Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier: a spectroscopic and molecular modelling study

The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan in the Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absence of the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trp are centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solvent exposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition of substrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of the oxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with the experimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenching of Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trp and does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cys and evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122 is masked by the substrate, likely through a substrate-induced conformational change.     

The mitochondrial oxoglutarate carrier: cysteine-scanning mutagenesis of transmembrane domain IV and sensitivity of Cys mutants to sulfhydryl reagents.

Using a functional mitochondrial oxoglutarate carrier mutant devoid of Cys residues (C-less carrier), each amino acid residue in transmembrane domain IV and flanking hydrophilic loops (from T179 to S205) was replaced individually with Cys. The great majority of the 27 mutants exhibited significant oxoglutarate transport in reconstituted liposomes as compared to the activity of the C-less carrier. In contrast, Cys substitution for G183, R190, Q198, and Y202, in either C-less or wild-type carriers, yielded molecules with complete loss of oxoglutarate transport activity. G183 and R190 could be partially replaced only by Ala and Lys, respectively, whereas Q198 and Y202 were irreplaceable with respect to oxoglutarate transport. Of the single-Cys mutants tested, only T187C, A191C, V194C, and N195C were strongly inactivated by N-ethylmaleimide and by low concentrations of methanethiosulfonate derivatives. Oxoglutarate protects Cys residues at positions 187, 191, and 194 against reaction with N-ethylmaleimide. These positions as well as the residues found to be essential for the carrier activity, except Y202 which is located in the extramembrane loop IV-V, reside on the same face of transmembrane helix IV, probably lining part of a water-accessible crevice or channel between helices of the oxoglutarate carrier.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

Effects of orally administrated amino acids on myofibrillar proteolysis in chicks

We examined the effects of orally administrated amino acids on myfibrillar proteolysis in food-deprived chicks. Plasma N(tau)-methylhistidine concentration, as an index of myofibrillar proteolysis, was decreased by the administration of Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg but not by Asp, Val, Phe, Tyr or His to chicks. Orally administrated Cys was fatal to chicks. These results indicate that oral Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg administration suppressed myofibrillar proteolysis in chicks.     

Structural basis of hematopoietic prostaglandin D synthase activity elucidated by site-directed mutagenesis

Hematopoietic prostaglandin (PG) D synthase (PGDS) is the first identified vertebrate ortholog in the Sigma class of the glutathione S-transferase (GST) family and catalyzes both isomerization of PGH(2) to PGD(2) and conjugation of glutathione to 1-chloro-2, 4-dinitrobenzene. We introduced site-directed mutations of Tyr(8), Arg(14), Trp(104), Lys(112), Tyr(152), Cys(156), Lys(198), and Leu(199), which are presumed to participate in catalysis or PGH(2) substrate binding based on the crystallographic structure. Mutants were analyzed in terms of structure, GST and PGDS activities, and activation of the glutathione thiol group. Of all the mutants, only Y8F, W104I, K112E, and L199F showed minor but substantial differences in their far-UV circular dichroism spectra from the wild-type enzyme. Y8F, R14K/E, and W104I were completely inactive. C156L/Y selectively lost only PGDS activity. K112E reduced GST activity slightly and PGDS activity markedly, whereas K198E caused a selective decrease in PGDS activity and K(m) for glutathione and PGH(2) in the PGDS reaction. No significant changes were observed in the catalytic activities of Y152F and L199F, although their K(m) for glutathione was increased. Using 5,5'-dithiobis(2-nitrobenzoic acid) as an SH-selective agent, we found that only Y8F and R14E/K did not accelerate the reactivity of the glutathione thiol group under the low reactivity condition of pH 5.0. These results indicate that Lys(112), Cys(156), and Lys(198) are involved in the binding of PGH(2); Trp(104) is critical for structural integrity of the catalytic center for GST and PGDS activities; and Tyr(8) and Arg(14) are essential for activation of the thiol group of glutathione.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

Mutations in Intron 1 and Intron 22 Inversion Negative Haemophilia A Patients from Western India

