Changes in hydrocarbons and elemental distribution in peloids during maturation processes (Secovlje Salina Nature Park Slovenia).

In Secovlje Salina Nature Park, the therapeutic mud matured in the natural sedimentary environmental site. This work aimed to determine the influence of the peloid maturation process on the hydrocarbon and elemental distributions, as well as changes in morphology. For this purpose, the sample before and after maturation was examined using various methods. n-Alkanes were the most abundant among saturated hydrocarbons in both immature and mature peloid samples. The results showed that the maturation mainly influenced the change in distribution and concentration (from 378 to 1958 ppm) of n-alkanes. The organic matter (OM) of the immature peloid sample was characterized by a slight prevalence of long-chain and odd carbon-numbered n-alkanes, maximizing at n-C27. However, mature peloid's OM showed a similar share of short-, mid- and long-chain n-alkanes with a slight dominance of short-chain members, maximizing at n-C16. The origin of short-chain and even carbon-numbered n-alkanes was attributed to microbial precursors (e.g., Leptolyngbyaceae). Hopanes were considerably more dominant compared to steranes in both peloids. The hopane series of immature peloid was characterized by the dominance of 22,29,30-trinor-hop-5(6)-ene (C27 hopene), as well as the presence of C30-hop-22(29)-ene (diploptene), which are widespread in cyanobacterial species. The aromatic fraction of immature peloid pointed to the predominance of polycyclic aromatic hydrocarbons (PAHs). As peloid aging progressed, the sample was richer in methyl-branched alkanes, carboxylic acids, their methyl esters, and thermodynamically more stable hopanes and steranes. The presence of elements with toxicological relevance during maturation was reduced below the limits prescribed in most of the directives for cosmetic products. It specifically refers to: As, Ni and Se. A higher concentration of total sulfur in the mature peloid can be related to gypsum precipitation in the summer and/or more intensive microbial activity.

T cell activation up-regulates cyclic nucleotide phosphodiesterases 8A1 and 7A3.

Agents that increase intracellular cAMP inhibit the activation and function of T cells and can lead to cell death. Recently, it has been postulated that cAMP inhibits T cell function in large part by acting as a brake on the T cell receptor and costimulatory receptor pathways. Therefore, for full activation of the T cell to occur, this inhibitory influence must be removed. One likely mechanism for accomplishing this is by up-regulation and/or activation of specific cyclic nucleotide phosphodiesterases (PDEs), and such a mechanism for one phosphodiesterase, PDE7A1, has been reported. In this paper, we extend this mechanism to another isozyme variant of the same PDE family, PDE7A3. We also report the full-length sequence of human PDE8A1 and show that it also is induced in response to a combination of T cell receptor and costimulatory receptor pathway activation. However, the time course for induction of PDE8A1 is slower than that of PDE7A1. The basal level measured and, therefore, the apparent fold induction of PDE7A1 mRNA and protein depend in large part on the method of isolation of the T cells. On the other hand, regardless of the isolation method, the basal levels of PDE7A3 and PDE8A1 are very low and fold activation is much higher. Constitutively expressed PDE8A1 and PDE7A3 also have been isolated from a human T cell line, Hut78.

Cloning and characterization of PDE7B, a cAMP-specific phosphodiesterase.

A member of the phosphodiesterase (PDE)7 family with high affinity and specificity for cAMP has been identified. Based on sequence homologies, we designate this PDE as PDE7B. The full-length cDNA of PDE7B is 2399 bp, and its ORF sequence predicts a protein of 446 amino acids with a molecular mass of 50.1 kDa. Comparison of the predicted protein sequences of PDE7A and PDE7B reveals an identity of 70% in the catalytic domain. Northern blotting indicates that the mRNA of PDE7B is 5.6 kb. It is most highly expressed in pancreas followed by brain, heart, thyroid, skeletal muscle, eye, ovary, submaxillary gland, epididymus, and liver. Recombinant PDE7B protein expressed in a Baculovirus expression system is specific for cAMP with a K(m) of 0.03 microM. Within a series of common PDE inhibitors, it is most potently inhibited by 3-isobutyl-1-methylxanthine with an IC(50) of 2.1 microM. It is also inhibited by papaverine, dipyridamole, and SCH51866 at higher doses. PDE7A and PDE7B exhibit the same general pattern of inhibitor specificity among the several drugs tested. However, differences in IC(50) for some of the drugs suggest that isozyme selective inhibitors can be developed.

