by affinity purification on matrix-bound avidin after affinity labelling with the disulfide of 3'(2')-O-biotinyl-thioinosine triphosphate, differential labelling with iodo[2-14C]acetic acid after affinity labelling with the disulfide of 3'(2')-O-biotinyl-thioinosine triphosphate was carried out. Tryptic digestion and FPLC purification led to the isolation of a radioactive carboxymethyl derivative of the cysteine-containing peptide ANACNSVIR. This peptide is equivalent to the cDNA-derived sequence 468-476 of Ca2+-ATPase [Brandl et al. (1986) Cell 44, 597-607] and is located between the phosphorylation site, Asp351, and Lys515, a part of the putative purine binding subsite of ATP. Although the carboxymethylation of Cys471 is hindered by (biotinyl-s6ITP)2, the strong dilution of the specific radioactivity of iodo[2-14C]acetic acid in the isolated peptide 468-476 argues against its direct interaction with the ATP analogue. It is therefore proposed that Cys471 undergoes ATP-dependent conformational changes.     

Lipoxygenase products of arachidonic acid modulate biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human neutrophils via phospholipase A2

When human neutrophils, previously labeled in their phospholipids with [14C]arachidonate, were stimulated with the Ca2+-ionophore, A23187, plus Ca2+ in the presence of [3H]acetate, these cells released [14C]arachidonate from membrane phospholipids, produced 5-hydroxy-6,8,11,14-[14C]eicosatetraenoic acid (5-HETE) and 14C-labeled 5S,12R-dihydroxy-6-cis,8,10-trans, 14-cis-eicosatetraenoic acid ([14C]leukotriene B4), and incorporated [3H]acetate into platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Ionophore A23187-induced formation of these radiolabeled products was greatly augmented by submicromolar concentrations of exogenous 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), 5-HETE, and leukotriene B4. In the absence of ionophore A23187, these arachidonic acid metabolites were virtually ineffective. Nordihydroguaiaretic acid (NDGA) and several other lipoxygenase/cyclooxygenase inhibitors (butylated hydroxyanisole, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 1-phenyl-2-pyrazolidinone) caused parallel inhibition of [14C]arachidonate release and [3H]PAF formation in a dose-dependent manner. Specific cyclooxygenase inhibitors, such as indomethacin and naproxen, did not inhibit but rather slightly augmented the formation of these products. Furthermore, addition of 5-HPETE, 5-HETE, or leukotriene B4 (but not 8-HETE or 15-HETE) to neutrophils caused substantial relief of NDGA inhibition of [3H]PAF formation and [14C]arachidonate release. As opposed to [3H]acetate incorporation into PAF, [3H]lyso-PAF incorporation into PAF by activated neutrophils was little affected by NDGA. In addition, NDGA had no effect on lyso-PAF:acetyl-CoA acetyltransferase as measured in neutrophil homogenate preparations. It is concluded that in activated human neutrophils 5-lipoxygenase products can modulate PAF formation by enhancing the expression of phospholipase A2.     

Inhibition of 20 alpha-hydroxysteroid dehydrogenase activity by follicle-stimulating hormone and androgens in cultured rat granulosa cells: a search for the mechanism of action

Alterations of progesterone metabolism and especially of 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity were studied in cultured rat granulosa cells following various treatments. The cells were incubated for up to 48 h with or without follicle-stimulating hormone (FSH), androgens, hydroxyflutamide, estrogens, chlorea toxin, and dibutyryl cAMP [Bu2 cAMP]. Subsequently, the cells were incubated for 3 h with [4-14 C] progesterone (0.5 microM). The progesterone utilization and accumulation of 20 alpha-reduced and 5 alpha-reduced metabolites were assessed following thin-layer chromatography separation of radiolabeled steroids. Both FSH (1 microgram/ml) and testosterone (0.5 microM) decreased the 20 alpha-HSD activity by decreasing the maximal velocity (by 52% and 37%, respectively) without changing significantly the Km value. The inhibition of 20 alpha-HSD was demonstrable following 12 and 24 h exposure to FSH and following 24 and 48 h exposure to testosterone. Effects comparable to that induced by testosterone were elicited by other androgens (androstenedione and 5 alpha-dihydrotestosterone), but not by estrogens (estradiol-17 beta and estrone). Hydroxyflutamide reversed testosterone-induced effects: the increase of endogenous progesterone accumulation and the decrease of 20 alpha-HSD activity. Both cholera toxin (0.001-10 micrograms/ml) and Bu2 cAMP (62.5-1000 micrograms/ml) caused a dose-dependent inhibition of 20 alpha-HSD activity. Present results indicate that: the inhibition of 20 alpha-HSD by both FSH and androgens may be of a noncompetitive nature; androgen action on 20 alpha-HSD may be a true androgenic, receptor-mediated effect; and cAMP may mediate the FSH action on 20 alpha-HSD activity.     

The N-terminal disulfide linkages of human insulin-like growth factor-binding protein-6 (hIGFBP-6) and hIGFBP-1 are different as determined by mass spectrometry.

The actions of insulin-like growth factors (IGFs) are modulated by a family of six high affinity binding proteins (IGFBPs 1-6). IGFBP-6 differs from other IGFBPs in having the highest affinity for IGF-II and in binding IGF-I with 20-100-fold lower affinity. IGFBPs 1-5 contain 18 conserved cysteines, but human IGFBP-6 lacks 2 of the 12 N-terminal cysteines. The complete disulfide linkages of IGFBP-6 were determined using electrospray ionization mass spectrometry of purified tryptic peptide complexes digested with combinations of chymotrypsin, thermolysin, and endoproteinase Glu-C. Numbering IGFBP-6 cysteines sequentially from the N terminus, the first three disulfide linkages are Cys1-Cys2, Cys3-Cys4, and Cys5-Cys6. The next two linkages are Cys7-Cys9 and Cys8-Cys10, which are analogous to those previously determined for IGFBP-3 and IGFBP-5. The C-terminal linkages are Cys11-Cys12, Cys13-Cys14, and Cys15-Cys16, analogous to those previously determined for IGFBP-2. Disulfide linkages of IGFBP-1 were partially determined and show that Cys1 is not linked to Cys2 and Cys3 is not linked to Cys4. Analogous with IGFBP-3, IGFBP-5, and IGFBP-6, Cys9-Cys11 and Cys10-Cys12 of IGFBP-1 are also disulfide-linked. The N-terminal linkages of IGFBP-6 differ significantly from those of IGFBP-1 (and, by implication, the other IGFBPs), which could contribute to the distinctive IGF binding properties of IGFBP-6.