On the Specificity of a Rennin-like Enzyme from Mucor pusillus

The specificity of a rennin-like enzyme from Mucor pusillus Lindt was determined using synthetic peptides and oxidized insulin B chain as substrates. The results indicate that the enzyme exhibits specificity against aromatic, bulky or hydrophobic amino acid residues at both sides of the splitting point. The susceptibility of peptide substrates increases with the increase of their molecular size, indicating the significance of secondary interaction for hydrolysis. Z-tetrapeptides such as (the arrows show the bond split) are found as efficient substrates for the enzyme. The main points of cleavage in oxidized insulin B chain are; Phe-Val (1–2), Ala-Leu (14–15), Leu-Tyr (15–16), Tyr-Leu (16–17), and Phe-Phe (24–25).

The specificity of the M. pusillus enzyme is almost identical with that of the rennin-like enzyme from Mucor miehei, and similar to those of usual acid proteinases possessing tryp- sinogen activating ability, except that the latter enzymes show specificity against basic amino acid residues at the carbonyl-side of the splitting point.     

Comparative specificity of microbial acid proteinases for synthetic peptides. 3. Relationship with their trypsinogen activating ability

The specificities of acid proteinases from Aspergillus niger, Aspergillus saitoi, Rhizopus chinensis, Mucor miehei, Rhodotorula glutinis, and Cladosporium sp., and that of swine pepsin, were determined and compared with ability of the enzymes to activate trypsinogen. Various oligopeptides containing l-lysine, Z-Lys-X-Ala, Z-Lys-(Ala)_m, Z-Lys-Leu-(Ala)₂, and Z-(Ala)_n-Lys-(Ala)₃ (X = various amino acid residues, m = 1–4, n = 1–2) were used as substrates. Of the enzymes which are able to activate trypsinogen, most split these peptides at the peptide bond formed by the carbonyl group of l-lysine. For the peptides to be susceptible to the enzymes it was essential that the chain extended for two or three amino acid residues on the C-terminal side of the catalytic point, and that a bulky or hydrophobic amino acid residue formed the imino-side of the splitting point. The rate of hydrolysis was markedly accelerated by elongation of the peptide chain with l-alanine on the N-terminal side of the catalytic point. Thus, of the substrates used, Z-(Ala)₂-Lys-(Ala)₃ was the most susceptible to the microbial acid proteinases possessing trypsinogen activating ability. On the other hand, M. miehei enzyme and pepsin, which do not activate trypsinogen, showed very little peptidase activity on the peptides.     

Purification and Properties of Hydroxycinnamic Acid Ester Hydrolase from Aspergillus japonicus

Hydroxycinnamic acid ester hydrolase from the wheat bran culture medium of Aspergillus japonicus was purified 255-fold by ammonium sulfate fractionation, DEAE-Sephadex treatment and column chromatographies on DEAE-Sephadex, CM-Sephadex and various other Sephadexes. The purified enzyme was free from tannase and found to be homogeneous on polyacrylamide disc gel electrophoresis. Its molecular weight was estimated to be 150,000 by gel filtration and 142,000 by SDS-gel electrophoresis. The isoelectric point of the enzyme was pH 4.80. As to its amino acid composition, aspartic acid and glycine were abundant. The optimum pH and temperature for the enzyme reaction were, respectively, 6.5 and 55°C when chlorogenic acid was used as a substrate. The enzyme was stable between pH 3.0 to 7.5 and inactivated completely by heat treatment at 70°C for 10 min.

All metal ions examined did not activate the enzyme, while Hg⁺⁺ reduced its activity. The enzyme was markedly inhibited by diisopropylfluorophosphate and an oxidizing reagent, iodine, although it was not affected so much by metal chelating or reducing reagents. The purified enzyme hydrolyzed not only esters of hydroxycinnamic acids such as chlorogenic acid, caffeoyl tartaric acid and p-coumaroyl tartaric acid, but also ethyl and benzyl esters of cinnamic acid. However, the enzyme did not act on ethyl esters of crotonic acid and acrylic acid or esters of hydroxybenzoic acids.     

