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1.
An aquatic perspective on the concepts of Ingestad relating plant nutrition to plant growth 总被引:2,自引:0,他引:2
Raven JA 《Physiologia plantarum》2001,113(3):301-307
Oil bodies are lipid storage organelles which have been analyzed biochemically due to the economic importance of oil seeds. Although oil bodies are structurally simple, the mechanisms involved in their formation and degradation remain controversial. At present, only two proteins associated with oil bodies have been described, oleosin and caleosin. Oleosin is thought to be important for oil body stabilization in the cytosol, although neither the structure nor the function of oleosin has been fully elucidated. Even less is known about caleosin, which has only recently been described [Chen et al. (1999) Plant Cell Physiol 40: 1079–1086; Næsted et al. (2000) Plant Mol Biol 44: 463–476]. Caleosin and caleosin-like proteins are not unique to oil bodies and are associated with an endoplasmatic reticulum subdomain in some cell types. Here we review the synthesis and degradation of oil bodies as they relate to structural and functional aspects of oleosin and caleosin. 相似文献
2.
Ascensión Reche Juan José Lazaro Rosario Hermoso Ana Chueca Julio López Gorgé 《Physiologia plantarum》1997,101(3):463-470
Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) binds its putative physiological activator thioredoxin f (Trx f ) at pH 7.9, the pH in the stroma of the illuminated chloroplast. Since Trx m , described as specific in NADP+ -malate dehydrogenase (NADPMDH) activation, appears in pea (Pisum sativum L.) also to be functional in FBPase modulation, we have here analyzed the effect of pH and the redox status of the chloroplast stroma in the pea FBPase binding of homologous Trx f and m . Both pea Trx were strongly bound by purified FBPase when they were preincubated at pH 7.9 with 2.5 m M dithiothreitol (DTT), but not when the reductant was omitted. As occurs with Trx f the Trx m /FBPase ratio of the complex was 4, but this was only observed with a Trx m /FBPase concentration ratio > 10 in the preincubation mixture. The FBPase-Trx m binding disappeared in the presence of 100 m M NaCl, even with 2.5 m M DTT at pH 7.9, with a concomitant appearance of different aggregation states of the FBPase subunit. A similar FBPase-Trx m complex was detected in the stromal solution when pea chloroplasts were lysed at pH 7.9 in the presence of DTT. No interaction was observed between NADP-MDH and Trx f or m , either in the presence or in the absence of DTT. Pea FBPase showed sigmoidal activation kinetics with pea Trx m , and an S0.5 of 133 n M versus 6.6 n M with pea Trx f . About 10-fold higher concentration of the former than that of the latter was required for obtaining maximum activity; however, the Vmax with Trx f was only 2-fold higher than that with Trx m . We conclude that pea FBPase binds and is activated by the homologous Trx m , even though to a lesser extent than with Trx f . We also deduce that in the light the conditions in the chloroplast stroma are optimal for forming an FBPase-Trx complex. 相似文献
3.
In plants, reactive oxygen species (ROS) are short-lived molecules produced through various cellular mechanisms in response
to biotic and abiotic stimuli. ROS function as second messengers for hormone signaling, development, oxygen deprivation, programmed
cell death, and plant–pathogen interactions. Recent research on ROS-mediated responses has produced stimulating findings such
as the specific sources of ROS production, molecular elements that work in ROS-mediated signaling and homeostasis, and a ROS-regulated
gene network (Neill et al., Curr Opin Plant Biol 5:388–395, 2002a; Apel and Hirt, Annu Rev Plant Biol 55:373–399, 2004; Mittler et al., Trends Plant Sci 9:490–498, 2004; Mori and Schroeder, Plant Physiol 135:702–708, 2004; Kwak et al., Plant Physiol 141:323–329, 2006; Torres et al., Plant Physiol 141:373–378, 2006; Miller et al., Physiol Plant 133:481–489, 2008). In this review, we highlight new discoveries in ROS-mediated abscisic acid (ABA) signaling.
