Biomedical subjects
G Sarath
Publications and source records attributed to G Sarath.
Characterization of recombinant soybean leghemoglobin a and apolar distal histidine mutants.
The cDNA for soybean leghemoglobin a (Lba) was cloned from a root nodule cDNA library and expressed in Escherichia coli. The crystal structure of the ferric acetate complex of recombinant wild-type Lba was determined at a resolution of 2.2 A. Rate constants for O2, CO and NO binding to recombinant Lba are identical with those of native soybean Lba. Rate constants for hemin dissociation and auto-oxidation of wild-type Lba were compared with those of sperm whale myoglobin. At 37 degrees C and pH 7, soybean Lba is much less stable than sperm whale myoglobin due both to a fourfold higher rate of auto-oxidation and to a approximately 600-fold lower affinity for hemin. The role of His61(E7) in regulating oxygen binding was examined by site-directed mutagenesis. Replacement of His(E7) with Ala, Val or Leu causes little change in the equilibrium constant for O2 binding to soybean Lba, whereas the same mutations in sperm whale myoglobin cause 50 to 100-fold decreases in K(O2). These results show that, at neutral pH, hydrogen bonding with His(E7) is much less important in regulating O2 binding to the soybean protein. The His(E7) to Phe mutation does cause a significant decrease in K(O2) for Lba, apparently due to steric hindrance of the bound ligand. The rate constants for O2 dissociation from wild-type and native Lba decrease significantly with decreasing pH. In contrast, the O2 dissociation rate constants for mutants with apolar E7 residues are independent of pH, suggesting that hydrogen bonding to the distal histidine residue in the native protein is enhanced under acid conditions. All of these results support the hypothesis that the high affinity of Lba for oxygen and other ligands is determined primarily by enhanced accessibility and reactivity of the heme group.
Purification of a protein phosphatase from chloroplast stroma capable of dephosphorylating the light-harvesting complex-II.
A protein phosphatase was purified from the stroma of Pea (Pisum sativum L.) chloroplasts that is capable of dephosphorylating synthetic phosphopeptides. Following chromatographic purification of greater than 400-fold, two-dimensional electrophoresis indicated that the stromal protein phosphatase is a 29-kD protein. A similar molecular size was determined for the protein-phosphatase activity using gel-permeation chromatography, indicating that the stromal protein phosphatase is probably a monomer. The purified enzyme was able to dephosphorylate synthetic phosphopeptides, which mimic the thylakoid light-harvesting complex II (LHC-II) N terminus, as well as LHC-II in thylakoid membranes, but did not dephosphorylate the major 64-kD phosphoprotein in the stroma. The stromal protein phosphatase did not discriminate between dephosphorylation of phosphothreonine and phosphoserine residues in synthetic peptide substrates, providing further evidence that this enzyme is distinct from the protein phosphatase localized in thylakoid membranes. The exact physiological role of the stromal protein phosphatase has yet to be determined, but it may function in the dephosphorylation of LHC-II.
Molecular cloning of the cowpea leghemoglobin II gene and expression of its cDNA in Escherichia coli. Purification and characterization of the recombinant protein.
Cowpea (Vigna unguiculata) nodules contain three leghemoglobins (LbI, LbII, and LbIII) that are encoded by at least two genes. We have cloned and sequenced the gene that encodes for LbII (lbII), the most abundant Lb in cowpea nodules, using total DNA as the template for PCR. Primers were designed using the sequence of the soybean lbc gene. The lbII gene is 679 bp in length and codes for a predicted protein of 145 amino acids. Using sequences of the cowpea lbII gene for the synthesis of primers and total nodule RNA as the template, we cloned a cDNA for LbII into a constitutive expression vector (pEMBL19+) and then expressed it in Escherichia coli. Recombinant LbII (rLbII) and native LbII (nLbII) from cowpea nodules were purified to homogeneity using standard techniques. Properties of rLbII were compared with nLbII by partially sequencing the proteins and by sodium dodecyl sulfate- and isoelectric focusing polyacrylamide gel electrophoresis, western-blot analysis using anti-soybean Lba antibodies, tryptic and chymotryptic mapping, and spectrophotometric techniques. The data showed that the structural and spectral characteristics of rLbII and nLbII were similar. The rLbII was reversibly oxygenated/deoxygenated, showing that it is a functional hemoglobin.