Synthesis and Use of New Semispecific Substrates for Trypsin-Catalyzed Peptide Bond Formation

Two series of semispecific acyl donors, hydroxyalkyl esters of Z-Ala-OH and TV-modified carboxamidomethyl (Cam) esters of Z-Xaa-OH (Xaa = Ala, Leu, Phe) were synthesized as substrates for trypsin-catalyzed peptide synthesis. It follows from the specificity constants of these compounds, that the carboxamidomethyl derivatives are well accepted by trypsin due to favourable S₂′ – P₂′ interactions. These new substrates can be successfully used for the trypsin-mediated formation of dipeptide amides. The synthesis outcome depends on the amino acid in the P₁ position, the ability of the leaving group to provide efficient interactions with the enzyme subsite and the hydrophobicity of the nucleophilic amino acid amide. The modified Cam esters give better peptide yields in comparison to the unmodified ones.     

Comparative specificity of microbial acid proteinases for synthetic peptides. Primary specificity with Z-tetrapeptides

The substrates Z-X

Leu-(Ala)₂ and

Z-Phe X-(Ala)₂ (Z = benzyloxycarbonyl, X = various amino acid residues) were synthesized in order to investigate the primary specificity of acid proteinases from molds and yeasts. Since these peptides are mainly susceptible to cleavage by the enzymes at the peptide bonds shown by the arrows, it was possible to determine the specificity with respect to the amino acid residues on both sides of the splitting point. Pepsin was used for comparison. The results indicated that the microbial acid proteinases exhibit specificity for aromatic or hydrophobic amino acid residues on both sides of splitting point in peptide substrates, as does pepsin. However, the microbial enzymes showed somewhat broader specificity than pepsin. The former enzymes, which possess trypsinogen-activating ability, show specificity for a lysine residue, while pepsin or Mucor rennin-like enzyme does not. Although pepsin is very specific for a tyrosine residue on the imino side of the splitting point, the microbial enzymes do not show such stringency.     

Design, synthesis and catalytic activity of a serine protease synthetic model

Summary The design, synthesis and catalytic properties of a cyclic branched peptide carrier that possesses the catalytic triad residues of the serine proteases is reported. The synthesis of the peptide model was totally completed on solid support using three different orthogonal amino protecting groups. Hydrolytic activity measurements against Suc-Ala-Ala-Ala-pNA substrate showed that it is hydrolysed by the peptide model to a small extent. Despite this small hydrolytic activity, it is the first time, to our knowledge, that hydrolysis of such a substrate is reported by an enzyme model compound. Contrary, this enzyme model peptide showed considerable activity against the Boc-Ala-pNP substrate (k _cat =0.414 min⁻¹ andK _m =0.228 mm). These results suggest that the designed carrier brings in appropriate contact the catalytic triad residues (Ser, His, Asp) resulting in the obtained hydrolytic activity.     

Isolation and characterization of a ferulic acid esterase (Fae1A) from the rumen fungus Anaeromyces mucronatus

Aims: A novel ferulic acid esterase gene from rumen fungus Anaeromyces mucronatus was cloned, heteroexpressed in Escherichia coli and characterized. Methods and Results: A total of 30 clones exhibiting activity on α‐naphthyl acetate (α‐NA) were isolated from an A. mucronatus YE505 cDNA library. Sequence analysis revealed that these clones represented two esterase‐coding sequences. The gene, fae1A, showed highest amino acid sequence identity to CE family 1 esterases from anaerobic micro‐organisms such as Orpinomyces sp., Ruminococcus albus and Clostridium thermocellum. The gene comprised 828 nucleotides encoding a polypeptide of 275 amino acids. The coding sequence was cloned into the pET30a expression vector and overexpressed in E. coli BL21 (DE3). Gene product Fae1A was found to exhibit activity against a number of substrates including naphthyl fatty acid esters, p‐nitrophenyl fatty acid esters and hydroxylcinnamic acid esters. Conclusions: Fae1A exhibited a lower K_m and higher catalytic efficiency (k_cat/K_m) on ferulic acid esters than on α‐NA or p‐nitrophenyl acetate, suggesting that it has a higher affinity for ethyl and methyl ferulate than for the acetyl esters. It releases ferulic acid and p‐coumaric acid from barley straw. Activity of Fae1A was inhibited by the serine‐specific protease inhibitor, phenylmethylsulfonyl fluoride, indicating that a serine residue plays a role in its activity. Significance and Impact of the Study: To our knowledge, this is the first report of characterization of carbohydrate esterase gene from the genus of Anaeromyces.     

