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 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 residue penultimate to the amino-terminal gly residue strongly affects two cotranslational protein modifications, N-myristoylation and N-acetylation

To examine the amino-terminal sequence requirements for cotranslational protein N-myristoylation, a series of site-directed mutagenesis of N-terminal region were performed using tumor necrosis factor as a nonmyristoylated model protein. Subsequently, the susceptibility of these mutants to protein N-myristoylation was evaluated by metabolic labeling in an in vitro translation system or in transfected cells. It was found that the amino acid residue at position 3 in an N-myristoylation consensus motif, Met-Gly-X-X-X-Ser-X-X-X, strongly affected the susceptibility of the protein to two different cotranslational protein modifications, N-myristoylation and N-acetylation; 10 amino acids (Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, and His) with a radius of gyration smaller than 1.80 A directed N-myristoylation, two negatively charged residues (Asp and Glu) directed N-acetylation, and two amino acids (Gly and Met) directed heterogeneous modification with both N-myristoylation and N-acetylation. The amino acid requirements at this position for the two modifications were dramatically changed when Ser at position 6 in the consensus motif was replaced with Ala. Thus, the amino acid residue penultimate to the N-terminal Gly residue strongly affected two cotranslational protein modifications, N-myristoylation and N-acetylation, and the amino acid requirements at this position for these two modifications were significantly affected by downstream residues.     

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.     

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 proteomics of N-terminal methionine cleavage

Methionine aminopeptidase (MAP) is a ubiquitous, essential enzyme involved in protein N-terminal methionine excision. According to the generally accepted cleavage rules for MAP, this enzyme cleaves all proteins with small side chains on the residue in the second position (P1'), but many exceptions are known. The substrate specificity of Escherichia coli MAP1 was studied in vitro with a large (>120) coherent array of peptides mimicking the natural substrates and kinetically analyzed in detail. Peptides with Val or Thr at P1' were much less efficiently cleaved than those with Ala, Cys, Gly, Pro, or Ser in this position. Certain residues at P2', P3', and P4' strongly slowed the reaction, and some proteins with Val and Thr at P1' could not undergo Met cleavage. These in vitro data were fully consistent with data for 862 E. coli proteins with known N-terminal sequences in vivo. The specificity sites were found to be identical to those for the other type of MAPs, MAP2s, and a dedicated prediction tool for Met cleavage is now available. Taking into account the rules of MAP cleavage and leader peptide removal, the N termini of all proteins were predicted from the annotated genome and compared with data obtained in vivo. This analysis showed that proteins displaying N-Met cleavage are overrepresented in vivo. We conclude that protein secretion involving leader peptide cleavage is more frequent than generally thought.     

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.     

Purification and characterization of the Streptococcus salivarius methionine aminopeptidase (MetAP)

Streptococcus salivarius methionine aminopeptidase (MetAP) was purified from a recombinant Escherichia coli strain containing the S. salivarius map gene, which codes for MetAP. S. salivarius map coded for a protein of 286 amino acids with a calculated molecular mass of 31,723 Da and a pI of 4.6. The native enzyme eluted from a Superdex column as a protein with a molecular mass of 30.6 kDa and cleaved N-terminal Met of peptide only when the penultimate amino acid was Gly, Ala, Ser, Val, Pro, or Thr. The enzyme was more active against tetrapeptides than tripeptides and did not recognize dipeptides. It required the presence of a metal cation for activity, with a preference for Co(2+) over Mn(2+). S. salivarius MetAP has a pH optimum of 8.0 and an optimal temperature at 50 degrees C. The S. salivarius protein had an extra sequence of 24 amino acids between two conserved aspartate residues involved in the coordination of the metal ion. A similar extra sequence is present in MetAP from other streptococci and from Lactococcus lactis, but not from other bacteria or eukaryotes.     

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.     