Soybean root nodule acid phosphatase.
Acid phosphatases are ubiquitous enzymes that exhibit activity against a variety of substrates in vitro, although little is known about their intracellular function. In this study, we report the isolation, characterization, and partial sequence of the major acid phosphatase from soybean (Glycine max L.) root nodules. The phosphatase was purified predominantly as a heterodimer with subunits of 28 and 31 kD; homodimers of both subunits were also observed and exhibited phosphatase activity. In addition to the general phosphatase substrate, p-nitrophenyl phosphate, the heterodimeric form of the enzyme readily hydrolyzed 5'-nucleotides, flavin mononucleotide, and O-phospho-L-Tyr. Low or negligible activity was observed with ATP or polyphosphate. Purified nodule acid phosphatase was stimulated by magnesium, inhibited by calcium and EDTA, and competitively inhibited by cGMP and cAMP with apparent Ki values of 7 and 12 microM, respectively. Partial N-terminal and internal sequencing of the nodule acid phosphatase revealed homology to the soybean vegetative storage proteins. There was a 17-fold increase in enzyme activity and a noticeable increase in protein levels detected by immunoblotting methods during nodule development. Both of these parameters were low in young nodules and reached a peak in mature, functional nodules, suggesting that this enzyme is important for efficient nodule metabolism.
Rice hemoglobins. Gene cloning, analysis, and O2-binding kinetics of a recombinant protein synthesized in Escherichia coli.
Although nonsymbiotic hemoglobins (Hbs) are found in different tissues of dicots and monocots, very little is known about hb genes in monocots and the function of Hbs in nonsymbiotic tissues. We report the cloning and analysis of two rice (Oryza sativa L.) hb genes, hb1 and hb2, that code for plant Hbs. Rice hb1 and hb2 genes contain four exons and three introns, as with all of the known plant hb genes. At least three copies of the hb gene were detected in rice DNA, and analysis of gene expression shows that hb1 and hb2 are expressed in leaves but only hb1 is expressed in roots. A cDNA for rice Hb1 was expressed in Escherichia coli, and the recombinant Hb (rHb1) shows an unusually high affinity for O2 because of a very low dissociation constant. The absorbance spectra of the ferric and deoxyferrous rHb1 indicate that, in contrast to symbiotic Hbs, a distal ligand is coordinated to the ligand-binding site. Mutation of the distal His demonstrates that this residue coordinates the heme Fe of ferric and deoxyferrous rHb1 and stabilizes O2 in oxy-rHb1. The biochemical properties of rice rHb1 suggest that this protein probably does not function to facilitate the diffusion of O2.
Kinetic analysis of the non-phosphorylated, in vitro phosphorylated, and phosphorylation-site-mutant (Asp8) forms of intact recombinant C4 phosphoenolpyruvate carboxylase from sorghum.
Steady-state kinetic analyses were performed on the non-phosphorylated, in vitro phosphorylated and phosphorylation-site mutant (Ser8-->Asp) forms of purified recombinant sorghum C4 phosphoenolpyruvate (P-pyruvate) carboxylase (EC 4.1.1.31) containing an intact N-terminus. Significant differences in certain kinetic parameters were observed between these three enzyme forms when activity was assayed at a suboptimal but near-physiological pH (7.3), but not at optimal pH (8.0). Most notably, at pH 7.3 the apparent Ki for the negative allosteric effector L-malate was 0.17 mM, 1.2 mM and 0.45 mM while the apparent Ka for the positive allosteric effector glucose 6-phosphate (Glc6P) at 1 mM P-pyruvate was 1.3 mM, 0.28 mM and 0.45 mM for the dephosphorylated, phosphorylated and mutant forms of the enzyme, respectively. These and related kinetic analyses at pH 7.3 show that phosphorylation of C4 P-pyruvate carboxylase near its N-terminus has a relatively minor effect on V and Km (total P-pyruvate) but has a dramatic effect on the extent of activation by Glc6P, type of inhibition by L-malate and, most especially, Ka (Glc6P) and Ki (L-malate). Thus, regulatory phosphorylation profoundly influences the interactive allosteric properties of this cytosolic C4-photosynthesis enzyme.