Peptide derived from the lipid binding domain of Group IB human pancreatic phospholipase A2 possesses antibacterial activity

Group 1B human pancreatic secretory phospholipase A₂ (hp-sPLA₂), a digestive enzyme synthesized by pancreatic acinar cells and present in pancreatic juice, do not have antibacterial activity towards Escherichia coli. Our earlier results suggest that the N-terminal first ten amino acid residues of hp-sPLA₂ constitute major portion of the membrane binding domain of full-length enzyme and is responsible for the precise orientation of enzyme on the membrane surface by inserting into the lipid bilayers (Pande et al. (2006) Biochemistry, 45,12436–12447). In this study we report the antibacterial properties of a peptide (AVWQFRKMIK-CONH₂; N10 peptide), which corresponds to the N-terminal first ten amino acid residues of hp-sPLA₂, against E. coli. Full-length hp-sPLA₂, which contains this peptide sequence as N-terminal α-helix, did not showed detectable antibacterial activity. Presence of physiological concentration of salt or preincubation of N10 peptide with soluble anionic polymer inhibits the antibacterial activity indicating the importance of electrostatic interaction in binding of peptide to bacterial membrane. Addition of peptide resulted in destabilization of outer as well as inner cytoplasmic membrane of E. coli suggesting bacterial membranes to be the main target of action. N10 peptide exhibits strong synergism with lysozyme and potentiates the antibacterial activity of lysozyme. The peptide was inactive against human erythrocyte. Our result shows for the first time that a peptide fragment of hp-sPLA₂ possesses antibacterial activity towards E. coli and at subinhibitory concentration and can potentiate the antibacterial activity of membrane active enzyme. These observations suggest that N10 peptide may play an important role in the antimicrobial activity of pancreatic juice.     

Enzymatic peptide synthesis by the recombinant proline-specific endopeptidase from Flavobacterium meningosepticum and its mutationally altered Cys-556 variant

Proline-specific endopeptidase (PSE) (EC 3.4.21.26) was investigated for its potential as a catalyst in peptide synthesis. Using an activated peptide ester or a peptide amide as the acyl component, the enzyme catalyzed kinetically controlled aminolysis and transpeptidation respectively, with various amino acid amides as acyl acceptors. To a certain extent the nucleophile preference reflected the amino acid preference in the S₁-position of the enzyme in peptide hydrolysis: the highest fractions of aminolysis were obtained using amino acid amides with hydrophobic side-chains (e.g. Leu-NH₂, Phe-NH₂). PSE also catalyzed the thermodynamically controlled condensation of short peptides with a free carboxyterminus and various amino acid amides. This enabled us to examine the acceptance of different acyl components in the substrate-binding site of the enzyme with regard to their amino acid composition: In the S₁ position proline was clearly favored, but alanine was also accepted, whereas the S₂ subsite accepted various amino acids rather unspecifically. Since PSE was shown to be extremely sensitive against water-miscible organic solvents, an alternative approach was used to increase yields in enzymatic peptide synthesis: a derivative of PSE in which the catalytic Ser-556 is converted to a Cys was constructed by protein engineering. This mutant (PSE_cys) exhibited a dramatically increased peptide ligase activity in aqueous solution.     

Structure–activity study of macropin,a novel antimicrobial peptide from the venom of solitary bee Macropis fulvipes (Hymenoptera: Melittidae)

A novel antimicrobial peptide, designated macropin (MAC‐1) with sequence Gly‐Phe‐Gly‐Met‐Ala‐Leu‐Lys‐Leu‐Leu‐Lys‐Lys‐Val‐Leu‐NH₂, was isolated from the venom of the solitary bee Macropis fulvipes. MAC‐1 exhibited antimicrobial activity against both Gram‐positive and Gram‐negative bacteria, antifungal activity, and moderate hemolytic activity against human red blood cells. A series of macropin analogs were prepared to further evaluate the effect of structural alterations on antimicrobial and hemolytic activities and stability in human serum. The antimicrobial activities of several analogs against pathogenic Pseudomonas aeruginosa were significantly increased while their toxicity against human red blood cells was decreased. The activity enhancement is related to the introduction of either l ‐ or d ‐lysine in selected positions. Furthermore, all‐d analog and analogs with d ‐amino acid residues introduced at the N‐terminal part of the peptide chain exhibited better serum stability than did natural macropin. Data obtained by CD spectroscopy suggest a propensity of the peptide to adopt an amphipathic α‐helical secondary structure in the presence of trifluoroethanol or membrane‐mimicking sodium dodecyl sulfate. In addition, the study elucidates the structure–activity relationship for the effect of d ‐amino acid substitutions in MAC‐1 using NMR spectroscopy. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Enantioselectivity of Enzymatic Cleavage Reaction of 13-Hydroperoxylinolenic Acid to C6-Aldehyde and C12-Oxo Acid in Tea Chloroplasts

