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The peroxisome proliferator-activated receptor gamma (PPARgamma) is a key regulator of terminal adipocyte differentiation. PPARdelta is expressed in preadipocytes, but the importance of this PPAR subtype in adipogenesis has been a matter of debate. Here we present a critical evaluation of the role of PPARdelta in adipocyte differentiation. We demonstrate that treatment of NIH-3T3 fibroblasts overexpressing PPARdelta with standard adipogenic inducers led to induction of PPARgamma2 expression and terminal adipocyte differentiation in a manner that was strictly dependent on simultaneous administration of a PPARdelta ligand and methylisobutylxanthine (MIX) or other cAMP elevating agents. We further show that ligands and MIX synergistically stimulated PPARdelta-mediated transactivation. In 3T3-L1 preadipocytes, simultaneous administration of a PPARdelta-selective ligand and MIX significantly enhanced the early expression of PPARgamma and ALBP/aP2, but only modestly promoted terminal differentiation as determined by lipid accumulation. Finally, we provide evidence that synergistic activation of PPARdelta promotes mitotic clonal expansion in 3T3-L1 cells with or without forced expression of PPARdelta. In conclusion, our results suggest that PPARdelta may play a role in the proliferation of adipocyte precursor cells, whereas activation of endogenous PPARdelta in 3T3-L1 cells appears to have only minor impact on the processes leading to terminal adipocyte differentiation.

The final step in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor is the transformation of the labile cyanohydrin into a stable storage form byO-glucosylation of (S)-p-hydroxymandelonitrile at the cyanohydrin function. The UDP-glucose:p-hydroxymandelonitrile-O-glucosyltransferase was isolated from etiolated seedlings of S. bicoloremploying Reactive Yellow 3 chromatography with UDP-glucose elution as the critical step. Amino acid sequencing allowed the cloning of a full-length cDNA encoding the glucosyltransferase. Among the few characterized glucosyltransferases, the deduced translation product showed highest overall identity to Zea maysflavonoid-glucosyltransferase (Bz-Mc-2 allele). The substrate specificity of the enzyme was established using isolated recombinant protein. Compared with endogenousp-hydroxymandelonitrile, mandelonitrile, benzyl alcohol, and benzoic acid were utilized at maximum rates of 78, 13, and 4%, respectively. Surprisingly, the monoterpenoid geraniol was glucosylated at a maximum rate of 11% compared withp-hydroxymandelonitrile. The picture that is emerging regarding plant glucosyltransferase substrate specificity is one of limited but extended plasticity toward metabolites of related structure. This in turn ensures that a relatively high, but finite, number of glucosyltransferases can give rise to the large number of glucosides found in plants.

We report here the cloning and optimized expression at 16 degrees C and the characterization of a Vitis vinifera UDP-glucose:flavonoid 3-O-glucosyltransferase, an enzyme responsible for a late step in grapevine anthocyanin biosynthesis. The properties of this and other UDP-glucose:flavonoid 3-O-glucosyltransferases, homologues of the product encoded by the maize Bronze-1 locus, are a matter of conjecture. The availability of a purified recombinant enzyme allowed for the unambiguous determination of the characteristics of a flavonoid 3-O-glucosyltransferase. Kinetic analyses showed that kcat for glucosylation of cyanidin, an anthocyanidin substrate, is 48 times higher than for glucosylation of the flavonol quercetin, whereas Km values are similar for both substrates. Activity toward other classes of substrates is absent. Cu2+ ions strongly inhibit the action of this and other glucosyltransferases; however, we suggest that this phenomenon in large part is due to Cu2+-mediated substrate degradation rather than inhibition of the enzyme. Additional lines of complementary biochemical data also indicated that in the case of V. vinifera, the principal, if not only, role of UDP-glucose:flavonoid 3-O-glucosyltransferases is to glucosylate anthocyanidins in red fruit during ripening. Other glucosyltransferases with a much higher relative activity toward quercetin are suggested to glucosylate flavonols in a distinct spatial and temporal pattern. It should be considered whether gene products homologous to Bronze-1 in some cases more accurately should be referred to as UDP-glucose:anthocyanidin 3-O-glucosyltransferases.