Peptide motif of the cattle MHC class I antigen BoLA-A11.
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Prostaglandin biosynthesis and subcellular localization of prostaglandin H synthase activity in the lone star tick, Amblyomma americanum.
We report on prostaglandin (PG) biosynthesis in the lone star tick, Amblyomma americanum. In vitro preparations of whole female ticks and internal tissues were competent to biosynthesize four PGs: PGA2/PGB2, PGD2, PGE2, PGF2 alpha. PGA2/PGB2 was the major product under optimal conditions. PG biosynthesis by whole tick and internal tissues were sensitive to incubation conditions including, protein concentration, time, temperature, pH, and presence of a co-factor cocktail composed of reduced glutathione, hydroquinone, and hemoglobin. Under standard assay conditions, 2 mg/ml protein were incubated at pH 8.0 for 2 min at 32 degrees C. PG biosynthesis was inhibited by indomethacin, a potent cyclooxygenase inhibitor in mammalian systems. Internal tissue preparations were fractionated into cytosolic and microsomal preparations by ultracentrifugation. PG biosynthetic activity was detected in both fractions. The subcellular distribution of PG biosynthetic activity in ticks is similar to other invertebrates, but quite different from mammals, in which PG biosynthetic activity is almost exclusively localized in the microsomal fractions. PGH synthase-2 was detected in the microsomal fraction on western blot analysis. These results suggest that the lone star tick is competent to biosynthesize PGs. These compounds may contribute to the success of tick feeding ecology by attenuating the defense responses of vertebrate hosts during lengthy feeding periods.
Cloning and sequence analysis of a cDNA encoding ferric leghemoglobin reductase from soybean nodules.
A cDNA encoding soybean (Glycine max [L.] Merr) ferric leghemoglobin reductase (FLbR), an enzyme that is postulated to play an important role in maintaining leghemoglobin in its functional ferrous state, has been cloned and characterized. A group of highly degenerate oligonucleotides deduced from the N-terminal amino acid sequence of FLbR was used to prime the polymerase chain reaction (PCR) on soybean nodule mRNA and cDNA. A full-length clone of FLbR cDNA was isolated by screening a lambda gt11 soybean nodule cDNA library using the specific PCR-amplified FLbR cDNA fragment as a probe. The cDNA contained about 1.8 kb and had a coding sequence for 523 amino acids with a predicted molecular mass of 55,729 D, which included a putative 30-residue signal peptide and a 493-residue mature protein. Computer-aided analysis of the deduced FLbR amino acid sequence showed considerable homology (varied from 20-50% with enzymes and species) to dihydrolipoamide dehydrogenase (EC 1.8.1.4), glutathione reductase (EC 1.6.4.2), mercuric reductase (EC 1.16.1.1), and trypanothione reductase (EC 1.6.4.8) in a superfamily of pyridine nucleotide-disulfide oxidoreductases from various organisms. Northern blot analysis using FLbR cDNA as a probe showed that the FLbR gene was expressed in soybean nodules, leaves, roots, and stems, with a greater level of expression in nodules and leaves than in roots and stems. Southern blot analysis of the genomic DNA showed the presence of two homologous FLbR genes in the soybean genome.
Phosphopeptides as substrates for thylakoid protein phosphatase activity.
Lack of a suitable substrate has been a major obstacle in studying the chloroplastic thylakoid membrane protein phosphatase activity. In this study, the suitability of synthetic phosphopeptides for this purpose was investigated. Phosphothreonine-containing phosphopeptides mimicking the N-terminal phosphorylation site of the major thylakoid phosphoprotein, the light-harvesting chlorophyll a/b-binding protein (LHCP-II), were dephosphorylated by isolated peak thylakoid membranes. Phosphopeptides representing unrelated sequences or in which the target phosphothreonine had been changed to a phosphoserine were not dephosphorylated. The dephosphorylation of phosphopeptides by thylakoid membranes was similar to the dephosphorylation of endogenous LHCP-II in its pH-dependence profile, sensitivity to inhibitors, and bivalent cation requirement. The same phosphopeptide analogs of the LHCP-II phosphorylation site inhibited endogenous LHCP-II dephosphorylation in isolated thylakoids, whereas the dephospho-analogs and nonsubstrate phosphopeptides had no effect. Collectively, these results suggest that phosphopeptides mimicking a thylakoid phosphoprotein dephosphorylation site can be exploited for further study of the thylakoid protein phosphatase activity.