A protease with strict specificity to lysyl peptide bonds like that of Achromobacter protease I was purified from a crude enzyme powder obtained from a culture filtrate of Achromobacter lyticus M497-1 and characterized. The purified enzyme had the following differences from protease I. The enzyme had an isoelectric point of 5.3, lower than the value of 6.9 for protease I. The amino acid composition of the enzyme had higher proportions of His, Glu, and Gly and lower proportions of Arg and Thr than protease I. The enzyme was unstable (30% residual activity) in the presence of 7 m urea (pH 8.0, 30°C, 20 min); protease I was resistant to the same conditions (80% residual activity). The k_cat/Km values for the hydrolysis of Tos-Lys-OMe and Lys-pNA by the enzyme were lower than those of protease I.     

Purification, characterization, and molecular cloning of organic-solvent-tolerant cholesterol esterase from cyclohexane-tolerant Burkholderia cepacia strain ST-200

Extracellular cholesterol esterase of Burkholderia cepacia strain ST-200 was purified from the culture supernatant. Its molecular mass was 37 kDa. The enzyme was stable at pH 5.5–12 and active at pH 5.5–6, showing optimal activity at pH 7.0 at 45°C. Relative to the commercially available cholesterol esterases, the purified enzyme was highly stable in the presence of various water-miscible organic solvents. The enzyme preferentially hydrolyzed long-chain fatty acid esters of cholesterol, except for that of cholesteryl palmitate. The enzyme exhibited lipolytic activity toward various p-nitrophenyl esters. The hydrolysis rate of p-nitrophenyl caprylate was enhanced 3.5- to 7.2-fold in the presence of 5–20% (vol/vol) water-miscible organic solvents relative to that in the absence of organic solvents. The structural gene encoding the cholesterol esterase was cloned and sequenced. The primary translation product was predicted to be 365 amino acid residues. The mature product is composed of 325 amino acid residues. The amino acid sequence of the product showed the highest similarity to the lipase LipA (87%) from B. cepacia DSM3959.     

Comparative study of various serine alkaline proteinases from microorganisms. Esterase activity against N-acylated peptide ester substrates

Hydrolyses of N-acylated peptide ester substrates by various serine alkaline proteinases from bacterial and mold origin were compared using Ac- or Z-(Ala)_m-X-OMe (m = 0-2 or 0-3; X = phenylalanine, alanine, and lysine) as esterase substrates. The results indicated that the esterase activities of these enzymes were markedly promoted by elongating the peptide chain from P₁ to P₂ or P₃ with alanine, irrespective of the kind of the amino acid residue at the P₁-position (amino acid residues in peptide substrates are numbered according to the system of Schechter and Berger (1)). The effect of the kind of amino acid residue at the P₂-position was further determined using Z-X-Lys-OMe (X = glycine, alanine, leucine, or phenylalanine) as esterase substrates. Alanine was the most efficient residue as X with subtilisins and Streptomyces fradiae Ib enzyme, while leucine or phenylalanine were most efficient with the enzymes from Streptomyces fradiae II, Aspergillus sojae, and Aspergillus melleus. All the serine alkaline proteinases tested in this study were sensitive to Z-Ala-Gly-PheCH₂Cl, the dependence of inhibition on the inhibitor concentration differed among the enzymes.     

