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.     

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 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.     

Arabidopsis-derived shrimp viral-binding protein, PmRab7 can protect white spot syndrome virus infection in shrimp

White spot syndrome virus is currently the leading cause of production losses in the shrimp industry. Penaeus monodon Rab7 protein has been recognized as a viral-binding protein with an efficient protective effect against white spot syndrome infection. Plant-derived recombinant PmRab7 might serve as an alternative source for in-feed vaccination, considering the remarkable abilities of plant expression systems. PmRab7 was introduced into the Arabidopsis thaliana T87 genome. Arabidopsis-derived recombinant PmRab7 showed high binding activity against white spot syndrome virus and a viral envelope, VP28. The growth profile of Arabidopsis suspension culture expressing PmRab7 (ECR21# 35) resembled that of its counterpart. PmRab7 expression in ECR21# 35 reached its maximum level at 5 mg g(-1) dry weight in 12 days, which was higher than those previously reported in Escherichia coli and in Pichia. Co-injection of white spot syndrome virus and Arabidopsis crude extract containing PmRab7 in Litopenaeus vannamei showed an 87% increase in shrimp survival rate at 5 day after injection. In this study, we propose an alternative PmRab7 source with higher production yield, and cheaper culture media costs, that might serve the industry's need for an in-feed supplement against white spot syndrome infection.     

Role of the domain encompassing Arg304-Ile328 in rat P2X2 receptor conformation revealed by alterations in complex glycosylation at Asn298

The final 25 amino acids of the ectodomain of the P2X receptors, immediately prior to the second TM (transmembrane domain) (pre-TM2: Arg(304)-Ile(328) in rat P2X(2)), are highly conserved. Whole-cell patch clamp recordings showed that single cysteine substitutions in the N-terminal half of pre-TM2 (Arg(304)-Ile(314)) led to loss of function at Arg(304), Leu(306), Lys(308) and Ile(312). Cysteine substitutions within this region also resulted in a significant reduction in the apparent molecular mass of receptors, due to loss of complex glycosylation at the nearby acceptor site Asn(298), which was not seen for the C-terminal portion of pre-TM2 (Asp(315)-Ile(328)). The reduction in complex glycosylation was not due to reduced cell-surface presentation, demonstrating that glycosylation at Asn(298) was acting as a sensor of subtle changes in receptor conformation within the pre-TM2 region. When this N-glycan site was repositioned closer to the plasma membrane by mutagenesis (N298S together with G299N, T300N, T301N or T303N), glycosylation was restored at G299N and T300N, but was impaired for T301N and completely absent for T303N. These results suggest that the region in the vicinity of Asp(315) is at the plasma membrane interface and that the N-terminal portion of pre-TM2 (Arg(304)-Ile(314)) is important for the correct conformation of the receptor at the extracellular face of the membrane.     

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.     

Comparison of the monomer structure of the FMN-binding protein from Desulfovibrio vulgaris obtained by NMR and molecular dynamics simulation approaches

Flavin mononucleotide (FMN)-binding proteins (FBPs) play an important role in the electron transport process in bacteria. In this study, the structures of the FBP from Desulfovibrio vulgaris (DvFBP) (Miyazaki F) were compared between those obtained experimentally by nuclear magnetic resonance (NMR) spectroscopy and those derived from molecular dynamics simulations (MDSs). A high-residue root of mean square deviation (RMSD) was observed in residues located at both sides of the wings (Gly22, Glu23, Asp24, Ala59, Arg60, Asp61, Glu62, Gly75, Arg76, Asn77, Gly78 and Pro79), while a low-residue RMSD was found in residues located in a hollow of the structure (Asn12, Glu13, Gly14, Val15, Val16, Asn30, Thr31, Trp32, Asn33, Ser34, Gly69, Ser70, Arg71 and Lys72). Inter-planar angles between the Phe7 and Iso and between the Phe7 and Trp106 residues were remarkably different between the MDS- and NMR-derived DvFBP structures. Distribution of the torsion angles around the covalent bonds in the aliphatic chain of FMN was similar in the MDS- and NMR-derived structures, except for those around the C1′–C2′ and C5′–O5′ bonds. Hydrogen bond formation between IsoO2 and the Gly49 or Gly50 peptide NH was formed in both the NMR- and MDS-derived structures. Overall, the MDS-derived structures were found to be considerably different from the NMR-derived structures, which must be considered when the photoinduced electron transfer in flavoproteins is analysed with MDS-derived structures.     