Mutation of conserved residues in the NADP(H)-binding domain of the proton translocating pyridine nucleotide transhydrogenase of Escherichia coli.

Possible NADP(H)-binding sites of the beta subunit of the pyridine nucleotide transhydrogenase of Escherichia coli were examined by site-directed mutagenesis. The sequence of the beta subunit at positions 314-350 showed several features typical of NADP(H)-binding sites. Mutation of betaGly314, the first glycine residue of the GXGXXV motif, and of betaArg350, which probably interacts with the 2'-phosphate of the substrate NADP(H), resulted in drastic loss of enzyme activity. The loss of activity in the betaArg350 mutants was not due to loss of ability to bind NADP(H). Several residues (betaVal319, betaGly337, betaHis345, and betaArg350) were mutated to make the sequence more similar to that of a NAD(H)-binding site. The introduction of multiple mutations resulted in improper assembly of the enzyme and decreased incorporation into the membrane. The GXGXXG motif, typical of beta alphabeta nucleotide-binding folds, in the sequence of the beta subunit at positions 274-279 was mutated without causing major changes in transhydrogenase activities. It is unlikely to be part of a nucleotide-binding domain. Deletion of the carboxy-terminal 32 amino acids of the beta subunit, a possible nucleotide-binding site, prevented assembly and incorporation of the truncated enzyme into the cytoplasmic membrane of E. coli.

The mechanism of hydride transfer between NADH and 3-acetylpyridine adenine dinucleotide by the pyridine nucleotide transhydrogenase of Escherichia coli.

The pyridine nucleotide transhydrogenase of Escherichia coli catalyzes the reversible transfer of hydride ion equivalents between NAD+ and NADP+ coupled to translocation of protons across the cytoplasmic membrane. Recently, transhydrogenation of 3-acetylpyridine adenine dinucleotide (AcPyAD+), an analog of NAD+, by NADH has been described using a solubilized preparation of E. coli transhydrogenase [Hutton, M., Day, J.M., Bizouarn, T., and Jackson, J.B. (1994) Eur. J. Biochem. 219, 1041-1051]. This reaction depended on the presence of NADP(H). We show that (a) this reaction did not require NADP(H) at pH 6 in contrast to pH 8; (b) the reaction occurred at pH 8 in the absence of NADP(H) in the mutant beta H91K and in a mutant in which six amino acids of the carboxy-terminus of the alpha subunit had been deleted; (c) the mutant transhydrogenases contained bound NADP+ and were in a conformation in which the beta subunit was digestible by trypsin; (d) the conformation of the beta subunit of the wild-type enzyme was made susceptible to trypsin digestion by NADP(H) or by placing the enzyme at pH 6 in the absence of NADP(H). It is concluded that reduction of AcPyAD+ by NADH does not involve NADPH as an intermediate and that the role of NADP(H) in this reaction at pH 8 is to cause the transhydrogenase to adopt a conformation favouring transhydrogenation between NADH and AcPyAD+.

Organization in the membrane of the N-terminal proton-translocating domain of the beta subunit of the pyridine nucleotide transhydrogenase of Escherichia coli.