Despite increased awareness and diagnostic facilities, 70–80% of the haemophilia A (HA) patients still remain undiagnosed in India. Very little data is available on prevalent mutations in HA from this country. We report fifty mutations in seventy one Indian HA patients, of which twenty were novel. Ten novel missense mutations [p.Leu11Pro (p.Leu-8Pro), p.Tyr155Ser (p.Tyr136Ser), p.Ile405Thr (p.Ile386Thr), p.Gly582Val (p.Gly563Val) p.Thr696Ile (p.Thr677Ile), p.Tyr737Cys (p.Tyr718Cys), p.Pro1999Arg (p.Pro1980Arg), p.Ser2082Thr (p.Ser2063Thr), p.Leu2197Trp (p.Leu2178Trp), p.Asp2317Glu (p.Asp2298Glu)] two nonsense [p.Lys396* (p.Lys377*), p.Ser2205* (p.Ser2186*)], one insertion [p.Glu1268_Asp1269ins (p.Glu1249_Asp1250)] and seven deletions [p.Leu882del (p.Leu863del), p.Met701del (p.Met682del), p.Leu1223del (p.Leu1204del), p.Trp1961_Tyr1962del (p.Trp1942_Tyr1943del) p.Glu1988del (p.Glu1969del), p.His1841del (p.His1822del), p.Ser2205del (p.Ser2186del)] were identified. Double mutations (p.Asp2317Glu; p.Thr696Ile) were observed in a moderate HA case. Mutations [p. Arg612Cys (p.Arg593Cys), p.Arg2326Gln (p.Arg2307Gln)] known to be predisposing to inhibitors to factor VIII (FVIII) were identified in two patients. 4.6% of the cases were found to be cross reacting material positive (CRM+ve). A wide heterogeneity in the nature of mutations was seen in the present study which has been successfully used for carrier detection and antenatal diagnosis in 10 families affected with severe to moderate HA.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

慈菇蛋白酶抑制剂A和B中Trp残基周围构象与酶抑制专一性的关系

通过定点诱变结合荧光光谱学方法研究了慈菇蛋白酶抑制剂A和B(APIA和APIB)Trp残基周围构象与酶抑制专一性之间的关系。研究表明APIB中的两个Trp残基 (93和 12 2位 )所处环境的疏水性要比APIA中的强。Trp定点诱变研究表明 ,在APIB中 ,Trp12 2 周围环境的疏水性要比Trp93 强。用Ser和Leu分别替代 82位Leu和 87位Arg ,使APIB中色氨酸荧光特性变得与APIA的基本相同 ,同时还发现其酶的抑制专一性也变得趋近APIA的 ,暗示Trp周围的构象与酶抑制剂的抑制专一性有关。     

The hydrophobic residues phenylalanine 184 and leucine 187 in the type-1 parathyroid hormone (PTH) receptor functionally interact with the amino-terminal portion of PTH-(1-34).

Recent mutagenesis and cross-linking studies suggest that three regions of the PTH-1 receptor play important roles in ligand interaction: (i) the extreme NH(2)-terminal region, (ii) the juxtamembrane base of the amino-terminal extracellular domain, and (iii) the third extracellular loop. In this report, we analyzed the second of these segments in the rat PTH-1 receptor (residues 182-190) and its role in functional interaction with short PTH fragment analogs. Twenty-eight singly substituted PTH-1 receptors were transiently transfected into COS-7 cells and shown to be fully expressed by surface antibody binding analysis. Alanine-scanning analysis identified Phe(184), Arg(186), Leu(187), and Ile(190) as important determinants of maximum binding of (125)I-labeled bovine PTH-(1-34) and (125)I-labeled bovine PTH-(3-34) and determinants of responsiveness to the NH(2)-terminal analog, PTH-(1-14) in cAMP stimulation assays. Alanine mutations at these four sites augmented the ability of the COOH-terminal peptide [Glu(22), Trp(23)]PTHrP-(15-36) to inhibit the cAMP response induced by PTH-(1-34). At Phe(184) and Leu(187), hydrophobic substitutions (e.g. Ile, Met, or Leu) preserved PTH-(1-34)-mediated cAMP signaling potency, whereas hydrophilic substitutions (e.g. Asp, Glu, Lys, or Arg) weakened this response by 20-fold or more, as compared with the unsubstituted receptor's response. The results suggest that hydrophobicity at positions occupied by Phe(184) and Leu(187) in the PTH-1 receptor plays an important role in determining functional interaction with the 3-14 portion of PTH.     