Importance of a hypervariable active-site residue in human Mu class glutathione transferases catalyzing the bioactivation of chemotherapeutic thiopurine prodrugs

Glutathione transferases (GSTs) catalyze the bioactivation of the thiopurine prodrugs azathioprine, cis-6-(2-acetylvinylthio)purine (cAVTP) and trans-6-(2-acetylvinylthio)guanine (tAVTG), thereby releasing the antimetabolites 6-mercaptopurine and 6-thioguanine. In the GST Mu class, GST M1-1 has the highest catalytic efficiency, whereas GST M2-2 and other enzymes are less active. In the evolution of Mu class GSTs, residue 210 appears hypervariable and has particular functional significance. We demonstrate that the catalytic activity of GST M1-1 with cAVTP or tAVTG is successively diminished when wild-type Ser-210 is mutated into Ala followed by Thr. Conversely, mutating wild-type Thr-210 in GST M2-2 into Ala and Ser enhanced the corresponding activities. Comparisons were also made with GST M2-2 distinguished by Gly or Pro in position 210, as well as wild-type GSTs M4-4 and M5-5. The results suggest that the hydroxyl group of Ser in position 210 stabilizes the transition state of the GST-catalyzed reaction. The low activity of GSTs containing Thr in position 210 is probably due to steric hindrance caused by the beta-methyl group of the side chain. The ratios of the different catalytic efficiencies were translated into differences in the Gibbs free energies of transition state stabilization. The effects of the mutations were qualitatively parallel for the alternative substrates, but vary significantly in magnitude. From the evolutionary perspective the data show that a point mutation can alternatively enhance or attenuate the activity with a particular substrate and illustrate the functional plasticity of GSTs.     

Molecular cloning and characterization of a cDNA encoding a novel cuticle protein in the silkworm,Bombyx mori

We have cloned the full length of a novel cDNA named Bombyx mori cuticle protein that contains an AlaAlaProAla/Val-repeat (BMCPA) from a cDNA library of integument in the larval silkworm. Both a typical tandem repeat (A-A-P-A/V) for cuticle protein and a unique tandem repeat with Ser, Ala, Gly, Pro, Val, Tyr and Thr were observed in the predicted amino acid sequence of the cDNA encoding BMCPA. Approximately 80% of the amino acids in BMCPA were composed of Ser, Ala, Gly, Pro, Val and Tyr. Northern-hybridization analysis indicated that BMCPA mRNA is expressed only in the larval epidermis and that the expression pattern of the BMCPA gene in the developmental stage was observed mainly at the larval stage. We propose BMCPA may be a novel component of cuticle, and may play an important role in the integument of the larval silkworm.     

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.     

Occurrence of a new corticotropin-like intermediate lobe peptide in salmon pituitary glands

A new corticotropin-like intermediate lobe peptide (CLIP) has been identified in the pituitary of chum salmon, Oncorhynchus keta. The newly isolated peptide is a tetracosa peptide, which is two residues longer than the predominant form, CLIP I, with the following amino acid sequence, H-ArgProIleLysValTyrAlaSerSerLeuGlu GlyGlyAspSerSerGluGlyThrPheProLeuGlnAlaOH. This peptide, named CLIP II is the fourth line of evidence in the teleost that the pituitary gland secretes two different forms of processed hormones, for which precursor molecules are coded on two separate genes. Together with the structures of α-melanotropin I and II, two putative ACTH molecules are proposed.     

Site Directed Mutagenesis of Schizosaccharomyces pombe Glutathione Synthetase Produces an Enzyme with Homoglutathione Synthetase Activity

Three different His-tagged, mutant forms of the fission yeast glutathione synthetase (GSH2) were derived by site-directed mutagenesis. The mutant and wild-type enzymes were expressed in E. coli DH5α and affinity purified in a two-step procedure. Analysis of enzyme activity showed that it was possible to shift the substrate specificity of GSH2 from Gly (k_m 0,19; wild-type) to β-Ala or Ser. One mutation (substitution of Ile471, Cy472 to Met and Val and Ala 485 and Thr486 to Leu and Pro) increased the affinity of GSH2 for β-Ala (k_m 0,07) and lowered the affinity for Gly (k_m 0,83), which is a characteristic of the enzyme homoglutathione synthetase found in plants. Substitution of Ala485 and Thr486 to Leu and Pro only, increased instead the affinity of GSH2 for Ser (k_m 0,23) as a substrate, while affinity to Gly was preserved (k_m 0,12). This provides a new biosynthetic pathway for hydroxymethyl glutathione, which is known to be synthesized from glutathione and Ser in a reaction catalysed by carboxypeptidase Y. The reported findings provide further insight into how specific amino acids positioned in the GSH2 active site facilitate the recognition of different amino acid substrates, furthermore they support the evolutionary theory that homoglutathione synthetase evolved from glutathione synthetase by a single gene duplication event.     

