Crystal structures of the Nicotiana glutinosa ribonuclease NT in complex with nucleoside monophosphates

Ribonuclease NT (RNase NT), induced upon tobacco mosaic virus (TMV) infection in Nicotiana glutinosa leaves, has a broad base specificity. The crystal structures of RNase NT in complex with either 5'-AMP, 5'-GMP, or 2'-UMP were determined at 1.8 A resolutions by molecular replacement. RNase NT consists of seven helices and seven beta strands, and the structure is highly similar to that of RNase NW, a guanylic acid preferential RNase from the N. glutinosa leaves, showing root mean square deviation (rmsd) of 1.1 A over an entire length of two molecules for Calpha atoms. The complex structures revealed that Trp42, Asn44, and Trp50 are involved in interactions with bases at B1 site (primary site), whereas Gln12, Tyr17, Ser78, Leu79, and Phe89 participate in recognition of bases at B2 site (subsite). The 5'-GMP and 5'-AMP bind both B1 and B2 sites in RNase NT, while 2'-UMP predominantly binds B1 site in the complex. The nucleotide binding modes in these complexes would provide a clue to elucidation of structural basis for the broad base specificity for RNase NT.     

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.     

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 residues in ribonuclease MC1 from bitter gourd seeds which are essential for uridine specificity

The ribonuclease MC1 (RNase MC1), isolated from seeds of bitter gourd (Momordica charantia), consists of 190 amino acids and is characterized by specific cleavage at the 5'-side of uridine. Site-directed mutagenesis was used to evaluate the contribution of four amino acids, Asn71, Val72, Leu73, and Arg74, at the alpha4-alpha5 loop between alpha4 and alpha5 helices for recognition of uracil base by RNase MC1. Four mutants, N71T, V72L, L73A, and R74S, in which Asn71, Val72, Leu73, and Arg74 in RNase MC1 were substituted for the corresponding amino acids, Thr, Leu, Ala, and Ser, respectively, in a guanylic acid preferential RNase NW from Nicotiana glutinosa, were prepared and characterized with respect to enzymatic activity. Kinetic analysis with a dinucleoside monophosphate, CpU, showed that the mutant N71T exhibited 7.0-fold increased K(m) and 2.3-fold decreased k(cat), while the mutant L73A had 14.4-fold increased K(m), although it did retain the k(cat) value comparable to that of the wild-type. In contrast, replacements of Val72 and Arg74 by the corresponding amino acids Leu and Ser, respectively, had little effect on the enzymatic activity. This observation is consistent with findings in the crystal structure analysis that Asn71 and Leu73 are responsible for a uridine specificity for RNase MC1. The role of Asn71 in enzymatic reaction of RNase MC1 was further investigated by substituting amino acids Ala, Ser, Gln, and Asp. Our observations suggest that Asn71 has at least two roles: one is base recognition by hydrogen bonding, and the other is to stabilize the conformation of the alpha4-alpha5 loop by hydrogen bonding to the peptide backbone, events which possibly result in an appropriate orientation of the alpha-helix (alpha5) containing active site residues. Mutants N71T and N71S showed a remarkable shift from uracil to guanine specificity, as evaluated by cleavage of CpG, although they did exhibit uridine specificity against yeast RNA and homopolynucleotides.     

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).     

Exogenous adenosine 3': 5'-monophosphate can release yeast from catabolite repression.

A new simple fast and reproducible purification procedure for the proteinase from rat liver mitochondria has been worked out. The specificity of cleavage of peptide bonds in glucagon, oxidized A and B chains of insulin and yeast proteinase B inhibitor by the proteinase of the inner mitochondrial membrane has been studied. The proteinase hydrolyzed three peptide bonds in glucagon, Tyr (13) - Leu (14), Trp (25) - Leu (26) and Phe (22) - Val (23) (minor cleavage site); none in the insulin A chain; one in the B chain of insulin, Tyr (16) - Leu (17); and three in the yeast proteinase B inhibitor, Phe (4) - Ile (5), Phe (20) - Leu (21) and Tyr (41) - Thr (42) (minor cleavage site).Thus, the mitochondrial proteinase cleaves peptide bonds at the carboxyl site of an aromatic amino acid and the amino site of a leucine, isoleucine, threonine or valine. The comparison with chymotrypsin A shows that the mitochondrial proteinase cleaves peptide bonds in a more restricted manner.     