Two β-glucan exohydrolases of apparent molecular masses 69,000 and 71,000 Da have been purified from extracts of 8-day germinated barley grains and are designated isoenzymes ExoI and ExoII, respectively. The sequences of their first 52 NH2-terminal amino acids show 64% positional identity. Both enzymes hydrolyze the(1, 3) -β-glucan, laminarin, but also hydrolyze (1,3;1,4)-β-glucan and 4-nitrophenyl β-D-glucoside. The complete sequence of 602 amino acid residues of the mature β-glucan exohydrolase isoenzyme ExoII has been deduced by nucleotide sequence analysis of a near full-length cDNA. Two other enzymes of apparent molecular mass 62,000 Da, designated βI and βII, were also purified from the extracts. Their amino acid sequences are similar to enzymes classified as β-glucosidases and although they hydrolyze 4-nitrophenyl β-glucoside, their substrate specificities and action patterns are more typical of polysaccharide exohydrolases of the(1, 4) -β-glucan glucohydrolase type. Both the β-glucan exohydrolase isoenzyme ExoI and the β-glucosidase isoenzyme βII release single glucosyl residues from the nonreducing ends of substrates and proton-NMR shows that anomeric configurations are retained during hydrolysis by both classes of enzyme. These results raise general questions regarding the distinction between polysaccharide exohydrolases and glucosidases, together with more specific questions regarding the functional roles of the two classes of enzyme in germinating barley grain.

Utilizing the ability of bacterial chaperonin 60 (GroEL) to functionally interact with chaperonin 10 (Cpn10) homologues in an ATP-dependent fashion, we have purified substantial amounts of mammalian, chloroplast, and thermophilic Cpn10 homologues from their natural host. In addition, large amounts of recombinant rat Cpn10 were produced in Escherichia coli and found to be identical to its authentic counterpart except for the lack of N-terminal acetylation. By comparing these two forms of Cpn10, it was found that acetylation does not influence the oligomeric structure of Cpn10 and is not essential for chaperone activity or mitochondrial import in vitro. In contrast, N-terminal acetylation proved crucial in the protection of Cpn10 against degradation by N-ethylmaleimide-sensitive proteases derived from organellar preparations of rat liver. The availability of large amounts of both affinity-purified and recombinant Cpn10 will facilitate not only further characterization of the eukaryotic folding machinery but also further scrutiny of the reported function of Cpn10 as early pregnancy factor.

Determination of the crystal structures of a 1,3-beta-D-glucanase (E.C. 3.2.1.39) and a 1,3-1,4-beta-D-glucanase (E.C. 3.2.1.73) from barley (Hordeum vulgare) (Varghese, J.N, Garrett, T. P. J., Colman, P. M., Chen, L., Høj, P. B., and Fincher, G. B. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 2785-2789) showed the spatial positions of the catalytic residues in the substrate-binding clefts of the enzymes and also identified highly conserved neighboring amino acid residues. Site-directed mutagenesis of the 1,3-beta-glucanase has now been used to investigate the importance of these residues. Substitution of glutamine for the catalytic nucleophile Glu231 (mutant E231Q) reduced the specific activity about 20,000-fold. In contrast, substitution of glutamine for the catalytic acid Glu288 (mutant E288Q) had less severe consequences, reducing kcat approximately 350-fold with little effect on Km. Substitution of two neighboring and strictly conserved active site-located residues Glu279 (mutant E279Q) and Lys282 (mutant K282M) led to 240- and 2500-fold reductions of Kcat, respectively, with small increases in Km. Thus, a tetrad of ionizable amino acids is required for efficient catalysis in barley beta-glucanases. The active site-directed inhibitor 2,3-epoxypropyl beta-laminaribioside was soaked into native crystals. Crystallographic refinement revealed all four residues (Glu231, Glu279, Lys282, and Glu288) to be in contact with the bound inhibitor, and the orientation of bound substrate in the active site of the glucanase was deduced.

