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 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.     

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.     

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.     

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.     

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.     

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.     

Cleavage specificity of cucumisin, a serine protease, with synthetic substrates

The substrate specificity of a plant serine protease, cucumisin (EC 3.4.21.25), was studied by the use of synthetic oligopeptides and peptidyl-pNA substrates. Since P1'-Ser, Ala, and Gly substrates were hydrolyzed rapidly, cucumisin appears to prefer a small side chain at the P1' position of the oligopeptide substrate. The k(cat)/Km for the hydrolysis of P1-Leu, Ala, Phe, and Glu substrates demonstrated that they were preferentially cleaved over P1-Lys, diaminopropionic acid (Dap), Gly, Val, and Pro substrates. From the digestion of peptidyl-pNAs, the specificity of the protease was determined to be broad, but the preferential cleavage sites were hydrophobic amino acid residues at the P1 position.     

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.     

Kinetics and mechanism of peptide synthesis in solution

The kinetics of the reaction of Boc-Xaa fluorophenyl esters (where Xaa = Ala, Val, Phe, Ser, Leu, Gly, Met, Pro, or Ile) with leucinamide was studied measuring changes in the fluorescence emission at 375 nm of the fluorophenyl chromophore accompanying the reaction. It was found that the experimental kinetic data couldn't be described by a simple scheme of the second order reaction. The measurements of the kinetic parameters of the reaction at various initial concentrations of reagents indicated that the reaction rate can be expressed as: v = kCNaCAEb, where k is the reaction rate constant, CN is the concentration of leucinamide, and LeuNH2, CAE is the concentration of fluorophenyl ester. The a and b reaction orders were close to 1/2 and 3/2 for Xaa = Ala, Val, Phe, Ser, or Leu, 1/2 and 1 for Gly, Met, or Pro, and 1 and 2 for Ile. The experimental equations for the reaction rate can theoretically be derived from a single scheme of chain reactions with various deactivation ways for active intermediates. The English version of the paper.     

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

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

Specificity of the wound-induced leucine aminopeptidase (LAP-A) of tomato activity on dipeptide and tripeptide substrates.

Wounding of tomato leaves results in the accumulation of an exoprotease called leucine aminopeptidase (LAP-A) that preferentially hydrolyzes amino acid-p-nitroanilide and -beta-naphthylamide substrates with N-terminal Leu, Met and Arg residues. To determine the substrate specificity of LAP-A on more natural substrates, the rates of hydrolysis of 60 dipeptide and seven tripeptide substrates were determined. For comparison, the specificities of the porcine and Escherichia coli LAPs were evaluated in parallel. Several marked differences in substrate specificities for the animal, plant and prokaryotic LAP enzymes were observed. Substrates with variable N-terminal (P1) residues (Xaa) were evaluated; these substrates had Leu or Gly in the penultimate (P1') position. The plant, animal, and prokaryotic LAPs hydrolyzed dipeptides with N-terminal nonpolar aliphatic (Leu, Val, Ile, and Ala), basic (Arg), and sulfur-containing (Met) residues rapidly, while P1 Asp or Gly were cleaved inefficiently from peptides. Significant differences in the cleavage of dipeptides with P1 aromatic residues (Phe, Tyr, and Trp) were noted. To systematically evaluate the impact of the P1' residue on cleavage of dipeptides, three series of dipeptides (Leu-Xaa, Gly-Xaa, and Arg-Xaa) were evaluated. The P1' residue strongly influenced hydrolysis of dipeptides and the magnitude of its effect was dependent on the P1 residue. P1' Pro, Asp, Lys and Gly slowed the hydrolysis rates of the tomato LAP-A, porcine LAP, and E. coli PepA markedly. Analysis six Arg-Gly-Xaa tripeptides showed that more diversity was tolerated in the P2' position. P2' Arg inhibited tripeptide cleavage by all three enzymes, while P2' Asp enhanced hydrolysis rates for the porcine and prokaryotic LAPs.     

Identification of coding polymorphisms in human circadian rhythm genes PER1, PER2, PER3, CLOCK, ARNTL, CRY1, CRY2 and TIMELESS in a multi-ethnic screening panel.