The proton-translocating transmembrane pyridine nucleotide transhydrogenase of Escherichia coli is composed of two types of subunits, alpha and beta. The beta subunit has several membrane-spanning segments in the N-terminal region followed by a cytosolic C-terminal domain bearing a binding site for NADP(H). The N-terminal region contains at least one residue involved in the process of transmembrane proton translocation. Using site-directed mutagenesis cysteine residues were introduced at selected sites into the N-terminal region of the beta subunit. The pattern of labelling of these residues with 3-(N-maleimidyl propionyl)biocytin and other sulfhydryl reagents has shown that a model in which the N-terminal region of the beta subunit spans the membrane in eight segments is more likely than a previously proposed six segment model (Holmberg et al. (1994) Biochemistry 33, 7691-7700). The preferred model accounts for the site of labelling of a glutamate residue (Glu124) in the N-terminal domain by N,N-dicyclohexylcarbodiimide.

Involvement of histidine-91 of the beta subunit in proton translocation by the pyridine nucleotide transhydrogenase of Escherichia coli.

The pyridine nucleotide transhydrogenase (EC 1.6.1.1) carries out transmembrane proton translocation coupled to transfer of a hydride equivalent between NAD+ and NADP+. Mutations were made in histidine-91 of the beta subunit of the pyridine nucleotide transhydrogenase of Escherichia coli. This amino acid is the only conserved charged residue in the transmembrane domains of this enzyme and thus potentially is involved in proton translocation by the transhydrogenase. The mutant beta H91N retained 80% of the hydride transfer activity while proton translocation was reduced to 7%. This behavior is consistent with a role for beta His91 in the proton translocation pathway. Other mutations at this residue affected the conformation of the enzyme. Thus, the enzyme in mutants beta H91C, beta H91T, and beta H91S was unable to undergo the conformational change that occurred on binding of the substrates NADP+ or NADPH. By contrast, the enzyme in the beta H91K mutant was present in the NADP(H)-induced conformation even in the absence of these substrates. Further evidence for the linkage between beta His91 and the conformation of the beta subunit was obtained by labeling the transmembrane domain of the beta subunit with [14C]N,N'-dicyclohexylcarbodiimide (DCCD). Labeling occurred most readily with the enzyme of beta H91K. It is concluded that beta His91 is a component of the proton translocation pathway of the transhydrogenase and that its state of protonation is probably linked to conformational changes induced by binding/debinding of substrates during the catalytic cycle of the enzyme.

Evidence for the presence of two pyridine nucleotide-binding sites on the beta subunit of the Escherichia coli pyridine nucleotide transhydrogenase.

The pyridine nucleotide transhydrogenase of Escherichia coli is composed of two types of subunits, alpha and beta. Trypsin digestion of the purified enzyme generates fragments of the alpha subunit. The beta subunit is uncleaved unless NADP(H) is present (Tong, R.C.W., Glavas, N.A. and Bragg. P.D. (1991) Biochim. Biophys. Acta 1080, 19-28). Purified transhydrogenase bound to either NAD- or NADP-agarose was treated with trypsin. The alpha subunit was cleaved to 16, 29 and 43 kDa fragments in both cases. The beta subunit remained bound to NAD-agarose but was released as two cleavage fragments (25 and 30 kDa) from NADP-agarose. The beta subunit of the transhydrogenase bound to NAD-agarose was cleaved by trypsin in the presence of NADP(H) to yield 25 and 30 kDa fragments of the beta subunit. These results suggest that the beta subunit contains two pyridine nucleotide-binding sites.

Prediction and site-specific mutagenesis of residues in transmembrane alpha-helices of proton-pumping nicotinamide nucleotide transhydrogenases from Escherichia coli and bovine heart mitochondria.