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.     

Express analysis of protein amino acid sequences. Primary structure of Penicillium chrysogenum 152A guanyl-specific ribonuclease

The complete amino acid sequence of Penicillium chrysogenum 152A guanyl-specific RNase has been established using automated Edman degradation of two non-fractionated peptide mixtures produced by tryptic and staphylococcal protease digests of the protein. The RNase contains 102 amino acid residues: His2, Arg3, Asp7, Asn8, Thr5, Ser11, Glu4, Gln2, Pro4, Gly11, Ala13, Cys4, Val8, Ile3, Leu3, Tyr9, Phe5 (Mr 10 747).     

Suppression of PmRab7 by dsRNA Inhibits WSSV or YHV Infection in Shrimp

Viral entry into host cells requires endocytosis machineries of the host for viral replication. PmRab7, a Penaeus monodon small GTPase protein, was investigated for its function in vesicular transport during viral infection. The double-stranded RNA of Rab7 was injected into a juvenile shrimp before challenging with white spot syndrome virus (WSSV) or yellow head virus (YHV). PmRab7 mRNA was specifically decreased at 48 h after dsRNA-Rab7 injection. Silencing of PmRab7 dramatically inhibited WSSV-VP28 mRNA and protein expression. Unexpectedly, the silencing of PmRab7 also inhibited YHV replication in the YHV-infected shrimp. These results suggested that PmRab7 is a common cellular factor required for WSSV or YHV replication in shrimp. Because PmRab7 should function in the endosomal trafficking pathway, its silencing prevents successful viral trafficking necessary for replication. Silencing of PmRab7 could be a novel approach to prevent both DNA virus (WSSV) and RNA virus (YHV) infection of shrimp.     

Modelling and predicting the binding mechanics of HIV P1053-0.5β antibody complex

Simulating antigen–antibody interactions is crucial in understanding the mechanics of antigen–antibody binding in medical science. In this study, molecular dynamics simulations are performed to analyse the dissociation of the P1053-0.5β antibody complex structure. The two-dimensional free energy profiles of the complex structure are extracted using the weighted histogram analysis method, and the binding pathway is then predicted using a modified form of the MaxFlux-PRM method. The simulation results suggest that 10 amino residues (i.e. Leu11, Val13, Asp34, Arg112, Thr101, Gly127, Val229, Ser231, Ile235 and Arg236) play a key role in relaxing the antibody structure, thereby facilitating the binding of the 0.5β antibody-P1053 peptide system.     

The phosphorylation at Thr 124 of simian virus 40 large T antigen is crucial for its oligomerization

SV40 large T antigen is phosphorylated at up to ten different amino acids clustered in an N-terminal and a C-terminal part of the polypeptide chain. The N-terminal phosphorylated residues include Ser 123 and Thr 124. We have analyzed the oligomerization, the complex formation with the cellular oncoprotein p53 and the DNA-binding properties of T antigen from two different SV40 transformed cell lines which have either an amino acid exchange at Ser 123 to Phe (W7) or Thr 124 to Ile (D29). In comparison to wild-type T antigen both mutant T antigens have a slightly reduced binding affinity for both binding sites, I and II, of SV40 DNA. Phosphorylation at both residues of T antigen is not essential for formation of the complex with p53. Only the phosphorylation at Thr 124 seems to be critical for the formation of high molecular mass oligomers. Our data support the hypothesis that the oligomerization of T antigen seems to be implicated in viral DNA replication.     

NH2-terminal sequences of DNA-, heparin-, and gelatin-binding tryptic fragments from human plasma fibronectin