An engineered change in the L-malate sensitivity of a site-directed mutant of sorghum phosphoenolpyruvate carboxylase: the effect of sequential mutagenesis and S-carboxymethylation at position 8.
A recombinant, site-directed mutant form of sorghum phosphoenolpyruvate carboxylase (PEPC), in which the phosphorylatable serine residue (Ser-8) was changed to cysteine (S8C), was chemically modified by iodoacetic acid and iodoacetamide for the purpose of testing the effect of introducing a negative charge at position 8. S-Carboxymethylation of the Cys-8 enzyme by iodoacetic acid decreased its sensitivity to L-malate from an I0.5 (50% inhibition) value of 0.12 to 0.35 mM at pH 7.3 when the active-site domain was protected during modification by the substrate phosphoenolpyruvate (PEP). In contrast, neither S-carboxymethylation of the wild-type enzyme nor modification of the mutant enzyme by iodoacetamide caused any change in the enzyme's sensitivity to L-malate. The modified, substrate-protected forms of the Ser-8 and S8C PEPCs had Km(total PEP) and Vmax values virtually identical to those of the unmodified control enzymes. Similar specific increases in the I0.5 value of L-malate have been reported previously for in vitro phosphorylated leaf and recombinant Ser-8 PEPCs, the site-directed mutant Asp-8 enzyme, and C4-leaf PEPC purified from light-adapted sorghum or maize (in vivo phospho-form). Therefore, these data from different but complementary experimental approaches provide convincing evidence that the effect of phosphorylation of Ser-8 on the L-malate sensitivity of sorghum C4-PEPC is caused by the introduction of negative charge into this N-terminal regulatory domain.
Site-directed mutagenesis of the phosphorylatable serine (Ser8) in C4 phosphoenolpyruvate carboxylase from sorghum. The effect of negative charge at position 8.
The properties of the dephospho and in vitro phosphorylated forms of recombinant sorghum phosphoenolpyruvate carboxylase have been compared with those of the authentic dark (dephospho) and light (phospho) leaf enzyme forms and two mutant enzymes in which the phosphorylatable serine residue (Ser8) has been changed by site-directed mutagenesis to Cys (S8C) or Asp (S8D). Kinetic analysis of the purified recombinant, mutant, and leaf enzyme forms at pH 8.0 indicated virtually identical Vmax, apparent Km (phosphoenolpyruvate), and half-maximal activation (glucose 6-P) values of about 44 units/mg, 1.1 mM, and 0.23 mM, respectively. In contrast, the Ser8, S8C, and dark leaf enzymes were about 3-fold more sensitive to inhibition by L-malate at pH 7.3 than the Ser8-P, S8D, and light leaf enzyme forms. These comparative results indicate that: (i) Ser8 is an important determinant in the regulation of sorghum phosphoenolpyruvate carboxylase activity by negative (L-malate), but not positive (glucose 6-phosphate) metabolite effectors, (ii) phosphorylation of this target residue can be functionally mimicked by Asp, but not Cys, and (iii) negative charge contributes to the effect of regulatory phosphorylation on this C4-photosynthesis enzyme.
Bivalent ACTH antagonists: influence of peptide and spacer components on potency enhancement.