Nicotinamide nucleotide transhydrogenase from bovine heart consists of a single polypeptide of 109 kD. The complete gene for this transhydrogenase was constructed, and the protein primary structure was determined from the cDNA. As compared to the previously published sequences of partially overlapping clones, three residues differed: Ala591 (previously Phe), Val777 (previously Glu), and Ala782 (previously Arg). The Escherichia coli transhydrogenase consists of an alpha subunit of 52 kD and a beta subunit of 48 kD. Alignment of the protein primary structure of the bovine trashydrogenase with that of the transhydrogenase from E. coli showed an identity of 52%, indicating similarly folded structures. Prediction of transmembrane-spanning alpha-helices, obtained by applying several prediction algorithms to the primary structures of the revised bovine heart and E. coli transhydrogenases, yielded a model containing 10 transmembrane alpha-helices in both transhydrogenases. In E. coli transhydrogenase, four predicted alpha-helices were located in the alpha subunit and six alpha-helices were located in the beta subunit. Various conserved amino acid residues of the E. coli transhydrogenase located in or close to predicted transmembrane alpha-helixes were replaced by site-specific mutagenesis. Conserved negatively charged residues in predicted transmembrane alpha-helices possibly participating in proton translocation were identified as beta Glu82 (Asp655 in the bovine enzyme) and beta Asp213 (asp787 in the bovine enzyme) located close to the predicted alpha-helices 7 and 9 of the beta subunit. beta Glu82 was replaced by Lys or Gln and beta Asp213 by Asn or His. However, the catalytic as well as the proton pumping activity was retained. In contrast, mutagenesis of the conserved beta His91 residue (His664 in the bovine enzyme) to Ser, Thr, and Cys gave an essentially inactive enzyme. Mutation of alpha His450 (corresponding to His481 in the bovine enzyme) to Thr greatly lowered catalytic activity without abolishing proton pumping. Since no other conserved acidic or basic residues were predicted in transmembrane alpha-helices regardless of the prediction algorithm used, proton translocation by transhydrogenase was concluded to involve a basic rather than an acidic residue. The only conserved cysteine residue, beta Cys260 (Cys834 in the bovine enzyme), located in the predicted alpha-helix 10 of the E. coli transhydrogenase, previously suggested to function as a redox-active dithiol, proved not to be essential, suggesting that redox-active dithiols do not play a role in the mechanism of transhydrogenase.

Site-directed mutagenesis of tyrosine residues at nicotinamide nucleotide binding sites of Escherichia coli transhydrogenase.

Nicotinamide nucleotide transhydrogenase (E.C.1.6.1.1) from Escherichia coli was investigated with respect to the role of specific conserved tyrosine residues of putative substrate-binding regions. The enzyme from E. coli is made up of two subunits, alpha (510 residues) and beta (462 residues). The corresponding enzyme from bovine mitochondria is a single polypeptide (1043 residues) whose N-terminal region corresponds to the alpha subunit and whose C-terminal region corresponds to the beta subunit. Tyrosines 245 and 1006 of the mitochondrial enzyme have been shown to react selectively with 5'-(p-fluorosulfonylbenzoyl)adenosine with inactivation of the enzyme. In E. coli these residues correspond to tyrosine 226 of the alpha subunit and tyrosine 431 of the beta subunit. In addition, tyrosine 315 of the beta subunit is of interest since mutation of an adjacent residue (glycine 314) leads to inactivation [Ahmad, S., Glavas, N. A., & Bragg, P. D. (1992) Eur. J. Biochem. 207, 733-739]. In order to assess the role of the aforementioned conserved tyrosine residues in the mechanism and structure of transhydrogenases, these were replaced by site-specific mutagenesis, using the cloned and overexpressed E. coli transhydrogenase genes [Clarke, D. M., & Bragg, P. D. (1985) J. Bacteriol. 162, 367-373]. Phenylalanine mutants of all three tyrosine residues showed approximately 50% activity or more with regard to catalytic activity assayed as reduction of 3-acetylpyridine-NAD+ by NADPH. These mutants were also active in proton pumping assayed as quenching of 9-methoxy-6-chloro-2-aminoacridine or quinacrine fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Assembly of multimeric proteins. Effect of mutations in the alpha-subunit on membrane assembly and activity of pyridine nucleotide transhydrogenase.