Crystal structures of the ribonuclease MC1 mutants N71T and N71S in complex with 5'-GMP: structural basis for alterations in substrate specificity

Ribonuclease MC1 (RNase MC1), isolated from bitter gourd seeds, is a uridine specific RNase belonging to the RNase T2 family. Mutations of Asn71 in RNase MC1 to the amino acids Thr (N71T) and Ser (N71S) in guanosine preferential RNases altered the substrate specificity from uridine specific to guanosine specific, as shown by the transphosphorylation of diribonucleoside monophosphates [Numata, T., et al. (2001) Biochemistry 40, 524-530]. To elucidate the structural basis for the alteration of substrate specificity, crystal structures of the RNase MC1 mutants N71T and N71S, free or complexed with 5'-GMP, were determined at resolutions higher than 2 A. In the N71T-5'-GMP and N71S-5'-GMP complexes, the guanine moiety was, as in the case of the uracil moiety bound to wild-type RNase MC1, firmly stabilized in the B2 site by an extensive network of hydrogen bonds and hydrophobic interactions. Structure comparisons showed that mutations of Asn71 to Thr or Ser cause an enlargement of the B2 site, which then make it feasible to insert a guanine base into the B2 site of mutants N71T and N71S. This binding further allows for hydrogen bonding interaction of the side chain hydroxyl groups of Thr71 or Ser71 with the N7 atom of the guanine base. The mode of guanine binding of mutants N71T and N71S was found to be essentially identical to that of a guanosine preferential RNase NW from Nicotiana glutinosa. In particular, hydrogen bonds between the N7 atom of the guanine base and the hydroxyl groups of the amino acids at position 71 (RNase MC1 numbering) were completely conserved in three guanosine preferential enzymes, thereby indicating that the hydrogen bond may play an essential role in guanine binding in guanosine preferential RNases in the RNase T2 family. Consequently, it can be concluded that amino acids at position 71 (RNase MC1 numbering) serve as one of the determinants for substrate specificity (or preference) in the RNase T2 fimily by changing the size and shape of the B2 site.     

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.     

Crystallographic study of mechanism of ribonuclease T1-catalysed specific RNA hydrolysis

Ribonuclease T1 (RNase T1) cleaves the phosphodiester bond of RNA specifically at the 3'-end of guanosine. 2'-guanosinemonophosphate (2'-GMP) acts as inhibitor for this reaction and was cocrystallized with RNase T1. X-Ray analysis provided insight in the geometry of the active site and in the parts of the enzyme involved in the recognition of guanosine. RNase T1 is globular in shape and consists of a 4.5 turns alpha-helix lying "below" a four-stranded antiparallel beta-sheet containing recognition center as well as active site. The latter is indicated by the position of phosphate and sugar residues of 2'-GMP and shows that Glu58, His92 and Arg77 are active in phosphodiester hydrolysis. Guanine is recognized by a stretch of protein from Tyr42 to Tyr45. Residues involved in recognition are peptide NH and C = O, guanine O6 and N1H which form hydrogen bonds and a stacking interaction of Tyr45 on guanine. Although, on a theoretical basis, many specific amino acid-guanine interactions are possible, none is employed in the RNase T1.guanine recognition.     

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.     