Drs. Daeshik Cho and June M. Kwak are the corresponding authors for this paper. 相似文献
4.
Jakubowicz M Jółkowska J Kwapiszewska G Krecisz A Czerski P 《Acta biochimica Polonica》2002,49(4):1037-1042
An important trait of tomato is the rate of fruit ripening, strongly dependent on ethylene production. The ripening-related ethylene synthesis in tomato is controlled mainly by 1-aminocyclopropane-1-carboxylate synthase LE-ACS2 and LE-ACS4 isoenzymes (Rottmann et al., 1991, J. Mol. Biol. 222: 937; Lincoln et al., 1993, J. Biol. Chem. 268: 19422; Barry et al., 2000, Plant Physiol. 123: 979). In spite of numerous reports on the LE-ACS2 and LE-ACS4 gene expression, only ones considered the genomic organisation each of these genes (Rottmann et al., 1991; Lincoln et al., 1993) reported one copy of each of these genes in tomato cv VF36. In this article we suggest that the genomic organisation of LE-ACS2 and LE-ACSS4 genes may depend on tomato cultivars and may differ from that described by the above authors. The results of Southern analyses of genomic DNAs from 17-day old seedlings (cultivars Jaga, Halicz, Betalux, New Yorker) imply that the genomic organisation of LE-ACS2 and LE-ACS4 genes in Polish cultivars differs from that reported for cv VF36. 相似文献
5.
Vanadium peroxidase catalyzes the extracellular assembly of Fucus zygote cell surface adhesive (Vreeland & Epstein 1996, Modern Meth. Plant Anal. 17, 95–116). Our goal is to identify the catalytic, self-associating and wall targeting functional domains of algal vanadium peroxidase to understand its role in algal propagule adhesion. As a first step, we truncated our recombinant Fucus vanadium peroxidase (GenBank AF053411) for catalytic domain identification. Recombinant constructs were prepared which reduced the C-terminal catalytic domain at either or both N- and C-terminal ends. Recombinant proteins were expressed in E. coli , refolded from cytoplasm and inclusion bodies and tested for vanadium-specific o-dianisidine peroxidase activity. Preliminary results demonstrated peroxidase activity when the 40 kDa catalytic domain was truncated on both ends to 24 kDa. Further terminal and internal truncation is needed to fully define the minimal catalytic unit, which could be as small as 15–20 kDa within the 73 kDa monomer. The very small catalytic unit in Fucus vanadium peroxidase is not unexpected considering the rigid bundled helical vanadate frame in the Curvularia fungal vanadium peroxidase (Macedo-Ribeiro et al. 1999, J. Biol. Inorg. Chem. 4, 209–219). We conclude that interactions between the N-terminal noncatalytic domain and the C-terminal catalytic domain, found in the crystalline Ascophyllum enzyme (Weyland et al. 1999, J. Mol. Biol. 293, 595–611), are unnecessary for peroxidase activity. Other conserved amino acids in the C-terminal half of Fucus vanadium peroxidase, peripheral to the helical core, could participate in protein surface functions such self-association and wall targeting. 相似文献
6.
Stéphanie Sobry rée Havelange Françoise Liners Pierre Van Cutsem 《Physiologia plantarum》2005,123(3):339-347
The pectin content in the meristem of Sinapis alba at flowering transition was assessed in electron microscopy using immunocytochemistry and the monoclonal antibody 2F4 that recognizes a Ca2+ -induced supramolecular conformation of homogalacturonans (Liners et al. Plant Physiol 91: 1419–1424, 1989; Liners et al. Plant Physiol 99: 1099–1104, 1992). The meristem's pectins were not recognized at all by the 2F4 monoclonal antibody unless they were enzymatically (PME: pectin methylesterase) or chemically (NaOH) de-esterified, indicating that native pectin must be largely esterified in the apical meristem. A striking but transient decrease of the homopolygalacturonic content of the meristem's cell walls occurred between 20 and 24 h after start of the inductive long day. These changes suggest a role for pectin in floral transition. 相似文献
7.