Chaperonin 10 (Cpn10) is one of only a few mitochondrial matrix proteins synthesized without a cleavable targeting signal. Using a truncated form of Cpn10 and synthetic peptides in mitochondrial import assays, we show that the N-terminal region is both necessary and sufficient for organellar targeting in vitro. To elucidate the structural features of this topogenic signal, peptides representing residues 1-25 of rat Cpn10 were synthesized with and without the naturally occurring N-terminal acetylation. 1H NMR spectroscopy in 20% CF3CH2OH,H2O showed that both peptides assume a stable helix-turn-helix motif and are highly amphiphilic in nature. Chemical shift and coupling constant data revealed that the N-terminal helix is stabilized by N-acetylation, whereas NOE and exchange studies were used to derive a three dimensional structure for the acetylated peptide. These findings are discussed with respect to a recent model predicting that targeting sequences forming a continuous alpha-helix of more than 11 residues cannot adopt a conformation necessary for proteolysis by the matrix located signal peptidases (Hammen, P. K., Gorenstein, D. G., and Weiner, H. (1994) Biochemistry 33, 8610-8617).

Catalytic amino acid residues in a 1,3-beta-D-glucan 3-glucanohydrolase (EC 3.2.1.39) and a homologous 1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) from barley have been investigated. To identify amino acids responsible for protonation of the glycosidic oxygen during hydrolysis, carbodiimide-mediated labeling of the enzymes with [14C]glycine ethyl ester was performed. This resulted in loss of activity and specific modification of the Glu288 residues in both enzymes. The stoichiometry of labeling was approximately 1:1, and modification was reduced in the presence of substrate analogues. Based on these data, the Glu288 residues are likely to be present at the active sites of the respective enzymes and may represent the catalytic acids in the hydrolytic reaction. The catalytic nucleophiles of the two enzymes were investigated by labeling with specific, mechanism-based epoxyalkyl-beta-oligoglucosides. Amino acid residues Glu232 and Glu231 were identified as the likely catalytic nucleophiles in the 1,3-1,4- and 1,3-beta-glucanases, respectively. Thus the position of the catalytic nucleophile and the putative proton donating amino acids in the two classes of beta-glucan endohydrolases are conserved. The acquisition of distinct substrate specificities in the evolution of these related enzymes may therefore not require the recruitment of novel catalytic amino acids but rather differences in their positioning at the active site and/or changes in substrate binding residues.

Bacillus amyloliquefaciens 1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) was modified by the mechanism-based, affinity-labeling reagent [14C](3,4)-epoxybutyl beta-D-cellobioside. Following partial inactivation a completely inactivated enzyme preparation containing 1.1 mol of covalently bound inhibitor/mol of protein was obtained by chromatography on a cellulosic matrix. The inactivated enzyme was digested with endoproteinase Glu-C and radioactive peptides purified by reversed-phase high performance liquid chromatography (HPLC). The affinity label was esterified exclusively to the gamma-carboxylate of Glu105 in the sequence Gly-Thr-Pro-Trp-Asp-Glu-Ile-Asp-Ile-Glu109. The sequence motif Glu-(Ile/Leu)-Asp-Ile is found in many glucanases and xylanases and may therefore serve to identify the catalytic nucleophile in beta-glycanases, which otherwise exhibit a low degree of sequence identity. The esterification of Glu105 by the affinity label abolished endoproteinase Glu-C-mediated hydrolysis of the Glu-Ile106 peptide bond. Identification of phenylthiohydantoin-Glu105 during automated sequence analysis was not possible unless the affinity label was liberated by prior base hydrolysis. These observations formed the basis for the development of a highly sensitive approach for the identification of catalytic carboxylates in polysaccharide hydrolases employing non-radioactive inhibitors, comparative HPLC mapping, electrospray mass spectrometry, and Edman degradation.

1,3-1,4-beta-D-Glucan 4-glucanohydrolases (EC 3.2.-1.73) from Bacillus subtilis and barley (Hordeum vulgare) with identical substrate specificities but unrelated primary structures have been probed with (R,S)-epoxyalkyl (-propyl, -butyl, -pentyl) beta-cellobiosides and with optically pure (3S)- and (3R)-3,4-cellobiosides as active site-directed inhibitors. The optimal aglycon length for inactivation differs for the two enzymes, and they are differentially inhibited by the pure epoxybutyl beta-cellobioside diastereoisomers. The (3S)-epoxybutyl beta-cellobioside inactivates the B. subtilis enzyme much more efficiently than does the (3R)-isomer, whereas the reverse is true for the barley enzyme. Both enzymes are inactivated by a mixture of the stereoisomers at a rate intermediate of that observed with the individual isomers. The two beta-glucan endohydrolases may therefore employ sterically different mechanisms to achieve glycoside bond hydrolysis in their common substrate. The efficiency and specificity of epoxide-based "suicide" inhibitors may be enhanced significantly by the use of inhibitors bearing only one stereoisomeric form of the epoxide group.