Polyproline, beta-turn helices. Novel secondary structures proposed for the tandem repeats within rhodopsin, synaptophysin, synexin, gliadin, RNA polymerase II, hordein, and gluten

Seven proteins each contain 8 to 52 tandem repeats of a unique class of oligopeptide. The consensus peptide for each is rhodopsin Tyr Pro Pro Gln Gly synaptophysin Tyr Gly Pro Gln Gly synexin Tyr Pro Pro Pro Pro Gly gliadin Tyr Pro Pro Pro Gln Pro RNA polymerase II Tyr Ser Pro Thr Ser Pro Ser hordein Phe Pro Gln Gln Pro Gln Gln Pro gluten Tyr Pro Thr Ser Pro Gln Gln Gly Tyr Although there is obvious variation of sequence and of length, the penta- to nonapeptides share an initial Tyr (or Phe) and have high Pro contents and abundant Gly, Gln, and Ser. We have evaluated helical models that both recognize the uniqueness of these sequence repeats and accommodate variations on the basic theme. We have developed a group of related helical models for these proteins with about three oligopeptide repeats per turn of 10-20 A. These models share several common features: Most of the phi dihedral angles are -54 degrees, to accommodate Pro at all positions except the first (Tyr). Except for the beta-turns, most psi dihedral angles are near +140 degrees as found in polyproline. Each oligopeptide has at least one beta-turn; several have two. Some contain a cis-Tyr, Pro peptide bond; a few have a cis-bond plus one beta-turn. Tyr side chains vary from totally exposed to buried within the helices and could move to accommodate either external hydrophobic interactions or phosphorylation. The several related structures seem to be readily interconverted without major change in the overall helical parameters, and therein may lie the key to their functions.     

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.     

Modes of binding of 2'-AMP to RNase T1. A computer modeling study.

The modes of binding of adenosine 2'-monophosphate (2'-AMP) to the enzyme ribonuclease (RNase) T1 were determined by computer modelling studies. The phosphate moiety of 2'-AMP binds at the primary phosphate binding site. However, adenine can occupy two distinct sites--(1) The primary base binding site where the guanine of 2'-GMP binds and (2) The subsite close to the N1 subsite for the base on the 3'-side of guanine in a guanyl dinucleotide. The minimum energy conformers corresponding to the two modes of binding of 2'-AMP to RNase T1 were found to be of nearly the same energy implying that in solution 2'-AMP binds to the enzyme in both modes. The conformation of the inhibitor and the predicted hydrogen bonding scheme for the RNase T1-2'-AMP complex in the second binding mode (S) agrees well with the reported x-ray crystallographic study. The existence of the first mode of binding explains the experimental observations that RNase T1 catalyses the hydrolysis of phosphodiester bonds adjacent to adenosine at high enzyme concentrations. A comparison of the interactions of 2'-AMP and 2'-GMP with RNase T1 reveals that Glu58 and Asn98 at the phosphate binding site and Glu46 at the base binding site preferentially stabilise the enzyme-2'-GMP complex.     

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.     

Primary structure of alpha and beta chains from the major hemoglobin component of the magpie goose (Anseranas semipalmata, Anatidae)

The amino acid sequence of the alpha and beta chains from the major hemoglobin component (HbA) of Australian Magpie Goose (Anseranas semipalmata) is given. The minor component with the alpha D chains was detected, but only found in low concentrations. By homologous comparison, Greylag Goose hemoglobin (Anser anser) and Australian Magpie Goose alpha chains differ by 13 amino acids or 17 nucleotide (4 two point mutations) exchanges, beta chains by 6 exchanges. Seven alpha 1 beta 1 contacts are modified by substitutions in positions alpha 30-(B11)Glu leads to Gln, alpha 34(B15)Thr leads to Gln, alpha 35(B16)-Ala leads to Thr, alpha 36(B17)Tyr leads to Phe, beta 55(D6)Leu leads to Ile, beta 119(GH2)Ala leads to Ser and beta 125(H3)Glu leads to Asp. Further, one alpha 1 beta 2 contact point was changed in beta 39(C5)Gln leads to Glu. Mutation in this position, except in two abnormal human hemoglobins, was not found in any species. Amino acid exchanges between hemoglobin of Australian Magpie Goose and other birds are discussed.     