Chemical Modification of Neutral Protease from Bacillus subtilis var. amylosacchariticus: Assignment of Tyrosyl Residues Iodinated

The neutral protease of Bacillus subtilis var. amylosacchariticus (B. amylosacchariticus) was iodinated with a 25-fold molar excess of iodine at pH 9.4 for 3 min at 0°C, by which treatment the proteolytic activity toward casein was markedly reduced, while the hydrolytic activity toward an N-blocked peptide substrate was rather increased. The modified enzyme was digested with Staphylococcus aureus V8 protease at pH 8.0 and the amino acid sequences of resultant peptides were compared with those obtained from the native enzyme. One of the peptides was found to have an amino acid sequence of Thr-Ala-Asn-Leu-Ile-Tyr-Glu, which corresponds to residue Nos. 153—159 of the enzyme, where Tyr-158 was identified to be mono-iodotyrosine. The other two peptides were those containing Tyr-21 which was mono- and di-iodinated, respectively. Referring to nitration experiments on the neutral protease and the active site structure of thermolysin, it was concluded that the iodination of Tyr-158 is mainly responsible for the activity changes of B. amylosacchariticus neutral protease.     

Purification of branched-chain amino acid aminotransferase from <Emphasis Type="Italic">Helicobacter pylori</Emphasis> NCTC 11637

Summary. Branched-chain amino acid aminotransferase was purified by several column chromatographies from Helicobacter pylori NCTC 11637, and the N-terminal amino acid sequence was analyzed. The enzyme gene was sequenced based on a putative branched-chain amino acid aminotransferase gene, ilvE of H. pylori 26695, and the whole amino acid sequence was deduced from the nucleotide sequence. The enzyme existed in a homodimer with a calculated subunit molecular weight (MW) of 37,539 and an isoelectric point (pI) of 6.47. The enzyme showed high affinity to 2-oxoglutarate (K _m = 0.085 mM) and L-isoleucine (K _m = 0.34 mM), and V _max was 27.3 μmol/min/mg. The best substrate was found to be L-isoleucine followed by L-leucine and L-valine. No activity was shown toward the D-enantiomers of these amino acids. The optimal pH and temperature were pH 8.0 and 37 °C, respectively.     

A Crystalline Aminopeptidase from Streptomyces peptidofaciens: Physico-chemical Properties and Characteristics as a Ca-Metalloprotease

A crystalline aminopeptidase obtained from the culture filtrate of Streptomyces peptidofaciens KY 2389 appeared to be homogeneous on ultracentrifugation and acrylamide gel electrophoresis. The sedimentation coefficient, s_{20, w}., was determined to be 2.6 S. The molecular weight was estimated to be approximately 19,000 by sedimentation equilibrium studies. The amino acid analyses indicated that the enzyme was composed of 147 amino acid residues and contained no sulfhydryl group. The isoelectric point was found to be around pH 7.4 by isoelectric focusing on ampholites.

The enzyme required Ca²⁺ for its maximal activity and was strongly inhibited by some metal-chelating agents such as ethylenediaminetetraacetic acid (EDTA) and o-phenanthroline. The EDTA-inactivated enzyme restored its activity almost completely by the addition of Ca²⁺ The crystalline preparation of aminopeptidase contained 1 g-atom of calcium and about 2 g-atoms of magnesium per mole of enzyme protein, and the calcium was essential for the activity of the enzyme.     

Purification and characterization of a newly screened microbial peptide amidase

A microbial peptide amidase was found in a limited screening and purified about 500-fold from Stenotrophomonas maltophilia. The native enzyme has a molecular mass of 38 kDa (gel filtration). The sequence of the first 16 amino acids was determined by Edman degradation. The isoelectric point was found to be around 5.8. The peptide amidase exhibited a pH optimum of 6.0 and a temperature optimum of about 39–45°C. The enzyme is stable in 50 mM TRIS/HCl, pH 7.5, at 30°C, and the residual activity was found to be above 90% after 1 week of incubation. The biocatalyst is not inhibited by potential inhibitors like Hg²⁺, EDTA, d-cycloserine or dithiothreitol and only weakly influenced by inhibitors of serine proteases. The peptide amidase deamidates selectively C-terminal amide groups in peptide amides without hydrolysing internal peptide bonds or amide functions in the side-chain of glutamine or asparagine. Unprotected amino acid amides are not hydrolysed. The enzyme is stereoselective with regard to l-enantiomers in the C-terminal position.     

