The influence of the side-chain sequence on the structure-activity correlations of immunomodulatory branched polypeptides. Synthesis and conformational analysis of new model polypeptides

New branched polypeptides were synthesized for a detailed study of the influence of the side-chain structure on the conformation and biological properties. The first subset of polypeptides were prepared by coupling of tetrapeptides to poly[L-Lys]. These polymers contain either DL-Ala3-X [poly[Lys-(X-DL-Ala3)n]] or X-DL-Ala3 [poly[Lys-(DL-Ala3-X)n] (n less than or equal to 1)] tetrapeptide side chains. Another group of branched polymers comprise a mixture of DL-Alam and of DL-Alam-X oligomeric branches in a random distribution [poly[Lys-(DL-Alam-Xi)] (i less than 1, m approximately 3)]. In each subset the X = Leu or Phe derivatives were prepared. The N-protected tetrapeptides were synthesized by conventional liquid phase methods and were coupled as active esters. The degree of racemization was found relatively high both for active esters and coupled derivatives, when optically active amino acids were in the C-terminal position of the tetrapeptides. In the case of the poly[Lys-(Leu-DL-Ala3)n] derivative, comparative experiments were carried out using various methodical alterations. The highest stereochemical homogeniety could be achieved when the tetrapeptide active ester was synthesized by the "backing off" method. CD spectra of poly[Lys-(Xi-DL-Alam)] (i less than 1, m approximately 3) and of poly[Lys-(X-DL-Ala3)n] were analyzed and compared to those of poly[Lys-(DL-Alam-Xi)] and of poly[Lys-(DL-Ala3-X)n]. All measurements were performed in water solutions of varying pH values and ionic strengths. The data obtained suggest that branched polypeptides containing a mixture of two different types of oligomeric side chains (DL-Alam and DL-Alam-Xi or Xi-DL-Alam) distributed randomly adopt an almost identical conformation to those that comprise only the respective tetrapeptide (DL-Ala3-X or X-DL-Ala3) branches. The results also indicate that the tendency to form an ordered structure is determined by the identity and the position of the chiral amino acid X (Phe or Leu) in the side chain.     

Methotrexate conjugate with branched polypeptide influences Leishmania donovani infection in vitro and in experimental animals

Methotrexate (MTX) has been coupled to various structurally related, polycationic (poly[Lys(DL-Ala(m))] (AK), poly[Lys(Ser(i)-DL-Ala(m))] (SAK), poly[Lys(DL-Ala(m)-Leu(i))] (ALK)), or amphoteric (poly[Lys(Glu(i)-DL-Ala(m))] (EAK)) synthetic branched polypeptides containing poly[L-Lys] backbone by the aid of BOP reagent. The average degree of MTX incorporation was found to be dependent on the charge properties of the polymer. Under the experimental conditions used, the molar substitution ratio achieved was higher for polycations (25%) than for the amphoteric polypeptide (10%). We have studied the effect of polycationic polypeptides on Leishmania donovani infection. Results demonstrated that MTX conjugates in which the drug is covalently attached to carrier have pronounced leishmanicid activity. In this communication we showed that (a) a branched polypeptide-methotrexate conjugate with a polycationic carrier (ALK) increases the effect of MTX against Leishmania donovani infection in mice; (b) the covalent bond between the carrier and methotrexate is essential for both in vivo and in vitro activity; and (c) the number of Leishmania donovani parasites in infected macrophages are markedly reduced in conjugate treated animals. In vitro observation might also indicate that the MTX conjugate exhibits an effect through an uptake by macrophages which is different from that of the free drug.     

A novel synthetic peptide polymer with cyclic RGD motifs supports serum-free attachment of anchorage-dependent cells

Cell adhesivity is a basic biological principle, which provides mechanisms for construction of multicellular organisms, tissue genesis, migration and individual cell survival. In vivo, the cell adhesive environment is provided by extracellular matrix molecules, neighboring cell surfaces and soluble factors delivered either by tissue cells or by blood circulation. The exact molecular composition of the microenvironment of a cell is not properly understood. The nondefined molecular composition of "native" adhesive components hinders their application when defined culture conditions are necessary, as, for an example, growing human cells for further clinical application. Applying large, substrate-coating molecules as backbones for carrying specific adhesive peptide motifs provides a relatively cheap, reproducible, and chemically defined group of synthetic adhesion molecules. Here, we report on the design, synthesis, and testing of a novel cyclic RGD-containing coating material, which promotes initial attachment, spreading, survival, and proliferation of a number of different cell types. The potent adhesive polypeptide-brush, composed of poly[Lys(DL-Ala(m))] branched chain polypeptide (AK) and multiple copies of cyclic(arginyl-glycyl-aspartyl-D-phenylalanyl-cysteine) pentapeptide prevents anoikis and supports cell attachment in the absence of serum or other biological additives. The defined conditions for cell maintenance make this material a promising candidate for coating artificial cell substrates even for therapeutic applications.     