Wangensteen OS Chueca A Hirasawa M Sahrawy M Knaff DB López Gorgé J 《Biochimica et biophysica acta》2001,1547(1):156-166
It has been proposed that a hydrophobic groove surrounded by positively charged amino acids on thioredoxin (Trx) serves as the recognition and docking site for the interaction of Trx with target proteins. This model for Trx-protein interactions fits well with the Trx-mediated fructose-1,6-bisphosphatase (FBPase) activation, where a protruding negatively charged loop of FBPase would bind to this Trx groove, in a process involving both electrostatic and hydrophobic interactions. This model facilitates the prediction of Trx amino acid residues likely to be involved in enzyme binding. Site-directed mutagenesis of some of these amino acids, in conjunction with measurements of the FBPase activation capacity of the wild type and mutated Trxs, was used to check the model and provided evidence that lysine-70 and arginine-74 of pea Trx m play an essential role in FBPase binding. The binding parameters for the interaction between chloroplast FBPase and the wild type pea Trxs f and m, as well as mutated pea Trx m, determined by equilibrium dialysis in accordance with the Koshland-Nemethy-Filmer model of saturation kinetics, provided additional support for the role of these basic Trx residues in the interaction with FBPase. These data, in conjunction with the midpoint redox potential (E(m)) determinations of Trxs, support the hydrophobic groove model for the interaction between chloroplast FBPase and Trx. This model predicts that differences in the FBPase activation capacity of Trxs arise from their different binding abilities. 相似文献
8.
Quantitative trait loci for polyamine content in an RFLP-mapped potato population and their relationship to tuberization 总被引:1,自引:0,他引:1
Peter J. Davies Ivan imko Suzanne M. Mueller G. Craig Yencho Candice Lewis Susan McMurry Mark A. Taylor Elmer E. Ewing 《Physiologia plantarum》1999,106(2):210-218
DNA-based genetic markers are now widely used by geneticists to locate genes for quantitative traits, and may also serve as a valuable tool for dissecting complex physiological phenomena. Van den Berg et al. (1996a QTL analysis of potato tuberization. Theor Appl Gen 93: 307–316), using restriction fragment length polymorphism (RFLP)-mapped populations of potato, detected eleven quantitative trait loci (QTLs) for tuberization. Taylor et al. (1992 Expression and sequence analysis of cDNAs induced during the early stages of tuberisation in different organs of the potato plant [ Solanum tuberosum L.]. Plant Mol Biol 20: 641–651) have identified one of the genes associated with tuberization as that for the enzyme S-adenosylmethionine decarboxylase (SAMdc), an enzyme of the polyamine biosynthetic pathway. Chromosomal loci for SAMdc and arginine decarboxylase were established on the potato and tomato chromosomal maps, respectively, by hybridizing cDNA probes for these genes to RFLP digests. The polyamine content of leaves from an RFLP-mapped potato population was analyzed by fluorescence detection following HPLC, with quantitation using an internal standard. The data were analyzed by the 'qGene' statistical program, and QTLs for polyamines were detected on seven chromosomes. At least six QTLs were found for spermine, two for spermidine, and two for putrescine. A spermidine QTL was on chromosome 5 linked to marker TG441 , very close to the place where SAMdc mapped. There was some congruence between QTLs for spermine and those previously detected for tuberization and dormancy, but relationships were not consistent. 相似文献
9.