The pyridine nucleotide transhydrogenase of Escherichia coli is an inner membrane protein of two different subunits (alpha and beta). It functions as a proton pump. The highly hydrophilic carboxy-terminal tail of ten amino acid residues in the alpha-subunit determines the correct folding and proper assembly of the beta-subunit leading to a functional enzyme. Premature termination of the alpha-subunit six amino acid residues from the carboxy-terminal end abolishes the activity completely. Although the two subunits are still assembled into the membrane, the conformation of the beta-subunit is perturbed. Systematic truncation and site-directed substitutions revealed that at least one positive charge in the carboxy-terminal region is required for efficient assembly of the two subunits to give a functional enzyme, while a phenylalanine residue, essential for activity, has no apparent effect on the extent of assembly of the two subunits.

Identification of N,N'-dicyclohexylcarbodiimide-reactive glutamic and aspartic acid residues in Escherichia coli transhydrogenase and the exchange of these by site-specific mutagenesis.

Pyridine nucleotide transhydrogenase (EC 1.6.1.1) from Escherichia coli was investigated with respect to the role of glutamic and aspartic acid residues reactive to N,N'-dicyclohexylcarbodiimide (DCCD) and potentially involved in the proton-pumping mechanism of the enzyme. The E. coli transhydrogenase consists of an alpha (510 residues) and a beta (462 residues) subunit. DCCD reacts with the enzyme to inhibit catalytic activity and proton pumping. This reagent modifies Asp alpha 232, Glu alpha 238, and Glu alpha 240 as well as amino acid residue(s) in the beta subunit. Using the cloned and overexpressed E. coli transhydrogenase genes (Clarke, D. M., and Bragg, P. D. (1985) J. Bacteriol. 162, 367-373), Asp alpha 232 and Glu alpha 238 were replaced independently by site-specific mutagenesis. In addition, Asp alpha 232, Glu alpha 238, and Glu alpha 240 were replaced to generate triple mutants. The specific catalytic activities of the mutant transhydrogenases alpha D232N, alpha D232E, alpha D232K, alpha D232H, alpha E238K, and alpha E238Q as well as of the triple mutants alpha D232N, alpha E238Q, alpha E240Q and alpha D232H, alpha E238Q, alpha E240Q were in the range of 40-90% of the wild-type activity. Proton-pumping activity was present in all mutants. Examination of the extent of subunit modification by [14C]DCCD revealed that the label was still incorporated into both alpha and beta subunits in the Asp alpha 232 mutants, but that the alpha subunit was not labeled in the triple mutants. Catalytic and proton-pumping activities were nearly insensitive to DCCD in the triple mutants. This suggests that loss of catalytic and proton-pumping activities is associated with modification of the aspartic and glutamic acid residues of the alpha subunit. In the presence of the substrate NADPH, the rate of modification of the beta subunit by [14C]DCCD was increased, and there was a greater extent of enzyme inactivation. By contrast, NADH and 3-acetylpyridine-NAD+ protected the catalytic activity of the transhydrogenase from inhibition by DCCD. The protection was particularly marked in the E238Q and E238K mutants. It is concluded that the Asp alpha 232, Glu alpha 238, and Glu alpha 240 residues are not essential for catalytic activity or proton pumping. The inactivation by DCCD is likely due to the introduction of a sterically hindering group that reacts with the identified acidic residues close to the NAD(H)-binding site.

A mutation at Gly314 of the beta subunit of the Escherichia coli pyridine nucleotide transhydrogenase abolishes activity and affects the NADP(H)-induced conformational change.

Escherichia coli RH1 contains a mutation causing complete loss of pyridine nucleotide transhydrogenase activity. A single base change in the chromosomal DNA resulted in the replacement of Gly314 of the beta subunit by a Glu residue. The mutant enzyme was partially purified and its trypsin cleavage products examined. The distinct pattern of polypeptides given by proteolysis of the normal transhydrogenase in the presence of NADP(H) was absent when the mutant enzyme was treated with trypsin. However, the beta subunit of the mutant enzyme retained its ability to bind to NAD-agarose. Further substitutions were made at Gly314 converting it to Ala, Val or Cys by the use of site-directed mutagenesis. All substitutions for Gly314 abolished the activity completely. The enzyme containing the Gly314----Ala mutation was studied in detail and behaved exactly as the enzyme containing the Gly314----Glu mutation. It is concluded that the mutation in the beta subunit abolished the NADP(H)-induced conformational change in the mutant enzyme. This conformational change, caused by NADP(H) binding, is required to cleave the normal beta subunit at Arg265 by trypsin. The genes encoding the pyridine nucleotide transhydrogenase were completely resequenced and several corrections have been made to the previously published sequence [Clarke et al. (1986) Eur. J. Biochem. 158, 647-653].