Tryptophan 500 and arginine 707 define product and substrate active site binding in soybean lipoxygenase-1

There is much debate whether the fatty acid substrate of lipoxygenase binds "carboxylate-end first" or "methyl-end first" in the active site of soybean lipoxygenase-1 (sLO-1). To address this issue, we investigated the sLO-1 mutants Trp500Leu, Trp500Phe, Lys260Leu, and Arg707Leu with steady-state and stopped-flow kinetics. Our data indicate that the substrates (linoleic acid (LA), arachidonic acid (AA)), and the products (13-(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acid (HPOD) and 15-(S)-hydroperoxyeicosatetraeonic acid (15-(S)-HPETE)) interact with the aromatic residue Trp500 (possibly pi-pi interaction) and with the positively charged amino acid residue Arg707 (charge-charge interaction). Residue Lys260 of soybean lipoxygenase-1 had little effect on either the activation or steady-state kinetics, indicating that both the substrates and products bind "carboxylate-end first" with sLO-1 and not "methyl-end first" as has been proposed for human 15-lipoxygenase.     

Importance of conserved N-domain residues Thr441, Glu442, Lys515, Arg560, and Leu562 of sarcoplasmic reticulum Ca2+-ATPase for MgATP binding and subsequent catalytic steps. Plasticity of the nucleotide-binding site

Nine single mutations were introduced to amino acid residues Thr441, Glu442, Lys515, Arg560, Cys561, and Leu562 located in the nucleotide-binding domain of sarcoplasmic reticulum Ca2+-ATPase, and the functional consequences were studied in a direct nucleotide binding assay, as well as by steady-state and transient kinetic measurements of the overall and partial reactions of the transport cycle. Some partial reaction steps were also examined in mutants with alterations to Phe487, Arg489, and Lys492. The results implicate all these residues, except Cys561, in high affinity nucleotide binding at the substrate site. Mutations Thr441 --> Ala, Glu442 --> Ala, and Leu562 --> Phe were more detrimental to MgATP binding than to ATP binding, thus pointing to a role for these residues in the binding of Mg2+ or to a difference between the interactions with MgATP and ATP. Subsequent catalytic steps were also selectively affected by the mutations, showing the involvement of the nucleotide-binding domain in these reactions. Mutation of Arg560 inhibited phosphoryl transfer but enhanced the E1PCa2 --> E2P conformational transition, whereas mutations Thr441 --> Ala, Glu442 --> Ala, Lys492 --> Leu, and Lys515 --> Ala inhibited the E1PCa2 --> E2P transition. Hydrolysis of the E2P phosphoenzyme intermediate was enhanced in Glu442 --> Ala, Lys492 --> Leu, Lys515 --> Ala, and Arg560 --> Glu. None of the mutations affected the low affinity activation by nucleotide of the phosphoenzyme-processing steps, indicating that modulatory nucleotide interacts differently from substrate nucleotide. Mutation Glu442 --> Ala greatly enhanced reaction of Lys515 with fluorescein isothiocyanate, indicating that the two residues form a salt link in the native protein.     

酸性和碱性酶稳定性机制及其识别

了解酸性和碱性酶稳定性机制并对其进行识别具有重要理论和实践意义。通过分析105条酸性酶和111条碱性酶序列的氨基酸组成, 结果表明: 酸性酶中Trp、Tyr、Thr和Ser的含量明显高于平均值, 而Glu、Lys、Met和Arg的含量则明显低于平均值; 碱性酶中Trp、Ala和Cys的含量略高于平均值, 而Lys、Arg和Glu的含量则略低于平均值; 酸性和碱性酶中Ala、Glu、Leu、Asn、Arg、Ser和Thr的含量存在较大差异。在此基础上, 发展了一种加权氨基酸组成的方法对两种酶进行识别, 其自一致性检验的识别精度可达86.1%, 5倍交叉验证的精度为83.3%。建立了一种基于序列识别酸性和碱性酶的新方法。     

The importance of four histidine residues in isocitrate lyase from Escherichia coli.