High-yield expression of pea thioredoxin m and assessment of its efficiency in chloroplast fructose-1,6-bisphosphatase activation. 总被引:1,自引:0,他引:1
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J Lpez Jaramillo A Chueca J P Jacquot R Hermoso J J Lzaro M Sahrawy J Lpez Gorg 《Plant physiology》1997,114(4):1169-1175
A cDNA clone encoding pea (Pisum sativum L.) chloroplast thioredoxin (Trx) m and its transit peptide were isolated from a pea cDNA library. Its deduced amino acid sequence showed 70% homology with spinach (Spinacia oleracea L.) Trx m and 25% homology with Trx f from pea and spinach. After subcloning in the Ndel-BamHI sites of pET-12a, the recombinant supplied 20 mg Trx m/L. Escherichia coli culture. This protein had 108 amino acids and was 12,000 D, which is identical to the pea leaf native protein. Unlike pea Trx f, pea Trx m showed a hyperbolic saturation of pea chloroplast fructose-1,6-bisphosphatase (FBPase), with a Trx m/ FBPase molar saturation ratio of about 60, compared with 4 for the Trx f/FBPase quotient. Cross-experiments have shown the ability of pea Trx m to activate the spinach chloroplast FBPase, results that are in contrast with those in spinach found by P. Schürmann, K. Maeda, and A. Tsugita ([1981] Eur J Biochem 116: 37-45), who did not find Trx m efficiency in FBPase activation. This higher efficiency of pea Trx m could be related to the presence of four basic residues (arginine-37, lysine-70, arginine-74, and lysine-97) flanking the regulatory cluster; spinach Trx m lacks the positive charge corresponding to lysine-70 of pea Trx m. This has been confirmed by K70E mutagenesis of pea Trx m, which leads to a 50% decrease in FBPase activation. 相似文献
10.
Purification and binding features of a pea fructose-1,6-bisphosphatase domain involved in the interaction with thioredoxin f 总被引:1,自引:0,他引:1
Rosario Hermoso Juan José Lázaro Ana Chueca Mariam Sahrawy Julio López-Gorgé 《Physiologia plantarum》1999,105(4):756-762
We previously demonstrated that a cluster in the available 150 Asn-170 Glu region of pea chloroplast fructose-1,6-bisphosphatase (FBPase) could be involved in its interaction with the physiological modulator thioredoxin (Trx). Using as template a cDNA coding for pea chloroplast FBPase, a DNA insert coding for a 19 amino acid fragment ( 149 Pro-167 Gly) was amplified by PCR. After insertion in the pGEX-4T vector-1, it was expressed in Escherichia coli as a fusion protein (GST-19) with the vector-coded glutathione transferase (GST). This protein appears in the supernatant of cell lysates, and was purified to homogeneity. After thrombin digestion, the 19 amino acid insert was isolated as a polypeptide which displayed a positive reaction against pea chloroplast FBPase antibodies. GST-19 linked to glutathione-Sepharose beads, but not the GST, strongly interacts with pea Trx f , suggesting that this binding depends on the 19 amino acid insert. ELISA and Western blot experiments also demonstrate the existence of a GST-19-Trx f interaction, as well as a negligible quantity of Trx f bound by the vector-coded GST. Putative competitive inhibition assays of FBPase activity carried out in the presence of increasing concentrations of the 19 amino acid insert do not demonstrate any enzyme inhibition. On the contrary, this protein fragment enhances the enzyme activity proportionally to its concentration in the assay mixture. This indicates that the FBPase-Trx f binding promotes some type of structural modification of the Trx molecule, or of the FBPase-Trx docking site, thus facilitating the reductive modulation of FBPase. 相似文献
11.