Characterization of the functional insulin binding epitopes of the full-length insulin receptor

Mutational analyses of the secreted recombinant insulin receptor extracellular domain have identified a ligand binding site composed of residues located in the L1 domain (amino acids 1-470) and at the C terminus of the alpha subunit (amino acids 705-715). To evaluate the physiological significance of this ligand binding site, we have transiently expressed cDNAs encoding full-length receptors with alanine mutations of the residues forming the functional epitopes of this binding site and determined their insulin binding properties. Insulin bound to wild-type receptors with complex kinetics, which were fitted to a two-component sequential model; the Kd of the high affinity component was 0.03 nM and that of the low affinity component was 0.4 nM. Mutations of Arg14, Phe64, Phe705, Glu706, Tyr708, Asn711, and Val715 inactivated the receptor. Alanine mutation of Asn15 resulted in a 20-fold decrease in affinity, whereas mutations of Asp12, Gln34, Leu36, Leu37, Leu87, Phe89, Tyr91, Lys121, Leu709, and Phe714 all resulted in 4-10-fold decreases. When the effects of the mutations were compared with those of the same mutations of the secreted recombinant receptor, significant differences were observed for Asn15, Leu37, Asp707, Leu709, Tyr708, Asn711, Phe714, and Val715, suggesting that the molecular basis for the interaction of each form of the receptor with insulin differs. We also examined the effects of alanine mutations of Asn15, Gln34, and Phe89 on insulin-induced receptor autophosphorylation. They had no effect on the maximal response to insulin but produced an increase in the EC50 commensurate with their effect on the affinity of the receptor for insulin.     

Targeting of hepatocellular carcinoma with glypican‐3‐targeting peptide ligand

Hepatocellular carcinoma is a common malignancy. The carcinoma cells express glypican‐3 (GPC‐3) on the cell membrane. GPC‐3 is also expressed in melanoma cells. Therefore, GPC‐3 might be a potential target for tumor imaging or therapy. Here, proteomic mass spectrometry was used to identify peptides that target GPC‐3‐expressing tumors. A mammalian expression vector expressing a FLAG‐GPC‐3 fusion protein was cloned for immunoprecipitation. With the use of liposomes, the vector was transfected into HepG2 (HepG2/FLAG‐GPC‐3) and HEK 293 cells, and the transfected cell lines were selected with geneticin. HepG2/FLAG‐GPC‐3 cells were used for immunoprecipitation of FLAG‐GPC‐3 fusion protein. Seven peptide candidates (L1–L7) were selected for GPC‐3‐targeting ligands by mass spectrometric analysis. The L5 peptide with 14 amino acids (Arg‐Leu‐Asn‐Val‐Gly‐Gly‐Thr‐Tyr‐Phe‐Leu‐Thr‐Thr‐Arg‐Gln) showed selective binding to the GPC‐3‐expressing tumor cells, as did a shortened L5 peptide (L5‐2) with seven amino acids (Tyr‐Phe‐Leu‐Thr‐Thr‐Arg‐Gln). These peptide ligands have potential as targeting moieties to GPC‐3‐expressing tumors for diagnostic and/or therapeutic purposes. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Contribution of Gln9 and Phe80 to substrate binding in ribonuclease MC1 from bitter gourd seeds.

