基于易错PCR的黄曲霉毒素解毒酶体外分子定向进化

运用定向进化-易错PCR方法,提高黄曲霉毒素解毒酶的活力及稳定性,并结合辣根过氧化物酶 (HRP)-隐性亮绿 (RBG) 快速高通量筛选系统,构建了库容约为104的突变体库。经过两轮易错PCR,最终分别获得了耐高温70 ℃突变酶A1773、pH 4.0稳定性的突变酶A1476,pH 4.0和pH 7.5均表现稳定性的突变酶A2863,其酶活力比野生酶分别提高了6.5倍、21倍和12.6倍。经序列分析表明,发现突变酶A1773发生了Glu127Lys和Gln613Arg突变;突变酶A2863发生了Gly73     

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 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.     

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.     

Further improvement of the thermal stability of a partially stabilized Bacillus subtilis 3-isopropylmalate dehydrogenase variant by random and site-directed mutagenesis.

A thermostabilized mutant of Bacillus subtilis 3-isopropylmalate dehydrogenase (IPMDH) obtained in a previous study contained a set of triple amino acid substitutions. To further improve the stability of the mutant, we used a random mutagenesis technique and identified two additional thermostabilizing substitutions, Thr22-->Lys and Met256-->Val, that separately endowed the protein with further stability. We introduced the two mutations into a single enzyme molecule, thus constructing a mutant with overall quintuple mutations. Other studies have suggested that an improved hydrophobic subunit interaction and a rigid type II beta-turn play important roles in enhancing the protein stability. Based on those observations, we successively introduced amino acid substitutions into the mutant with the quintuple mutations by site-directed mutagenesis: Glu253 at the subunit interface was replaced by Leu to increase the hydrophobic interaction between the subunits; Glu112, Ser113 and Ser115 that were involved in the formation of the turn were replaced by Pro, Gly and Glu, respectively, to make the turn more rigid. The thermal stability of the mutants was determined based on remaining activity after heat treatment and first-order rate constant of thermal unfolding, which showed gradual increases in thermal stability as more mutations were included.     

Chaperone function of mutant versions of alpha A- and alpha B-crystallin prepared to pinpoint chaperone binding sites.

A major stress protein, alpha-crystallin, functions as a chaperone. Site-directed mutagenesis has been used to identify regions of the protein necessary for chaperone function. In this work we have taken some of the previously described mutants produced and assessed their chaperone function by both a traditional heat-induced aggregation method at elevated temperature and using enzyme methods at 37 degrees C. In general the different assays gave parallel results indicating that the same property is being measured. Discrepancies were explicable by the heat lability of some mutants. Most mutants had full chaperone function showing the robust nature of alpha-crystallin. A mutant corresponding to a minor component of rodent alpha A-crystallin, alpha Ains-crystallin, had decreased chaperone function. Decreased chaperone function was also found for human Ser139--> Arg, Thr144-->Arg, Ser59-->Ala mutants of alpha B-crystallin and double mutants Ser45-->Ala/Ser59-->Ala, Lys103--> Leu/His104-->Ile, and Glu110-->His/His111-->Glu. A mutant Phe27-->Arg that was the subject of previous controversy was shown to be fully active at physiological temperatures.     

Upward shift of the pH optimum of Acremonium ascorbate oxidase

A gene encoding a thermostable Acremonium ascorbate oxidase (ASOM) was randomly mutated to generate mutant enzymes with altered pH optima. One of the mutants, which exhibited a significantly higher activity in the pH range 4.5-7 compared to ASOM, had a Gln183Arg substitution in the region corresponding to SBR1, one of the substrate binding regions of the zucchini enzyme. The other mutant with almost the same pH profile as Gln183Arg had a Thr527Ala substitution near the type 3 copper center and became more sensitive to azide than ASOM. Site-directed mutagenesis in the substrate binding regions with reference to the amino acid sequences of plant enzymes led to isolation of mutants shifted upward in the pH optimum; Val193Pro and Val193Pro/Pro190Ile increased the pH optimum by 1 and 0.5 units, respectively, while retaining the near-wild-type thermostability and azide sensitivity. The homology model of ASOM constructed from the zucchini enzyme coordinates suggested that replacement of Val193 by Pro could disturb the ion pair networks among Arg309, Glu192, Arg194 and Glu311. This perturbation could affect either the molecular recognition between the substrate and ASOM or the electron transfer from the substrate to the type 1 copper center, leading to the alkaline shift of the catalytic activity of the mutant enzyme. The other mutations, Val193Pro/Pro190Ile, could also induce similar structural perturbations involving the ion pair networks.     