A new acid carboxypeptidase,O-1, fromAspergillus oryzae

A new acid carboxypeptidase was purified fromAspergillus oryzae grown on solid bran culture medium. The purified enzyme was found to be homogeneous by disc gel electrophoresis at pH 9.4 and isoelectric focusing. The enzyme was termedA. oryzae acid carboxypeptidase O-1 with isoelectric point 4.08. The substrate specificity of the new enzyme was investigated with proangiotensin, angiotensin, and bradykinin. Even when the proline was present at the penultimate position of the peptide, the enzyme rapidly hydrolyzed the carboxyterminal Pro-X (X=amino acid) peptide bond. TheK _m andk _cat values for angiotension (–Pro⁷–Phe⁸) at pH 3.7 and 30°C were 0.2 mM and 1.7 sec^–1, respectively.     

Purification and Biochemical Characterization of Indole-3-acetyl-aspartic Acid Synthetase from Immature Seeds of Pea (Pisum sativum)

Indole-3-acetic acid (IAA) amidosynthetases catalyzing the ATP-dependent conjugation of IAA and amino acids play an important role in the maintenance of auxin homeostasis in plant cells. A new amidosynthetase, indole-3-acetic acid:l-aspartic acid ligase (IAA-Asp synthetase) involved in IAA-amino acid biosynthesis, was isolated via a biochemical approach from immature seeds of the pea (Pisum sativum L). The enzyme was purified to homogeneity by a three-step procedure, involving PEG 6000 fractionation, DEAE-Sephacel anion-exchange chromatography, and preparative PAGE, and characterized as a 70-kDa monomeric protein by analytical gel filtration and SDS-PAGE. Rabbit antiserum against recombinant AtGH3.5 cross-reacted with the pea IAA-Asp synthetase, and a single immunoreactive polypeptide band was observed at 70 kDa. The purified enzyme had an apparent isoelectric point at pH 4.7, the highest activity at pH 8.2, preferred Mg²⁺ as a cofactor, and was strongly activated by reducing agents. Similar to known recombinant GH3 enzymes, an IAA-Asp synthetase from pea catalyzes the conjugation of phytohormone acyl substrates to amino acids. The enzyme had the highest synthesizing activity on IAA, followed by 1-NAA, SA, 2,4-D, and IBA, whereas activities on l-Trp, IPA, PAA, (±)JA, and 2-NAA were not significant or not detected. Of 14 amino acids tested, the enzyme had the highest activity on Asp and lower activity on Ala and Lys. Glutamate was found to be a very poor substrate and no conjugating activity was observed on the rest of the amino acids. Steady-state kinetic analysis indicated that IAA and aspartate were preferred substrates for the pea IAA-Asp synthetase. The enzyme exhibited both higher affinities for IAA and Asp (K _m = 0.2 and 2.5 mM, respectively) and catalytic efficiencies (k _cat/K _m = 682,608.7 and 5080 s⁻¹ M⁻¹, respectively) compared with other auxins and amino acids examined. This study describes the first amidosynthetase isolated and purified from plant tissue and provides the foundation for future genetic approaches to explain the role of IAA-Asp in Pisum sativum physiology.     

Therapeutic benefits of 9-amino acid peptide derived from prothymosin alpha against ischemic damages

Prothymosin alpha (ProTα), a nuclear protein, plays multiple functions including cell survival. Most recently, we demonstrated that the active 30-amino acid peptide sequence/P₃₀ (amino acids 49–78) in ProTα retains its substantial activity in neuroprotection in vitro and in vivo as well as in the inhibition of cerebral blood vessel damages by the ischemic stress in retina and brain. But, it has remained to identify the minimum peptide sequence in ProTα that retains neuroprotective activity. The present study using the experiments of alanine scanning suggested that any amino acid in 9-amino acid peptide sequence/P₉ (amino acids 52–60) of P₃₀ peptide is necessary for its survival activity of cultured rat cortical neurons against the ischemic stress. In the retinal ischemia-perfusion model, intravitreous injection of P₉ 24 h after ischemia significantly inhibited the cellular and functional damages at day 7. On the other hand, 2,3,5-triphenyltetrazolium chloride (TTC) staining and electroretinogram assessment showed that systemic delivery with P₉ 1 h after the cerebral ischemia (1 h tMCAO) significantly blocks the ischemia-induced brain damages. In addition, systemic P₉ delivery markedly inhibited the cerebral ischemia (tMCAO)-induced disruption of blood vessels in brain. Taken together, the present study provides a therapeutic importance of 9-amino acid peptide sequence against ischemic damages.