STUDY OBJECTIVE: In this study, the exonic regions of the circadian rhythm genes PER1, PER2, PER3, CLOCK, ARNTL, CRY1, CRY2 and TIMELESS were re-sequenced and coding changes identified in a panel of 95 individuals varying in ethnicity. STUDY PARTICIPANTS: DNA screening panel consisting of 95 DNA samples (17 American Caucasians, 17 African Americans, 8 Ashkenazi Jews, 8 Chinese, 8 Japanese, 5 Mexican Indians, 8 Mexicans, 8 Northern Europeans, 8 Puerto Ricans, and 8 South Americans) selected from the Coriell Institute Human Variation Panel. RESULTS: In addition to coding changes already identified in the database dbSNP, novel coding changes were identified, including PER1: Pro37Ser, Pro351Ser, Gln988Pro, Ala998Thr; PER2: Leu83Arg, Leu157Leu, Thre174Ile, Phe400Phe, Pro822Pro, Ala828Thr, Ala861Val, Phe876Leu, Val883Met, Val903Ile, Ala923Pro; PER3: Pro67Pro, Val90Ile, His638His, Ala820Ala, Leu929Leu; ARNTL: Arg166Gln, Ser459Phe; CLOCK: Ala34Ala, Ser208Cys, Phe233Phe, Ser632Thr, Ser816Ser; TIMELESS: Met870Val and CRY2: His35His. No coding polymorphisms were identified in CRY1. CONCLUSIONS: Considerable genetic variation occurs within the coding region of the genes regulating circadian rhythm. Many of the non-synonymous coding polymorphisms could affect protein structure/function with the potential to affect molecular regulation of the sleep/wake cycle. Many of the potential functional effects could be ethnic group specific.     

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.     

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.     

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.     

Substrate specificity of ascidian sperm trypsin-like proteases,spermosin and acrosin

In order to investigate systematically the substrate or subsite specificity of two sperm proteases; acrosin and spermosin (a novel trypsin-like protease) of the ascidian, Halocynthia roretzi, the effects of peptidyl-argininals on the purified enzymes as well as on fertilization were examined. Among four benzyloxycarbonyl (Z)-Leu-X-argininals (X = Pro, Leu, Ser, and Gly), Z-Leu-Pro-argininal showed the strongest inhibition toward the spermosin activity. On the P3 site specificity, Val-Pro-argininal derivatives showed a stronger inhibition than a Leu-Pro-argininal derivative, suggesting the preference of Val rather than Leu residue at the P3 position. Similar results were obtained by analyzing the hydrolyzing activity of the fluorogenic peptide substrates: it hydrolyzed Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide (MCA) most efficiently, and Boc-Asp(O-benzyl)-Pro-Arg-MCA was the next best substrate, but Gly-Pro-Arg (or Lys)-MCAs were hardly hydrolyzed. On the other hand, acrosin was found to prefer Leu or Pro residue rather than Gly or Ser residue at the P2 position as revealed by comparing the Ki values of peptidyl-argininals. Detailed kinetic analysis on the inhibitory abilities of peptidyl-argininals toward the purified enzymes and the ascidian fertilization suggested that both acrosin and spermosin are involved in ascidian fertilization. © 1996 Wiley Liss, Inc.     

Differential effect of amino acid residues on the stability of double helices formed from polyribonucleotides and its possible relation to the evolution of the genetic code

The interaction of amino acid residues with polyribonucleotides was characterized by measurements of melting temperatures (tm) for poly(A).poly(U) and poly(I).poly(C) as functions of the concentrations of various amino acid amides. The amides of hydrophilic amino acids lead to a continuous increase of tm with increasing concentration, whereas amides of hydrophobic amino acids induce a decrease of tm at low concentrations (approximately 1 mM) followed by an increase at higher concentrations. Analysis of the data by a simple site model provides the affinity of each ligand for the double helix relative to that for the single strands. This parameter decreases in the order Ala greater than Gly greater than Ser greater than Asn greater than Pro greater than Met, Val greater than Ile, Leu for poly(A).poly(U) and Ala, Gly, Ser greater than Asn greater than Pro greater than Val greater than Ile, Met, Leu for poly(I).poly(C). The special effects of hydrophobic amino acids may be related to the similarity of the codons for these amino acids. A simple model for assignment of codons to amino acids is proposed.     

Design, chemical synthesis and kinetic studies of trypsin chromogenic substrates based on the proteinase binding loop of Cucurbita maxima trypsin inhibitor (CMTI-III)

A series of trypsin chromogenic substrates with formula: Y-Ala-X-Abu-Pro-Lys-pNA, where X = Gly, Ala, Abu, Val, Leu, Phe, Ser, Glu and Y = Ac, H; pNA = p-nitroanilide was synthesized. The Cucurbita maxima trypsin inhibitor CMTI-III molecule was used as a vehicle to design the trypsin substrates. To evaluate the influence of position P(4) on the substrate-enzyme interaction, kinetic parameters of newly synthesized substrates with bovine beta-trypsin were determined. The increasing hydrophobicity of the amino acid residue (Gly, Ala, Abu, Val) introduced in position P(4) significantly enhanced the substrate specificity (k(cat)/K(m)) which was over 8 times higher for the last residue than that for the first one. The introduction of residues with more hydrophilic side chain (Glu, Ser) in this position reduced the value of this parameter. These results correspond well with those obtained using molecular dynamics of bovine beta-trypsin with monosubstituted CMTI-I analogues, indicating that in both trypsin substrate and inhibitor position 4 plays an important role in the interaction with the enzyme.