Long distance interactions within the potassium channel pore are revealed by molecular diversity of viral proteins

Kcv is a 94-amino acid protein encoded by chlorella virus PBCV-1 that corresponds to the pore module of K(+) channels. Therefore, Kcv can be a model for studying the protein design of K(+) channel pores. We analyzed the molecular diversity generated by approximately 1 billion years of evolution on kcv genes isolated from 40 additional chlorella viruses. Because the channel is apparently required for virus replication, the Kcv variants are all functional and contain multiple and dispersed substitutions that represent a repertoire of allowed sets of amino acid substitutions (from 4 to 12 amino acids). Correlations between amino acid substitutions and the new properties displayed by these channels guided site-directed mutations that revealed synergistic amino acid interactions within the protein as well as previously unknown interactions between distant channel domains. The effects of these multiple changes were not predictable from a priori structural knowledge of the channel pore.     

Pyruvate carboxylase: isolation of the biotin-containing tryptic peptide and the determination of its primary sequency

The biotin-containing tryptic peptides of pyruvate carboxylase from sheep, chicken, and turkey liver mitochondria have been isolated and their primary structures determined. The amino acid sequences of the 19 residue peptides from chicken and turkey are identical and share a common sequence of 14 residues around biocytin with the 24-residue peptide isolated from sheep. The sequences obtained were: residue 1 → 11 Avian: Gly Ala Pro Leu Val Leu Ser Ala Met Biocytin Met Sheep: Gly Gln Pro Leu Val Leu Ser Ala Met Biocytin Met residues 12 → 19 or 24 Avian: Glu Thr Val Val Thr Ala Pro Arg Sheep: Glu Thr Val Val Thr Ser Pro Val Thr Glu Gly Val Arg A sensitive radiochemical assay for biotin was developed based on the tight binding of biotin by avidin. The ability of zinc sulfate to precipitate, without dissociating, the avidin-biotin complex provided a convenient procedure for separating free and bound biotin, and hence, for back-titrating a standard amount of avidin with [¹⁴C]biotin.     

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.     

青霉素G酰化酶β亚基Ser^579,Arg^580的定点突变研究

According to the comparison of amino acid sequence between PGA (Penicillin G Acylase) and PBPs (Penicillin Binding Protein), We suggest that No. 565-595 peptide fragment in beta-subunit of PGA may be a substrate-binding site of enzyme. Plasmid pTZGA was constructed by cloning the 2.6 kb PGA gene of pWGA into phagemid pTZ18U The technique of site-specific mutagenesis was used to study the role of residue No. 579 (Ser) and No. 580 (Arg) of PGA. Four kinds of mutants were obtained (Ser579-->Gly579, Arg580-->Gly580, Arg580-->Glu580, Arg580-->Lys580), both Glu580 and Gly580 mutants showed no activity of enzyme and Lys580 mutant remained 30% and Gly579 mutant kept 70% activity of wilde type. The same protein expression of four mutants according to the results of ELISA indicate that mutation does not affect the expression of PGA, but Arg580 residue may be essential for substrate-binding or catalysis of PGA.     

Conformational rearrangement of beta-lactoglobulin upon interaction with an anionic membrane

The recent availability of the SHV-1 beta-lactamase crystal structure provides a framework for the understanding of the functional role of amino acid residues in this enzyme. To that end, we have constructed by site-directed mutagenesis 18 variants of the SHV beta-lactamase: an extended spectrum group: Gly238Ser, Gly238Ser-Glu240Lys, Asp104Lys-Gly238Ser, Asp104Lys-Thr235Ser-Gly238Ser, Asp179Asn, Arg164His, and Arg164Ser; an inhibitor resistant group: Arg244Ser, Met69Ile, Met69Leu, and Ser130Gly; mutants that are synergistic with those that confer resistance to oxyimino-cephalosporins: Asp104Glu, Asp104Lys, Glu240Lys, and Glu240Gln; and structurally conserved mutants: Thr235Ser, Thr235Ala and Glu166Ala. Among the extended spectrum group the combination of high-level ampicillin and cephalosporin resistance was demonstrated in the Escherichia coli DH10B strains possessing the Gly238Ser mutation: Gly238Ser, Gly238Ser-Glu240Lys, Asp104Lys-Gly238Ser, and Asp104Lys-Thr235Ser-Gly238Ser. Of the inhibitor resistant group, the Ser130Gly mutant was the most resistant to ampicillin/clavulanate. Using a polyclonal anti-SHV antibody, we assayed steady state protein expression levels of the SHV beta-lactamase variants. Mutants with the Gly238Ser substitution were among the most highly expressed. The Gly238Ser substitution resulted in an improved relative k(cat)/K(m) value for cephaloridine and oxyimino-cephalosporins compared to SHV-1 and Met69Ile. In our comparative survey, the Gly238Ser and extended spectrum beta-lactamase variants containing this substitution exhibited the greatest substrate versatility against penicillins and cephalosporins and greatest protein expression. This defines a unique role of Gly238Ser in broad-spectrum beta-lactam resistance in this family of class A beta-lactamases.     