NH₂-terminal sequence analysis was performed on subregions of human plasma fibronectin including 24,000-dalton (24K) DNA-binding, 29,000-dalton (29K) gelatin-binding, and 18,000-dalton (18K) heparin-binding tryptic fragments. These fragments were obtained from fibronectin after extensive trypsin digestion followed by sequential affinity purification on gelatin-Sepharose, heparin-agarose, and DNA-cellulose columns. The gelatin-binding fragment was further purified by gel filtration on Sephadex G-100, and the DNA-binding and heparin-binding fragments were further purified by high-performance liquid chromatography. The 29K fragment had the following NH₂-terminal sequence: AlaAlaValTyrGlnProGlnProHisProGlnProPro (Pro)TyrGlyHis HisValThrAsp(His)(Thr)ValValTyrGly(Ser) ?(Ser)?-Lys. The NH₂-terminal sequence of a 50K, gelatin-binding, subtilisin fragment by L. I. Gold, A. Garcia-Pardo, B. Prangione, E. C. Franklin, and E. Pearlstein (1979, Proc. Nat. Acad. Sci. USA76, 4803–4807) is identical to positions 3–19 (with the exception of some ambiguity at position 14) of the 29K fragment. These data strongly suggest that the 29K tryptic fragment is included in the 50K subtilisin fragment, and that subtilisin cleaves fibronectin between the Ala²Val³ residues of the 29K tryptic fragment. The 18K heparin-binding fragment had the following NH₂-terminal sequence: (Glu)AlaProGlnProHisCysIleSerLysTyrIle LeuTyrTrpAspProLysAsnSerValGly?(Pro) LysGluAla?(Val)(Pro). The 29K gelatin-binding and 18K heparin-binding fragments have proline-rich NH₂-terminal sequences suggesting that they may have arisen from protease-sensitive, random coil regions of fibronectin corresponding to interdomain regions preceding macromolecular-binding domains. Both of these fragments contain the identical sequence ProGlnProHis, a sequence which may be repeated in other interdomain regions of fibronectin. The 24K DNA-binding fragment has the following NH₂-terminal sequence: SerAspThrValProSerProCysAspLeuGlnPhe ValGluValThrAspVal LysValThrIleMetTrpThrProProGluSerAla ValThrGlyTyrArgVal AspValCysProValAsnLeuProGlyGluHisGly Gln(Cys)LeuProIleSer. The sequence of positions 9–22 are homologous to positions 15–28 of the α chain of DNA-dependent RNA polymerase from Escherichia coli. The homology observed suggests that this stretch of amino acids may be a DNA-binding site.     

Partial glycosylation of Asn2181 in human factor V as a cause of molecular and functional heterogeneity. Modulation of glycosylation efficiency by mutagenesis of the consensus sequence for N-linked glycosylation.

Coagulation factor V (FV) circulates in two forms, FV1 and FV2, having slightly different molecular masses and phospholipid-binding properties. The aim was to determine whether this heterogeneity is due to the degree of glycosylation of Asn(2181). FVa1 and FVa2 were isolated and digested with endoglycosidase PNGase F. As judged by Western blotting, the FVa2 light chain contained two N-linked carbohydrates, whereas FVa1 contained three. Wild-type FV and three mutants, Asn(2181)Gln, Ser(2183)Thr, and Ser(2183)Ala, were expressed in COS1 cells, activated by thrombin, and analyzed by Western blotting. Wild-type FVa contained the 71 kDa-74 kDa doublet, whereas the Asn(2181)Gln and Ser(2183)Ala mutants contained only the 71 kDa light chain. In contrast, the Ser(2183)Thr mutant gave a 74 kDa light chain. This demonstrated that the third position in the Asn-X-Ser/Thr consensus affects glycosylation efficiency, Thr being associated with a higher degree of glycosylation than Ser. The Ser(2183)Thr mutant FVa was functionally indistinguishable from plasma-purified FVa1, whereas Asn(2181)Gln and Ser(2183)Ala mutants behaved like FVa2. Thus, the carbohydrate at Asn(2181) impaired the interaction between FVa and the phospholipid membrane, an interpretation consistent with a structural analysis of a three-dimensional model of the C2 domain and the position of a proposed phospholipid-binding site. In conclusion, we show that the FV1-FV2 heterogeneity is caused by differential glycosylation of Asn(2181) related to the presence of a Ser rather than a Thr at the third position in the consensus sequence of glycosylation.     

Novel polymorphisms in ovine prion protein gene

The aim of this study was to identify the PRNP polymorphisms outside the standard codons 136, 154 and 171 in 1110 sheep with no clinical sign of scrapie from all 18 Turkish native sheep breeds and compare our results with published data on ovine PRNP polymorphism from other regions of the world. Among the 22 amino acid polymorphisms and three silent mutations, 10 were novel for ovine PRNP: p.Gly94Gly, p.Leu128Ile, p.Met132Leu, p.Ser135Arg, p.Met137Val, p.Asn146Lys, p.Arg159Arg, p.Tyr160Asn, p.Gln163His and p.Thr193Ser. These data reveal that sheep breeds close to the historic center of small ruminant domestication have remained highly diverse in the prion gene locus, with distinctive genetic similarities to both Asian and European sheep breeds.     