An improved procedure is reported for large-scale preparation of photosystem I (PS-I) vesicles from thylakoid membranes of barley (Hordeum vulgare L.). The PS-I vesicles contain polypeptides of molecular masses 82, 18, 16, 14, and 9 kDa in an apparent molar ratio of 4:2:2:1:2. The 18-, 16-, and 9-kDa polypeptides were purified to homogeneity after exposure of the PS-I vesicles to chaotropic agents. The isolated 9-kDa polypeptide binds 65-70% of the zero-valence sulfur of denatured PS-I vesicles, and the remaining 30-35% is bound to P700-chlorophyll a-protein 1. The N-terminal amino acid sequence (29 residues) of the 9-kDa polypeptide was determined. Comparison with the nucleotide sequence of the chloroplast genome of Marchantia polymorpha (Ohyama, K., Fukuzawa, H., Kohchi, T., Shirai, H., Sano, T., Sano, S., Umesono, K., Shiki, Y., Takeuchi, M., Chang, Z., Aota, S.-i., Inokuchi, H., and Ozeki, H. (1986) Nature 322, 572-574) and of Nicotiana tabacum (Shinozaki, K., Ohme, M., Tanaka, M., Wakasugi, T., Hayashida, N., Matsubayashi, T., Zaita, W., Chunwongse, J., Obokata, J., Yamaguchi-Shinozaki, K., Ohto, C., Torazawa, K., Meng, B. Y., Sugita, M., Deno, H., Kamogashira, T., Yamada, K., Kusuda, J., Takaiwa, F., Kato, A., Tohdoh, N., Shimada, H., and Sugiura, M. (1986) EMBO J. 5, 2043-2049) identified the chloroplast gene encoding the 9-kDa polypeptide. We designate this gene psaC. The complete amino acid sequence deduced from the psaC gene identifies the 9-kDa PS-I polypeptide as a 2[4Fe-4S] protein. Since P700-chlorophyll a-protein 1 carries center X, the 9-kDa polypeptide carries centers A and B. A hydropathy plot permits specific identification of the cysteine residues which coordinate centers A and B, respectively. Except for the loss of the N-terminal methionine residue, the primary translation product of the psaC gene is not proteolytically processed. P700-chlorophyll a-protein 1 binds 4 iron atoms and 4 molecules of acid-labile sulfide/molecule of P700. Each of the two apoproteins of P700-chlorophyll a-protein 1 contains the sequence Phe-Pro-Cys-Asp-Gly-Pro-Gly-Arg-Gly-Gly-Thr-Cys (Fish, L. E., Kuck, U., and Bogorad, L. (1985) J. Biol. Chem. 260, 1413-1421). The stoichiometry of the component polypeptides of PS-I indicates the presence of four copies of this sequence per molecule of P700. Center X may be composed of two [2Fe-2S] centers bound to the 8 cysteine residues contained in these four segments.

Germination and growth of barley (Hordeum vulgare L.) in the presence of 59Fe2+ or 35SO4(2-) allows heavy incorporation of both isotopes into the thylakoid membranes and into isolated photosystem I particles. Analysis of 59Fe-labeled preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under mild conditions demonstrates that a minimum of four iron atoms/P700 is carried on P700-chlorophyll a-protein 1. When isolated from 35S-labeled preparations, P700-chlorophyll a-protein 1 binds zero valence 35S, which is converted into acid-labile [35S]sulfide by dithiothreitol reduction. Isolated photosystem I particles contain 14 acid-labile sulfide atoms and 10 iron atoms for each molecule of P700 and are composed of polypeptides of 110, 18, 15, 10, and 8 kDa of which the 10-kDa component is loosely bound. Under the electrophoretic conditions used, none of the low molecular weight polypeptides could be shown to be specifically associated with iron or acid-labile sulfide. Carboxymethylation of cysteine residues shows a high cysteine content in the 8-kDa polypeptide and an intermediate content in the 110- and 18-kDa polypeptides, whereas the 15-kDa polypeptide is devoid of sulfur amino acids. The experiments with the 59Fe-labeled thylakoids reveal other labeled polypeptides not associated with photosystem I, namely cytochrome f and possibly cytochromes b6 and b559.