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.     

Paramecium bursaria chlorella virus-1 encodes an unusual arginine decarboxylase that is a close homolog of eukaryotic ornithine decarboxylases

Paramecium bursaria chlorella virus (PBCV-1) is a large double-stranded DNA virus that infects chlorella-like green algae. The virus encodes a homolog of eukaryotic ornithine decarboxylase (ODC) that was previously demonstrated to be capable of decarboxylating l-ornithine. However, the active site of this enzyme contains a key amino acid substitution (Glu for Asp) of a residue that interacts with the delta-amino group of ornithine analogs in the x-ray structures of ODC. To determine whether this active-site change affects substrate specificity, kinetic analysis of the PBCV-1 decarboxylase (PBCV-1 DC) on three basic amino acids was undertaken. The k(cat)/K(m) for l-arginine is 550-fold higher than for either l-ornithine or l-lysine, which were decarboxylated with similar efficiency. In addition, alpha-difluoromethylarginine was a more potent inhibitor of the enzyme than alpha-difluoromethylornithine. Mass spectrometric analysis demonstrated that inactivation was consistent with the formation of a covalent adduct at Cys(347). These data demonstrate that PBCV-1 DC should be reclassified as an arginine decarboxylase. The eukaryotic ODCs, as well as PBCV-1 DC, are only distantly related to the bacterial and plant arginine decarboxylases from their common beta/alpha-fold class; thus, the finding that PBCV-1 DC prefers l-arginine to l-ornithine was unexpected based on evolutionary analysis. Mutational analysis was carried out to determine whether the Asp-to-Glu substitution at position 296 (position 332 in Trypanosoma brucei ODC) conferred the change in substrate specificity. This residue was found to be an important determinant of substrate binding for both l-arginine and l-ornithine, but it is not sufficient to encode the change in substrate preference.     

Anomalous rates of evolution of pancreatic polypeptide and peptide tyrosine-tyrosine (PYY) in a tetraploid frog, Xenopus laevis (Anura:Pipidae)

The South African clawed frog Xenopus laevis is believed to have arisen as a result of a tetraploidization event occurring approximately 30 million years ago. Two molecular forms of pancreatic polypeptide (PP) have been isolated from an extract of the pancreas of this species and two molecular forms of peptide tyrosine-tyrosine (PYY) from the intestine. Despite the fact that the amino acid sequence of PP has, in general, been very poorly conserved during the evolution of tetrapods (only Pro(5), Pro(8), Gly(9), Ala(12), Tyr(27), Arg(33) and Arg(35) are invariant among species studied so far), the two Xenopus PPs differ by only a single amino acid substition (Asp(22)-->Glu). In contrast the two molecular forms of PYY differ by six amino acid substitutions (Glu(15)-->Gln, Thr(18)-->Ala, Leu(21)-->Met, Ile(22)-->Thr, Ile(28)-->Val, Val(31)-->Ile). The data imply that strong evolutionary pressure is acting to conserve the functional domain in both genes encoding PP and so suggest that PP may have an important physiological role in amphibians (although the nature of this role has yet to be determined). The more rapid mutation of the functional domain in the genes encoding PYY, a peptide whose amino acid sequence has been quite well conserved in tetrapods and whose physiological significance is well established, suggests that one of the PYY genes may be evolving towards a new function or towards becoming a pseudogene.     

Characterization of an alkaline subtilopeptidase type Pfizer.

The physiochemical properties, amino acid composition and profile of the the tryptic peptides for an alkaline subtilopeptidase type Pfizer have been determined. The enzyme is stable in the pH range from 5 to 10, has a pH optimum of 9.5 to 10, and is relatively stable for a period of 2 h up to a temperature of 50C. Homogeneity was demonstrated by electrophoretic techniques and the mobilities indicated on isoelectric point of 8.7. The molecular weight was found to be 25,000 by gel filtration. The amino acid composition was found to be Ala32, Arg4, Aspgamma8, Glu15, Gly29, His4, Ile9, Leu13, Lys11, Met5, Phe4, Pro14, Ser31, Thr17, Tyr9, Val22, a total of 247 amino acid residues. The enzyme does not contain either disulfide bonds or cysteine, and lacks tryptophan as well. The N-terminal end-group residue is alanine: the C-terminal amino acid is arginine. Tryptic hydrolysis of the enzyme produced 15 peptides which were separated by gradient elution on Dowex 50-X2. The amino acid composition of each appropriately purified tryptic peptide was established.     