Genetic,structural, and functional analysis of pathogenic variations causing methylmalonyl-CoA epimerase deficiency

Human methylmalonyl-CoA epimerase (MCEE) catalyzes the interconversion of d-methylmalonyl-CoA and l-methylmalonyl-CoA in propionate catabolism. Autosomal recessive pathogenic variations in MCEE reportedly cause methylmalonic aciduria (MMAuria) in eleven patients. We investigated a cohort of 150 individuals suffering from MMAuria of unknown origin, identifying ten new patients with pathogenic variations in MCEE. Nine patients were homozygous for the known nonsense variation p.Arg47* (c.139C > T), and one for the novel missense variation p.Ile53Arg (c.158T > G). To understand better the molecular basis of MCEE deficiency, we mapped p.Ile53Arg, and two previously described pathogenic variations p.Lys60Gln and p.Arg143Cys, onto our 1.8 Å structure of wild-type (wt) human MCEE. This revealed potential dimeric assembly disruption by p.Ile53Arg, but no clear defects from p.Lys60Gln or p.Arg143Cys. We solved the structure of MCEE-Arg143Cys to 1.9 Å and found significant disruption of two important loop structures, potentially impacting surface features as well as the active-site pocket. Functional analysis of MCEE-Ile53Arg expressed in a bacterial recombinant system as well as patient-derived fibroblasts revealed nearly undetectable soluble protein levels, defective globular protein behavior, and using a newly developed assay, lack of enzymatic activity - consistent with misfolded protein. By contrast, soluble protein levels, unfolding characteristics and activity of MCEE-Lys60Gln were comparable to wt, leaving unclear how this variation may cause disease. MCEE-Arg143Cys was detectable at comparable levels to wt MCEE, but had slightly altered unfolding kinetics and greatly reduced activity. These studies reveal ten new patients with MCEE deficiency and rationalize misfolding and loss of activity as molecular defects in MCEE-type MMAuria.     

Conformation in solution of porcine brain natriuretic peptide determined by combined use of nuclear magnetic resonance and distance geometry

The conformation in solution of porcine brain natriuretic peptide was determined by combined use of NMR spectroscopy and distance geometry. A set of 157 inter-proton-distance constraints was derived from the two-dimensional NOE spectra, and further a set of three hydrogen bond constraints was obtained from analysis of the temperature dependence of labile protons. The five structures with minimal violations were selected after performing distance-geometry calculations starting from 40 random initial conformations. The distance-geometry structures were further refined by the use of restrained energy minimization and restrained molecular dynamics. This structure shows a compact conformation with the carboxy-terminal region, Asn21-Tyr26, folded back to the disulfide-linked loop region, Cys4-Cys20. The characteristics of the conformation determined are as follows: conformations of the three segments interposed by glycine residues, which are Arg7-Ile12, Ser14-Leu18 and Cys20-Arg25, were well defined and the segments Arg7-Ile12 and Cys20-Arg25 are rather close to each other and nearly parallel. The biological significance of these local conformations is discussed on the basis of comparisons with those of atrial natriuretic peptide reported by Kobayashi et al.     

Structure of Somatostatin Isolated from Bovine Retina

Abstract: Somatostatin-like immunoreactivity (SLI) from bovine retina was purified and its structure determined. Retinal tissue (1868 g) extracted with 3% acetic acid yielded 18.6 nmol SLI. This peptide was purified by chromatography on an affinity column made with anti-somatostatin antiserum, a reverse-phase C-18 HPLC column, and three sequential applications on a reverse-phase phenyl HPLC column. The peptide was purified 103,000-fold from the initial extract with an overall yield of 14.4%. Amino acid sequence determination by an automatic Edman degradation technique revealed the sequence to be as follows: Ser - Ala - Asn - Ser - Asn - Pro - Ala - Met - Ala - Pro - Arg - Glu - Arg - Lys - Ala - Gly - (Cys) - Lys - Asn - Phe - Phe - Trp - Lys - Thr - (Phe, Thr, Ser, Cys). The apparent identity of this peptide with somatostatin octacosapeptide (S28) purified from other mammalian tissue indicates the phylogenetic conservation of its structure and facilitates the use of the retina as a model system for studying the neurotransmitter function of somatostatin.