By site-directed mutagenesis, substitutions were made for His-184 (H-184), H-197, H-266, and H-306 in Escherichia coli isocitrate lyase. Of these changes, only mutations of H-184 and H-197 appreciably reduced enzyme activity. Mutation of H-184 to Lys, Arg, or Leu resulted in an inactive isocitrate lyase, and mutation of H-184 to Gln resulted in an enzyme with 0.28% activity. Nondenaturing polyacrylamide gel electrophoresis demonstrated that isocitrate lyase containing the Lys, Arg, Gln, and Leu substitutions at H-184 was assembled poorly into the tetrameric subunit complex. Mutation of H-197 to Lys, Arg, Leu, and Gln resulted in an assembled enzyme with less than 0.25% wild-type activity. Five substitutions for H-266 (Asp, Glu, Val, Ser, and Lys), four substitutions for H-306 (Asp, Glu, Val, and Ser), and a variant in which both H-266 and H-306 were substituted for showed little or no effect on enzyme activity. All the H-197, H-266, and H-306 mutants supported the growth of isocitrate lyase-deficient E. coli JE10 on acetate as the sole carbon source; however, the H-184 mutants did not.     

The intrinsic fluorescence of the recombinant human leukocyte interferon-alpha A and fibroblast interferon-beta 1

The environment of Trp residues of the recombinant human interferons has been studied by the analysis of the emission spectra of native and denatured proteins at pH 1.5-8.5 and temperature 10-65 degrees C in the presence and absence of the anionic, cationic and neutral to charge contact quenchers--KI, CsCl and acrylamide, respectively. The obtained data allow to suppose that in IFN-alpha A and IFN-beta 1 Trp141 interacts with Arg145 and one or several from the following residues--Phe124, Ile127, Tyr130, Leu131, whereas Trp77--with Arg33 and Phe36, Phe78, Leu81 or Leu82 (Ile81 or Val82 for IFN-beta 1).     

Ydj1p二聚体中β14~β15与domain-III分离的拉伸分子动力学模拟研究

分子伴侣Hsp40是一种以二聚体的形式调控非天然多肽折叠的热激蛋白。本文通过拉伸分子动力学研究了酵母Hsp40家族成员Ydj1p二聚体中β14-β15与domain-Ⅲ的分离过程,深入探讨了影响Ydj1p二聚体稳定性的重要残基和相互作用力。研究表明,残基Thr366、Asp368、Cys370、Leu372和Phe375在Ydj1P二聚体的形成过程中发挥着重要的作用。其中,β14-β15中的残基Thr366和Asp368分别通过与domain-Ⅲ内的残基Asp291、Trp292和Trp292、Lys294之间形成的氢键,Asp368通过与domain-Ⅲ内的残基Lys314形成盐桥,Cys370、Leu372和Phe375则是通过与domain-Ⅲ形成疏水作用力来稳定Ydj1p二聚体结构。     

Select nutrients in the ovine uterine lumen. I. Amino acids, glucose, and ions in uterine lumenal flushings of cyclic and pregnant ewes