Thermal denaturation of eukaryotic class 1 translation termination factor eRF1. Relationship between stability of the eRF1 molecule and alteration of functional activity of its mutants

Thermal denaturation of eukaryotic class-1 translation termination factor eRF1 and its mutants was examined using differential scanning microcalorimetry (DSK). Changes of free energy caused by mutants in the N domain of human eRF1 were calculated. Melting of eRF1 molecule composed of three individual domains is cooperative. Some amino acid substitutions did not affect protein thermostability and in some other cases even slightly stabilize the protein globule. These imply that these amino acid residues are not involved in maintenance of the 3D structure of human eRF1. Thus, in Glu55Asp, Tyr125Phe, Asn61Ser, Glu55Arg, Glu55A1a, Asn61Ser + Ser64Asp, Cys127Ala and Ser64Asp mutants selective inactivation of release activity is not caused by a destabilization of protein 3D structure and, most likely, is associated with local stereochemical changes introduced by substitutions of amino acid side chains in the functionally essential sites of N-domain molecule. Some residues (Asn129, Phe131) as shown by calorimetric measurements are essential for preservation of stable protein structure, but at the same time they affect selective stop codon recognition probably via their neighboring amino acids. Recognition of UAG and UAA stop codons in vitro is more sensitive to preservation of protein stability than the UGA recognition.     

Reverse polarization in amino acid and nucleotide substitution patterns between human-mouse orthologs of two compositional extrema.

Genome-wide analysis of sequence divergence patterns in 12,024 human-mouse orthologous pairs reveals, for the first time, that the trends in nucleotide and amino acid substitutions in orthologs of high and low GC composition are highly asymmetric and polarized to opposite directions. The entire dataset has been divided into three groups on the basis of the GC content at third codon sites of human genes: high, medium, and low. High-GC orthologs exhibit significant bias in favor of the replacements, Thr --> Ala, Ser --> Ala, Val --> Ala, Lys --> Arg, Asn --> Ser, Ile --> Val etc., from mouse to human, whereas in low-GC orthologs, the reverse trends prevail. In general, in the high-GC group, residues encoded by A/U-rich codons of mouse proteins tend to be replaced by the residues encoded by relatively G/C-rich codons in their human orthologs, whereas the opposite trend is observed among the low-GC orthologous pairs. The medium-GC group shares some trends with high-GC group and some with low-GC group. The only significant trend common in all groups of orthologs, irrespective of their GC bias, is (Asp)(Mouse) --> (Glu)(Human) replacement. At the nucleotide level, high-GC orthologs have undergone a large excess of (A/T)(Mouse) --> (G/C)(Human) substitutions over (G/C)(Mouse) --> (A/T)(Human) at each codon position, whereas for low-GC orthologs, the reverse is true.     

Alanine scanning mutagenesis of oxygen-containing amino acids in the transmembrane region of the Na,K-ATPase.

Oxygen-containing amino acids in the transmembrane region of the Na, K-ATPase alpha subunit were studied to identify residues involved in Na+ and/or K+ coordination by the enzyme. Conserved residues located in the polar face of transmembrane helices were selected using helical wheel and topological models of the enzyme. Alanine substitution of these residues were introduced into an ouabain-resistant sheep alpha1 isoform and expressed in HeLa cells. The capacity to generate essential Na+ and K+ gradients and thus support cell growth was used as an initial indication of the functionality of heterologous enzymes. Enzymes carrying alanine substitution of Ser94, Thr136, Ser140, Gln143, Glu144, Glu282, Thr334, Thr338, Thr340, Ser814, Tyr817, Glu818, Glu821, Ser822, Gln854, and Tyr994 supported cell growth, while those carrying substitutions Gln923Ala, Thr955Ala, and Asp995Ala did not. To study the effects of these latter replacements on cation binding, they were introduced into the wild-type alpha1 sheep isoform and expressed in mouse NIH3T3 cells where [3H]ouabain binding was utilized to probe the heterologous proteins. These substitutions did not affect ouabain, K+, or Na+ binding. Expression levels of these enzymes were similar to that of control. However, the level of Gln923Ala-, Thr955Ala-, or Asp995Ala-substituted enzyme at the plasma membrane was significantly lower than that of the wild-type isoform. Thus, these substitutions appear to impair the maturation process or targeting of the enzyme to the plasma membrane, but not cation-enzyme interactions. These results complete previous studies which have identified Ser755, Asp804, and Asp808 as absolutely essential for Na+ and K+ transport by the enzyme. Thus, it is significant that most transmembrane conserved-oxygen-containing residues in the Na,K-ATPase can be replaced without substantially affecting cation-enzyme interactions to the extent of preventing enzyme function. Consequently, other chemical groups, aromatic rings or backbone carbonyls, should be considered in models of cation-binding sites.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.