Javier López Jaramillo Ana Chueca Mariam Sahrawy & Julio López Gorgé 《The Plant journal : for cell and molecular biology》1998,15(2):155-163
Two hybrid thioredoxins (Trx) have been constructed from cDNA clones coding for pea chloroplast Trxs m and f. The splitting point was the AvaII site situated between the two cysteines of the regulatory cluster. One hybrid, Trx m/f, was purified from Escherichia coli-expressed cell lysates as a high yielding 12 kDa protein. Western blot analysis showed a positive reaction with antibodies against pea Trxs m and f and, like the parenteral pea Trx m, displayed an acidic pI (5.0) and a high thermal stability. In contrast, the opposite hybrid Trx f/m appeared in E. coli lysates as inclusion bodies, where it was detected by Western blot against pea Trx f antibodies as a 40 kDa protein. Trx f/m was very unstable, sensitive to heat denaturation, and could not be purified. Trx m/f showed a higher affinity for pea chloroplast fructose-1,6-bisphosphatase (FBPase) and a smaller Trx/FBPase saturation ratio than both parenterals; however, the FBPase catalytic rate was lower than that with Trxs m and f. Surprisingly, the hybrid Trx m/f appeared to be incompetent in the activation of pea NADP-malate dehydrogenase. Computer-assisted models of pea Trxs m and f, and of the chimeric Trx m/f, showed a change in the orientation of the α4-helix in the hybrid, which could explain the kinetic modifications with respect to Trxs m and f. We conclude that the stability of Trxs lies on the N-side of the regulatory cluster, and is associated with the acidic character of this fragment and, as a consequence, with the acidic pI of the whole molecule. In contrast, the ability of FBPase binding and enzyme catalysis depends on the structure on the C-side of the regulatory cysteines. 相似文献
12.
Chloroplast fructose 1,6-bisphosphatase (FBPase) is activated by reduction of a regulatory disulfide through thioredoxin f (Trx f). In the course of this reduction a transient mixed disulfide is formed linking covalently Trx f with FBPase, which possesses three Cys on a loop structure, two of them forming the redox-active disulfide bridge. The goal of this study was to identify the Cys involved in the transient mixed disulfide. To stabilize this reaction intermediate, mutant proteins with modified active sites were used. We identified Cys-155 of the FBPase as the one engaged in the formation of the mixed disulfide intermediate with Cys-46 of Trx f. 相似文献
13.
Majerová et al. (Plant Mol Biol, 2011) have recently reported that a considerable fraction of cytosines at tobacco telomeres is methylated. Although the data presented
in this report indicate that tobacco telomeric sequences undergo certain levels of DNA methylation, it is not clear whether
the methylated sequences are at telomeres, at internal chromosomal loci or at both. 相似文献
14.
Astaxanthin accumulation by green microalgae is a natural phenomenon known as red snows and blood rains. The fact that astaxanthin synthesis requires oxygen, NADPH and Fe2+ led Cunningham and Gantt [Annu. Rev. Plant Physiol. Plant Mol. Biol. 49 (1998) 557–583] to propose that a cytochrome P450-dependent enzyme might be involved in the transformation of β-carotene to astaxanthin. In Haematococcus only esterified astaxanthin molecules accumulate, but it is not determined whether a fatty acid synthesis should occur simultaneously to allow pigment accumulation. The aim of this contribution was to answer these two questions using specific inhibitors of β-carotene (norflurazon) and fatty acid (cerulenin) synthesis, and of cytochrome P450 enzyme activity (ellipticine). 相似文献
15.
The maize cysteine protease complex, which required SDS for its activation in vitro, is a 179 kDa trimeric complex (P-I)3 of a cysteine protease (P) [EC 3.4.22] and a cysteine protease inhibitor (I), cystatin [Yamada et al. (1998) Plant Cell Physiol. 39: 106, Yamada et al. (2000) Plant Cell Physiol. 41: 185]. Here, we show the mechanism of the SDS-dependent activation of the trimeric (P-I) complex and stabilization of the activated protease by its specific substrates. The cystatin-free cysteine protease isolated by preparative SDS-PAGE was still specifically activated by SDS, and its profile of SDS-dependency was exactly the same as that of the trimeric (P-I) complex. It is, therefore, evident that an SDS-dependent conformational change of the protease itself, rather than the release of cystatin from the complex, is crucial for the activation. Pre-treatment analysis with SDS revealed that SDS was required for the initiation of the activation of the trimeric (P-I) complex. Furthermore, we found that once the protease was activated, if there was no substrate, it was rapidly inactivated under optimum conditions of proteolysis, and showed that such inactivation was not due to autolysis of the protease. In contrast, addition of specific substrates prevented the inactivation, and thus we presumed that the activity of the cysteine protease is regulated by both activation by conformational change and rapid inactivation after consumption of substrates. 相似文献
16.