Nutrients in uterine secretions are essential for development and survival of conceptuses (embryo and associated extraembryonic membranes) during pregnancy; however, little is known about changes in the amounts of specific nutrients in the uterine fluids of cyclic and pregnant ruminants. This study determined quantities of glucose, amino acids, glutathione, calcium, sodium, and potassium in uterine lumenal fluid from cyclic (Days 3-16) and pregnant (Days 10-16) ewes. Total recoverable glucose, Arg, Gln, Leu, Asp, Glu, Asn, His, beta-Ala, Tyr, Trp, Met, Val, Phe, Ile, Lys, Cys, Pro, glutathione, calcium, and sodium were greater in the uterine fluid of pregnant compared with cyclic ewes between Days 10 and 16. In cyclic ewes, only modest changes in the total amounts of glucose, Asn, Cit, Tyr, Trp, Met, Val, Cys, glutathione, calcium, and potassium were detected between Days 3 and 16. However, in pregnant ewes, amounts of glucose, Arg, Gln, Glu, Gly, Cys, Leu, Pro, glutathione, calcium, and potassium in uterine fluids increased 3- to 23-fold between Days 10 and 14 and remained high to Day 16. Of particular interest were increases in glucose, Arg, Leu, and Gln in uterine flushings of pregnant ewes between Days 10 and 16 of pregnancy. Total amounts of His, ornithine, Lys, Ser, Thr, Ile, Phe, Trp, Met, and Cit in uterine fluids also increased, but to a lesser extent during early pregnancy. These novel results indicate activation of pregnancy-associated mechanisms for transport of nutrients into the uterine lumen, and they provide a framework for future studies of nutrients, including glucose, amino acids, and glutathione, required to activate nutrient-sensing cell signaling pathways for growth, development, and survival of conceptuses, as well as for optimization of culture media for in vitro studies of conceptus development.     

Lysozyme modification by the fenton reaction and gamma radiation

A comparative study was performed on lysozyme modification after exposure to Fenton reagent (Fe(II)/H2 O2) or hydroxyl radicals produced by y radiation. The conditions were adjusted to obtain, with both systems, a 50% loss of activity of the modified ensemble. Gamma radiation modified almost all types of amino acid residues in the enzyme, with little specificity. The modification order was Tyr > Met = Cys > Lys > Ile + Leu > Gly > Pro = Phe > Thr + Ala > Trp = Ser > Arg > Asp + Glu, with 42 mol of modified residues per initial mole of native enzyme. In contrast, when the enzyme was exposed to the Fenton reaction, only some types of amino acids were modified. Furthermore, a smaller number of residues (13.5) were damaged per initial mole of enzyme. The order of the modified residues was Tyr > Cys > Trp > Met His > Ile + Leu > Val > Arg. These results demonstrate that the modifications elicited by these two free radical sources follow different mechanisms. An intramolecular free radical chain reaction is proposed to play a dominant role in the oxidative modification of the protein promoted by gamma radiation.     

Site-directed mutagenesis of active site residues of phosphite dehydrogenase

Phosphite dehydrogenase (PTDH) catalyzes the unusual oxidation of phosphite to phosphate with the concomitant reduction of NAD(+) to NADH. PTDH shares significant amino acid sequence similarity with D-hydroxy acid dehydrogenases (DHs), including strongly conserved catalytic residues His292, Glu266, and Arg237. Site-directed mutagenesis studies corroborate the essential role of His292 as all mutants of this residue were completely inactive. Histidine-selective inactivation studies with diethyl pyrocarbonate provide further evidence regarding the importance of His292. This residue is most likely the active site base that deprotonates the water nucleophile. Kinetic analysis of mutants in which Arg237 was changed to Leu, Lys, His, and Gln revealed that Arg237 is involved in substrate binding. These results agree with the typical role of this residue in D-hydroxy acid DHs. However, Glu266 does not play the typical role of increasing the pK(a) of His292 to enhance substrate binding and catalysis as the Glu266Gln mutant displayed an increased k(cat) and unchanged pH-rate profile compared to those of wild-type PTDH. The role of Glu266 is likely the positioning of His292 and Arg237 with which it forms hydrogen bonds in a homology model. Homology modeling suggests that Lys76 may also be involved in substrate binding, and this postulate is supported by mutagenesis studies. All mutants of Lys76 display reduced activity with large effects on the K(m) for phosphite, and Lys76Cys could be chemically rescued by alkylation with 2-bromoethylamine. Whereas a positively charged residue is absolutely essential for activity at the position of Arg237, Lys76 mutants that lacked a positively charged side chain still had activity, indicating that it is less important for binding and catalysis. These results highlight the versatility of nature's catalytic scaffolds, as a common framework with modest changes allows PTDH to catalyze its unusual nucleophilic displacement reaction and d-hydroxy acid DHs to oxidize alcohols to ketones.