Primary sequence determination of a Kunitz inhibitor isolated from Delonix regia seeds

A serine proteinase inhibitor was purified from Delonix regia seeds a Leguminosae tree of the Caesalpinioideae subfamily. The inhibitor named DrTI, inactivated trypsin and human plasma kallikrein with K(i )values 2.19x10(-8) M and 5.25 nM, respectively. Its analysis by SDS-PAGE 10-20% showed that the inhibitor is a protein with a single polypeptide chain of M(r) 22 h Da. The primary sequence of the inhibitor was determined by Edman degradation, thus indicating that it contained 185 amino acids and showed that it belongs to the Kunitz type family; however, its reactive site did not contain Arg or Lys at the putative reactive site (position 63, SbTI numbering) or it was displaced when compared to other Kunitz-type inhibitors.     

Conjugation of epitope peptides with SH group to branched chain polymeric polypeptides via Cys(Npys)

Since bioconjugates may play an important role as therapeutics in the future, the development of new and effective conjugation strategies is necessary. For the attachment of peptide-like molecules to carriers, there are two main coupling methods involving amide or disulfide bonds. Conjugation through an amide bond can be achieved in several well-defined ways known from peptide chemistry. However, the formation of disulfide bridges between cysteine-containing peptides and carrier molecules still has some problems. In this paper, we describe a novel approach in which the carrier polypeptide is modified by 3-nitro-2-pyridinesulfenyl (Npys)-protected cysteine and this derivative has been applied for conjugation of Cys-containing epitope peptides with poly(L-lysine)-based branched polypeptides. Considering the stability of Npys group in the presence of pentafluorophenol, Boc-Cys(Npys)-OPfp dervivative was selected for introduction to the N-terminal of branches of polypeptides backbone. The branches of the polymers were built up from oligo(DL-alanine) (poly[Lys(DL-Ala(m))], AK) and elongated by an optically active amino acid [poly[Lys(X(i)-DL-Ala(m))], XAK]. We found that the nature of X (Glu, Ser, Thr) has great influence on the incorporation of the protected cysteine residue. Herpes simplex virus and adenovirus epitope peptides were conjugated to Boc-Cys(Npys)-modified polypeptides. Results indicate that the incorporation of epitope peptides depends on the number of Npys group on the polymers as well as on the presence/absence of Boc-protecting group on the Cys residue. This new class of Cys(Npys)-derivatized branched polypeptides is stable for a couple of months and suitable for effective preparation of epitope peptide conjugates possessing increased water solubility.     

DNA-binding region of the simian virus 40 tumor antigen.

The simian virus 40 (SV40) tumor (T) antigen was purified by immunoaffinity chromatography and cleaved with small amounts of trypsin, and the resulting fragments were subjected to SV40 DNA cellulose chromatography. A 44,000-molecular-weight fragment (44K fragment) from the left end of the molecule and a 30K fragment mapping from approximately Lys 131 to Lys 371 bound to the column and were eluted with 1 M NaCl. In a second series of experiments, T antigen was immunoprecipitated with hamster anti-T serum or various monoclonal antibodies and partially digested with trypsin. Fragments that were solubilized by this treatment were tested for DNA-binding activity by using an SV40 DNA fragment-binding assay. A 17K fragment which originated from the amino-terminal region of the polypeptide had no apparent binding activity in this assay. On the other hand, larger fragments (76K, 46K, and 30K) whose amino termini were mapped around Lys 131 did display DNA-binding activity. Finally, complexes consisting of SV40 DNA and T-antigen fragments were precipitated in the DNA-binding assay with monoclonal antibodies that recognize the central region of the protein; however, antibodies with specificities to the amino- or carboxy-terminal regions were inactive. These results strongly suggest that the DNA-binding region of T antigen lies approximately between Lys 131 and Lys 371, corresponding to 0.51 and 0.37 map units on the DNA.     