UDP-sugar contents were measured using high performance liquid chromatography and gas chromatography during the cell cycle in a synchronous culture of Catharanthus roseus (L.) G. Don. UDP-glucose, UDP-galactose, UDP-glucuronic acid, UDP-xylose and UDP-arabinose could be determined, and 75–90% of the UDP-sugars were UDP-glucose. The contents of UDP-glucose and UDP-galactose increased in the late G2-M and the late S-M phases, respectively, whereas UDP-glucoronic acid and UDP-arabinose increased in amount in the G1 phase. These changes in the levels of UDP-sugars during the cell cycle generally correlated well with the changes in cell wall constituents and in the activities of the enzyme involved in synthesis and interconversion of UDP-sugars reported by S. Amino et al. (Physiol. Plant. 1985. 64: 111–117). 相似文献
17.
Light-regulated phosphorylation of maize phosphoenolpyruvate carboxykinase plays a vital role in its activity 总被引:2,自引:0,他引:2
Qing Chao Xiao-Yu Liu Ying-Chang Mei Zhi-Fang Gao Yi-Bo Chen Chun-Rong Qian Yu-Bo Hao Bai-Chen Wang 《Plant molecular biology》2014,85(1-2):95-105
Phosphoenolpyruvate carboxykinase (PEPCK)—the major decarboxylase in PEPCK-type C4 plants—is also present in appreciable amounts in the bundle sheath cells of NADP-malic enzyme-type C4 plants, such as maize (Zea mays), where it plays an apparent crucial role during photosynthesis (Wingler et al., in Plant Physiol 120(2):539–546, 1999; Furumoto et al., in Plant Mol Biol 41(3):301–311, 1999). Herein, we describe the use of mass spectrometry to demonstrate phosphorylation of maize PEPCK residues Ser55, Thr58, Thr59, and Thr120. Western blotting indicated that the extent of Ser55 phosphorylation dramatically increases in the leaves of maize seedlings when the seedlings are transferred from darkness to light, and decreases in the leaves of seedlings transferred from light to darkness. The effect of light on phosphorylation of this residue is opposite that of the effect of light on PEPCK activity, with the decarboxylase activity of PEPCK being less in illuminated leaves than in leaves left in the dark. This inverse relationship between PEPCK activity and the extent of phosphorylation suggests that the suppressive effect of light on PEPCK decarboxylation activity might be mediated by reversible phosphorylation of Ser55. 相似文献
18.
Despite mitochondria and chloroplasts having their own genome, 99% of mitochondrial proteins (Rehling et al., Nat Rev Mol
Cell Biol 5:519–530, 2004) and more than 95% of chloroplast proteins (Soll, Curr Opin Plant Biol 5:529–535, 2002) are encoded by nuclear DNA, synthesised in the cytosol and imported post-translationally. Protein targeting to these organelles
depends on cytosolic targeting factors, which bind to the precursor, and then interact with membrane receptors to deliver
the precursor into a translocase. The molecular chaperones Hsp70 and Hsp90 have been widely implicated in protein targeting
to mitochondria and chloroplasts, and receptors capable of recognising these chaperones have been identified at the surface
of both these organelles (Schlegel et al., Mol Biol Evol 24:2763–2774, 2007). The role of these chaperone receptors is not fully understood, but they have been shown to increase the efficiency of protein
targeting (Young et al., Cell 112:41–50, 2003; Qbadou et al., EMBO J 25:1836–1847, 2006). Whether these receptors contribute to the specificity of targeting is less clear. A class of chaperone receptors bearing
tetratricopeptide repeat domains is able to specifically bind the highly conserved C terminus of Hsp70 and/or Hsp90. Interestingly,
at least of one these chaperone receptors can be found on each organelle (Schlegel et al., Mol Biol Evol 24:2763–2774, 2007), which suggests a universal role in protein targeting for these chaperone receptors. This review will investigate the role
that chaperone receptors play in targeting efficiency and specificity, as well as examining recent in silico approaches to find novel chaperone receptors. 相似文献
19.