Subunit interactions involved in the assembly of pyridine nucleotide transhydrogenase in the membranes of Escherichia coli.

The pyridine nucleotide transhydrogenase (PNT) of Escherichia coli consists of two different subunits (alpha and beta) and assembles as a tetramer (alpha 2 beta 2) in the inner membrane. The pnt genes from E. coli have been cloned on a multicopy plasmid resulting in high level expression of the enzyme activity. We have studied the influence of the different segments of the polypeptide chains of the alpha and beta subunits on the assembly and function of the enzyme by constructing a series of deletion mutants for both of the subunits. Our results show that the assembly of the beta subunit is contingent upon the insertion of the alpha subunit into the membrane, while the alpha subunit can assemble independently of the beta subunit. All deletions constructed for the cytosolic portion of the alpha subunit gave no incorporation of the alpha subunit and, as a consequence, of the beta subunit, also. Of the four membrane-spanning regions of the alpha subunit, the last two were indispensable, while the deletion of the first two still allowed the association of alpha as well as of the beta subunit with the membrane. However, the enzyme was not functional. The two subunits were also loosely associated as mild detergent treatment released them from the membrane in contrast with the wild-type enzyme. Deletions within the beta subunit had little effect on the assembly of the alpha subunit, although less was incorporated. All deletions involving the cytosolic portion of the beta subunit resulted in loss of incorporation into the membrane. Of the eight membrane-spanning regions of the beta subunit, the deletion of regions 2-3, 2-4, 2-6, and 2-7 yielded significant association of both the subunits with the membrane. However, none of these mutants assembled a functional enzyme, and again the two subunits were loosely associated with the membrane. Based on the stringent requirement of the cytosolic portions of alpha and beta subunits for assembly, a model is proposed that suggests interactions between these two regions must occur prior to assembly.

Topological analysis of the pyridine nucleotide transhydrogenase of Escherichia coli using proteolytic enzymes.

The pyridine nucleotide transhydrogenase of Escherichia coli has an alpha 2 beta 2 structure (alpha: Mr, 54,000; beta: Mr, 48,700). Hydropathy analysis of the amino acid sequences suggested that the 10 kDa C-terminal portion of the alpha subunit and the N-terminal 20-25 kDa region of the beta subunit are composed of transmembranous alpha-helices. The topology of these subunits in the membrane was investigated using proteolytic enzymes. Trypsin digestion of everted cytoplasmic membrane vesicles released a 43 kDa polypeptide from the alpha subunit. The beta subunit was not susceptible to trypsin digestion. However, it was digested by proteinase K in everted vesicles. Both alpha and beta subunits were not attacked by trypsin and proteinase K in right-side out membrane vesicles. The beta subunit in the solubilized enzyme was only susceptible to digestion by trypsin if the substrates NADP(H) were present. NAD(H) did not affect digestion of the beta subunit. Digestion of the beta subunit of the membrane-bound enzyme by trypsin was not induced by NADP(H) unless the membranes had been previously stripped of extrinsic proteins by detergent. It is concluded that binding of NADP(H) induces a conformational change in the transhydrogenase. The location of the trypsin cleavage sites in the sequences of the alpha and beta subunits were determined by N- and C-terminal sequencing. A model is proposed in which the N-terminal 43 kDa region of the alpha subunit and the C-terminal 30 kDa region of the beta subunit are exposed on the cytoplasmic side of the inner membrane of E. coli. Binding sites for pyridine nucleotide coenzymes in these regions were suggested by affinity chromatography on NAD-agarose columns.