Carrier design: new generation of polycationic branched polypeptides containing OH groups with prolonged blood survival and diminished in vitro cytotoxicity.

For the construction of macromolecule-drug conjugates, it is important to provide rational basis to the selection of proper carrier. With respect to the importance of the side-chain structure and charge of the branched polypeptides in biological properties, we have prepared a new class of branched polypeptides with single or multiple hydroxyl groups and studied their solution conformation, in vitro cytotoxicity, biodistribution, and immunoreactivity. For comparative studies, polypeptides were designed to contain serine at various positions of the side chains, varying also the number. Ser was attached to the end of oligo(DL-Ala) side chains grafted to polylysine resulting polypeptides with the general formula poly[Lys(Ser(i)-DL-Ala(m))], (SAK). Ser was also coupled directly to the polylysine backbone poly[Lys(Ser(i))] (S(i)K) and then elongated by polymerization of N-carboxy-DL-Ala anhydride resulting poly[Lys(DL-Ala(m)-Ser(i))] (ASK). An additional polymer was also prepared, but instead of the oligo(DL-Ala) branches, oligo(DL-Ser) side chains were introduced (poly[Lys(DL-Ser(m))], SK). The presence of hydroxyl groups resulted in compounds with improved of water solubility. CD spectra of polypeptides showed significant differences correlating with the position and numbers of Ser residues in the side chains. Under physiological conditions, polycationic polypeptides assumed ordered secondary structure (S(i)K and LSK) or partially unordered conformation (SK, SAK, and ASK). Data of selected polymers demonstrate that these polycationic compounds are essentially nontoxic in vitro on normal rat liver or mouse spleen cells and have no cytostatic effect on mouse colorectal carcinoma C26 cells. The blood clearance and biodistribution of these derivatives were greatly dependent on the position and number of Ser residues in the branches and possess a rather extended blood survival in mice. Polypeptides were taken up predominantly by the liver and kidney (S(i)K, LSK, and ASK) or kidney and lung (SK and SAK). The best survival in the blood was found with SAK, representing the first polycationic branched polypeptide, which show extended blood clearance. The relative position of Ser residue had also a marked influence on the immunogenicity of polypeptides. The characteristics of the antibody response to polypeptide containing Ser at the end of the branches (SAK) or adjacent to the polylysine backbone (ASK) was also dependent on the genetic background of the mouse strains. We also found that these compounds have no effect on to the SRBC-specific humoral immune response, indicating the lack of nonspecific immunostimulatory potential. In conclusion, these studies suggest that synthetic branched polypeptides with Ser can be considered as candidates for constructing suitable conjugates for drug/epitope delivery. It is not only due to the presence of hydroxyl group to be used for oxime chemistry but also to their beneficial biological features.     

Porcine spleen cathepsin H hydrolyzes oligopeptides solely by aminopeptidase activity

Cathepsin H purified from porcine spleens was studied for its specificity against various peptide and denatured protein substrates. The enzyme degraded all peptide substrates exclusively by an aminopeptidase activity. The enzyme preferentially released NH2-terminal amino acid residues with large hydrophobic (Phe, Trp, Leu, and Tyr) or basic (Arg and Lys) side chains. Amino acids containing small or polar side chains were not released. Peptides with a proline in the NH2-terminal or penultimate positions were not hydrolyzed either. Large polypeptides such as reduced and carboxymethylated soybean trypsin inhibitor and aldolase were not degraded. These results indicate that cathepsin H is an exopeptidase but not an endopeptidase. We propose that the biological role of this enzyme is the degradation of tissue proteins in lysosomes by its aminopeptidase activity.     

Purification and characterization of an enkephalin aminopeptidase from rat brain membranes