This work was done to test claims (Sangwan and Singh, Physiol. Plant. 73: 21–26) that the developing endosperm of wheat ( Triticum aestivum L.) contains a cytosolic and a plastidic fructose- 1,6-bisphosphatase (EC 3.1.3.11; FBPase). Repetition of the procedure of Sangwan and Singh with extracts of developing endosperm of Triticum aestivum cv. Mercia produced two peaks of apparent FBPase activity on elution from DEAE-cellulose. Both peaks showed high activity of pyrophosphate:fructose-6-phos-phate 1-phosphotransferase [EC 2.7.1.90; PFK(PPi )]. The apparent FBPase activity in both peaks was stimulated by 20 μ M fructose-2,6-bisphosphate and inhibited by antibodies to PFK(PPi ). Antibody to plastidic FBPase did not react positively in an immunoblot analysis with any protein of Mr comparable to that of known FBPase in either peak. It is argued that the ability of each peak to convert fructose-1,6-bisphosphate to fructose-6-phosphate was due to PFK(PPi ). and that there remains no substantiated evidence for the presence of a plastidic FBPase in the developing endosperm of wheat. 相似文献
20.
Aspartate transcarbamylase (EC 2.1.3.2) from E. coli is a multimeric enzyme consisting of two catalytic subunits and three regulatory subunits whose activity is regulated by subunit interactions. Differential scanning calorimetric (DSC) scans of the wild-type enzyme consist of two peaks, each comprised of at least two components, corresponding to denaturation of the catalytic and regulatory subunits within the intact holoenzyme (Vickers et al., J. Biol. Chem. 253 (1978) 8493; Edge et al., Biochemistry 27 (1988) 8081). We have examined the effects of nine single-site mutations in the catalytic chains. Three of the mutations (Asp-100-Gly, Glu-86-Gln, and Arg-269-Gly) are at sites at the C1: C2 interface between c chains within the catalytic subunit. These mutations disrupt salt linkages present in both the T and R states of the molecule (Honzatko et al., J. Mol. Biol. 160 (1982) 219; Krause et al., J. Mol. Biol. 193 (1987) 527). The remainder (Lys-164-Ile, Tyr-165-Phe, Glu-239-Gln, Glu-239-Ala, Tyr-240-Phe and Asp-271-Ser) are at the C1: C4 interface between catalytic subunits and are involved in interactions which stabilize either the T or R state. DSC scans of all of the mutants except Asp-100-Gly and Arg-269-Gly consisted of two peaks. At intermediate concentrations, Asp-100-Gly and Arg-269-Gly had only a single peak near the Tm of the regulatory subunit transition in the holoenzyme, although their denaturational profiles were more complex at high and low protein concentrations. The catalytic subunits of Glu-86-Gln, Lys-164-Ile and Asp-271-Ser appear to be significantly destabilized relative to wild-type protein while Tyr-165-Phe and Tyr-240-Phe appear to be stabilized. Values of delta delta G degree cr, the difference between the subunit interaction energy of wild-type and mutant proteins, evaluated as suggested by Brandts et al. (Biochemistry 28 (1989) 8588) range from -3.7 kcal mol-1 for Glu-86-Gln to 2.4 kcal mol-1 for Tyr-165-Phe. 相似文献