Amino acid sequences of two tryptic peptides from D(-)-beta-hydroxybutyrate dehydrogenase radiolabeled at essential carboxyl and sulfhydryl groups

D(-)beta-hydroxybutyrate dehydrogenase (BDH) purified from bovine heart mitochondria contains essential thiol and carboxyl groups. A tryptic BDH peptide labeled at an essential thiol with [3H]N-ethylmaleimide (NEM), and another tryptic peptide labeled at an essential carboxyl with N,N'-dicyclohexyl [14C]carbodiimide (DCCD), were isolated and sequenced. The peptide labeled with [3H]NEM had the sequence Met.Glu.Ser.Tyr.Cys*.Thr.Ser. Gly.Ser.Thr.Asp.Thr.Ser.Pro.Val.Ile.Lys. The label was at Cys. The same peptide was isolated from tryptic digests of BDH labeled at its nucleotide-binding site with the photoaffinity labeling reagent, arylazido- -[3-3H] alanyl-NAD. These results suggest that the essential thiol of BDH is located at its nucleotide-binding site, and agree with our previous observation that NAD and NADH protect BDH against inhibition by thiol modifiers. The [14C]DCCD-labeled peptide had the sequence Glu.Val.Ala.Glu*.Val. Asn. Leu.Trp.Gly.Thr.Val.Arg. DCCD appeared to modify the glutamic acid residue marked by an asterisk. Sequence analogies between these peptides and other proteins have been discussed.     

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.     

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.     

Substrate specificity of cucumisin on synthetic peptides

The substrate specificity of cucumisin [EC 3.4.21.25] was identified by the use of the synthetic peptide substrates Leu(m)-Pro-Glu-Ala-Leu(n) (m = 0-4, n = 0-3). Neither Pro-Glu-Ala-Leu (m = 0) nor Leu-Pro-Glu-Ala (n = 0) was cleaved by cucumisin, however other analogus peptides were cleaved between Glu-Ala. The hydrolysis rates of Leu(m)-Pro-Glu-Ala-Leu increased with the increase of m = 1 to 2 and 3, but was however, essentially same with the increase of m = 3 to 4. Similarly, the hydrolysis rates of Leu-Leu-Pro-Glu-Ala-Leu(n) increased with the increase of n = 0 to 1 and 2, but was essentially same with the increase of n = 2 to 3. Then, it was concluded that cucumisin has a S5-S3' subsite length. In order to identify the substrate specificity at P1 position, Leu-Leu-Pro-X-Ala-Leu (X; Gly, Ala, Val, Leu, Ile, Pro, Asp, Glu, Lys, Arg, Asn, Gln, Phe, Tyr, Ser, Thr, Met, Trp, His) were synthesized and digested by cucumisin. Cucumisin showed broad specificity at the P1 position. However, cucumisin did not cleave the C-terminal side of Gly, Ile, Pro, and preferred Leu, Asn, Gln, Thr, and Met, especially Met. Moreover, the substrates, Leu-Leu-Pro-Glu-Y-Leu (Y; Gly, Ala, Ser, Leu, Val, Glu, Lys, Phe) were synthesized and digested by cucumisin. Cucumisin did not cleave the N-terminal side of Val but preferred Gly, Ser, Ala, and Lys especially Ser. The specificity of cucumisin for naturally occurring peptides does not agree strictly with the specificity obtained by synthetic peptides at the P1 or P1' position alone, but it becomes clear that the most of the cleavage sites on naturally occurring peptides by cucumisin contain suitable amino acid residues at P1 and (or) P1' positions. Moreover, cucumisin prefers Pro than Leu at P2 position, indicating that the specificity at P2 position differs from that of papain.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Conformation analysis of the N-glycosylation site Asn-X-Thr/Ser in glycoproteins

Theoretical conformational analysis of oligopeptides CH3CO-Asn-X-Thr-NHCH3 (X = Gly, Ala, Pro), modelling N-glycosylation site, and their glycosylated derivatives CH3CO-(GlcNAc beta 1-4GlcNAc beta 1) Asn-X-Thr-NHCH3 has been carried out. Active conformations of the site are found, corresponding to structural prerequisities of N-glycosylation: Asn residue's position in beta-turn and hydrogen bond formation between side chains of Asn and Thr/Ser residues. In this case the L conformation of the central residue X is most probable. Since Pro residue does not possess this conformation, sequences with X = Pro are not glycosylated. It is shown that glycosylation of the above-mentioned sites is accompanied by reorientation of the Asn residue's side chains.