The antagonist potency of a series of bivalent adrenocorticotropic hormone (ACTH) peptides was examined using suspensions of inner zone rat adrenocortical cells. Bivalent antagonists were prepared by bis(maleimide) covalent cross-linking of carboxyl terminal cysteine sulfhydryl groups of synthetic ACTH peptides, Cys25 ACTH(7-25) and Cys39 ACTH(7-39). Antagonist potency enhancement was defined by shifts in ACTH(1-39) concentration-steroidogenic response curves relative to monovalent antagonist analogs. The EC50 values measured in the presence of 0.5 microM monovalent antagonist were 110 +/- 28 pM for Cys25 ACTH(7-25)-S-N-ethylsuccinimide and 44 +/- 9 pM for Cys39 ACTH(7-39)-S-N-ethylsuccinimide. Some bivalent ACTH antagonists displayed much greater antagonist potency than their monovalent analogs, which supports the findings of Stolz and Fauchere (Helv Chim Acta 71: 1421-1428, 1988). The level of potency enhancement, however, was found to be dependent upon the spacer used to link receptor binding domains and the length of the ACTH peptide used in bivalent antagonist synthesis. The most potent inhibitor, bis(Cys25 ACTH(7-25)-S-succinimidopropionyl)2-hydroxy-1,3-propanediamine, was 28 times more potent than its monovalent analog, Cys25 ACTH(7-25)-S-N-ethylsuccinimide. However, a bivalent Cys25 ACTH(7-25) peptide containing two bis(succinimidopropionyl)2-hydroxy-1,3-propanediamine spacers that had been linked end-to-end via dithioerythritol showed no potency enhancement. Cys25 ACTH(7-25) based peptides containing one receptor binding domain and having the structure peptide-spacer-cysteine displayed no enhancement in antagonist potency. Bivalent Cys39 ACTH(7-39) linked by bis(succinimidopropionyl)2-hydroxy-1,3-propanediamine spacer exhibited only 4-fold enhancement in antagonist potency relative to Cys39 ACTH(7-39)-S-N-ethylsuccinimide. We therefore conclude that the potency enhancement observed with bivalent ACTH peptides: (1) is optimal with spacers less than approximately 40 A in length, (2) is not due to direct interactions between the spacer and cell surface, and (3) is dependent on the length of the ACTH peptide component. In addition, these results indicate that electrostatic interaction between bivalent ACTH peptides and plasma membrane lipids does not adequately account for the potency enhancements observed.
High-performance liquid chromatographic separation of leghemoglobins from soybean root nodules.
A crude fraction of soybean nodule ferri-leghemoglobin was absorbed onto a commercial DEAE HPLC column at pH 8.0, and resolved into eight isoprotein fractions. The identity of the leghemoglobins were determined by their order of elution from the DEAE column and by isoelectric focusing, using isoprotein standards isolated by conventional procedures. The three isoproteins of the c complex, c1, c2, c3, were not resolved. Unexpected heme containing proteins eluted just after leghemoglobin a and the c complex. These components possessed proteins similar to leghemoglobin a and the c complex, respectively, as judged by isoelectric focusing and absorbance spectra of the ferri and ferrous forms. The components designated leghemoglobin a' and leghemoglobin c' were also differentiated from leghemoglobin a and c by reverse-phase HPLC in a C18 column. Amounts of protein for the DEAE HPLC column ranged from 10 micrograms to 20 mg and sample volumes ranged from 2 to 250 microliters. The time required for chromatography varied depending on the gradient used, but never exceeded 40 min for samples up to 5 mg protein or 120 min for samples containing 5 to 20 mg protein. Due to the sensitivity of detection at 403 nm and leghemoglobins being the predominant chromophore at that wavelength, it was possible to quantitate levels of individual leghemoglobins in samples extracted from single nodules (ca. 15 to 65 mg fresh weight tissue). Quantitation was performed by interfacing the spectrophotometer output (10 mV) to a microcomputer and using commercially available software.
Bacteroids Are Stable during Dark-Induced Senescence of Soybean Root Nodules.
Physiological and biochemical markers of metabolic competence were assayed in bacteroids isolated from root nodules of control, dark-stressed, and recovered plants of Glycine max Merr. cv ;Woodworth.' Nitrogenase-dependent acetylene reduction by the whole plant decreased to 8% of control rates after 4 days of dark stress and could not be detected in plants dark stressed for 8 days. However, in bacteroids isolated anaerobically, almost 50% of initial acetylene reduction activity remained after 4 days of dark stress but was totally lost after 8 days of dark stress. Bacteroid acetylene reduction activity recovered faster than whole plant acetylene reduction activity when plants were dark stressed for 8 days and returned to a normal light regimen. Significant changes were not measured in bacteroid respiration, protein content, sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles, or in bacteroid proteolytic activity throughout the experiment. Immunoblots of bacteroid extracts revealed the presence of nitrogenase component II in control, 4-day dark-stressed, and 8-day dark-stressed plants that were allowed to recover under a normal light regimen, but not in 8-day dark-stressed plants. Our data indicate that dark stress does not greatly affect bacteroid metabolism or induce bacteroid senescence.