Ribonuclease MC1 (RNase MC1) isolated from bitter gourd (Momordica charantia) seeds specifically cleaves phosphodiester bonds on the 5'-side of uridine. The crystal structures of RNase MC1 in complex with 2'-UMP or 3'-UMP reveal that Gln9, Asn71, Leu73, and Phe80 are involved in uridine binding by hydrogen bonding and hydrophobic interactions [Suzuki et al. (2000) Biochem. Biophys. Res. Commun. 275, 572-576]. To evaluate the contribution of Gln9 and Phe80 to uridine binding, Gln9 was replaced with Ala, Phe, Glu, or His, and Phe80 with Ala by site-directed mutagenesis. The kinetic properties of the resulting mutant enzymes were characterized using cytidylyl-3',5'-uridine (CpU) as a substrate. The mutant Q9A exhibited a 3.7-fold increased K(m) and 27.6-fold decreased k(cat), while three other mutations, Q9F, Q9E, and Q9H, predominantly affected the k(cat) value. Replacing Phe80 with Ala drastically reduced the catalytic efficiency (k(cat)/K(m)) with a minimum K(m) value equal to 8 mM. It was further found that the hydrolytic activities of the mutants toward cytidine-2',3'-cyclic monophosphate (cCMP) were reduced. These results demonstrate that Gln9 and Phe80 play essential roles not only in uridine binding but also in hydrolytic activity. Moreover, we produced double Ala substituted mutants at Gln9, Asn71, Leu73, and Phe80, and compared their kinetic properties with those of the corresponding single mutants. The results suggest that these four residues may contribute to uridine binding in a mutually independent manner.     

Computer modeling studies on the subsite interactions of ribonuclease T1.

The modes of binding of pGp,ApG,CpG and UpG to the enzyme ribonuclease T1 were determined by computer modeling. Essentially two binding modes are possible for all the four ligands--one with the 3'-phosphate group occupying the phosphate binding site (substrate mode of binding) and the second with the 5'-phosphate group occupying the phosphate binding site (inhibitor mode of binding). The latter binding mode is energetically favoured over the former and in this mode the base (G) and the 5'-phosphate moieties occupy the same sites on the enzyme as 5'-GMP when bound to RNase T1. The ribose moiety of pGp adopts a C3'-endo pucker form when bound to the enzyme and the glycosyl torsion angle will be in -syn range as 5'-GMP in the RNase T1-5'-GMP complex. Based on these results, a mechanism for the release of the product subsequent to cleavage of the substrate by the enzyme has been proposed. The amino acid residues Asn98 and Tyr45 are shown to form the subsites for the phosphate and the base respectively on the 5'-side of the guanine occupying the primary binding site. These studies also provide a stereochemical explanation for the specificity of the 1N subsite for adenine.     

Parallel control of hepatic proteolysis by phenylalanine and phenylpyruvate through independent inhibitory sites at the plasma membrane.

Intracellular protein degradation in the rat hepatocyte is regulated by 7 amino acids of which Leu, Gln, and Tyr play major roles. Although Phe is known to inhibit proteolysis as effectively as Tyr at high concentrations, it has not been regarded as an active regulator because of its rapid hydroxylation to Tyr. We now show that proteolytic responses to Phe during liver perfusion differ strikingly from effects of the multiphasic regulators Leu, Gln, and Tyr in eliciting mirror image responses at half-normal and normal plasma concentrations. Since response curves to phenylpyruvate and Phe were identical, we considered the possibility that phenylpyruvate mediated its anomalous inhibition intracellularly. However, when phenylpyruvate was produced from phenyllactate intracellularly at a rate providing the same rate of transamination (and intracellular concentration) as that derived from the uptake of phenylpyruvate, no response was obtained. Hence, the effect of phenylpyruvate was not initiated within the cell but more likely from the plasma membrane. Comparable evidence for Phe is less direct. Recent findings indicate that recognition sites for Leu and Gln are located at the plasma membrane. Since Phe augments the concerted inhibition by Leu and Gln at 4-fold normal levels, Phe is probably recognized in close proximity to them. However, the failure of phenylpyruvate to substitute for Phe in this interaction suggests that proteolytic inhibition by phenylpyruvate and Phe is mediated through similar, although independent, plasma membrane sites.