A membrane-bound aminopeptidase was purified from rat brain, and its activity was assayed by high-pressure liquid chromatography with Met-enkephalin as the substrate. The enzyme was extracted with 1% Triton X-100 and purified by chromatography, successively on DEAE-Sepharose CL-6B, Bio-Gel HTP, and Sephadex G-200 columns. The overall purification was about 1200-fold, with 25% yield. The purified enzyme showed one band on disc gel electrophoresis and two bands on sodium dodecyl sulfate electrophoresis with molecular weights of 62 000 and 66 000. The aminopeptidase has a pH optimum of 7.0, a Km of 0.28 mM, and a Vmax of 45 mumol (mg of protein)-1 min-1 for Met-enkephalin. It releases tyrosine from Met-enkephalin, but it does not split the byproduct. It does not hydrolyze gamma- or beta-endorphin, or dynorphin, but it does hydrolyze neutral and basic aminoacyl beta-naphthylamides. The enzyme is inhibited by the aminopeptidase inhibitors amastatin, bestatin, and bestatin-Gly. Its properties, such as its subcellular localization, substrate specificity, pH optimum, and molecular weight, distinguish it from leucine aminopeptidase, aminopeptidase A, aminopeptidase B, aminopeptidase M, and the soluble aminopeptidase for enkephalin degradation.     

Purification and Characterization of Two Soluble C1−-Activated Arginyl Aminopeptidases from Human Brain and Their Endopeptidase Action on Neuropeptides

Two closely related Cl(-)-activated arginyl aminopeptidases (I and II) were purified from a soluble extract of postmortem human cerebral cortex by anion-exchange chromatography and preparative gel electrophoresis. The electrophoretic mobility of II was approximately 80% that of I; the molecular mass of both enzymes was approximately 70 kilodaltons (kDa) (gel filtration). The aminopeptidase action of I and II on aminoacyl-7-amido-4-methylcoumarin (AMC) substrates was restricted to the Arg and Lys derivatives. Both enzymes had significant endopeptidase activity, hydrolysing several biologically active peptides including neurotensin, bradykinin, angiotensin-I, substance P, luliberin, and somatostatin at internal bonds. Other peptides [Leu-enkephalin, proctolin, thyroliberin, adrenocorticotropin18-39 (ACTH18-39), ACTH11-24, and dynorphin (1-13)] were not appreciably hydrolysed. The amino- and endopeptidase activities had pH optima at 6.5 and 7, respectively, and were both inhibited by metal ion chelators and sulphydryl group blocking agents. The aminopeptidase activity was stimulated 20-fold by Cl- ions, whereas the endopeptidase activity was unaffected by the latter. Km values for neurotensin degradation were 20 microM (I) and 37 microM (II) and for Arg-AMC hydrolysis they were 167 microM (I) and 125 microM (II). The endopeptidase activity was not inhibited by the aminopeptidase inhibitors arphamenine or bestatin (IC50 = 9 nM and 0.1 microM, respectively, with Arg-AMC substrate).     

Enhanced enzymatic stability and antitumor activity of daunorubicin-GnRH-III bioconjugates modified in position 4

Here, we report on the synthesis, enzymatic stability, and antitumor activity of novel bioconjugates containing the chemotherapeutic agent daunorubicin attached through an oxime bond to various gonadotropin-releasing hormone-III (GnRH-III) derivatives. In order to increase the enzymatic stability of the bioconjugates (in particular against chymotrypsin), (4)Ser was replaced by N-Me-Ser or Lys(Ac). A compound in which (4)Lys was not acetylated was also prepared, with the aim of investigating the influence of the free ε-amino group on the biochemical properties. The in vitro cytostatic effect of the bioconjugates was determined on MCF-7 human breast, HT-29 human colon, and LNCaP human prostate cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their stability/degradation (1) in human serum, (2) in the presence of rat liver lysosomal homogenate, and (3) in the presence of digestive enzymes (trypsin, chymotrypsin, and pepsin) was analyzed by liquid chromatography in combination with mass spectrometry. The results showed that (1) all synthesized bioconjugates had in vitro cytostatic effect, (2) they were stable in human serum at least for 24 h, and (3) they were hydrolyzed in the presence of lysosomal homogenate. All compounds were stable in the presence of (1) pepsin and (2) trypsin (except for the (4)Lys containing bioconjugate). In the presence of chymotrypsin, all bioconjugates were digested; the degradation rate strongly depending on their structure. The bioconjugates in which (4)Ser was replaced by N-Me-Ser or Lys(Ac) had the highest enzymatic stability, making them potential candidates for oral administration. In vivo tumor growth inhibitory effect of two selected bioconjugates was evaluated on orthotopically developed C26 murine colon carcinoma bearing mice. The results indicated that the compound containing Lys(Ac) in position 4 had significantly higher antitumor activity than the parent bioconjugate.     

Photoaffinity labeling of terminal deoxynucleotidyl transferase. 2. Identification of peptides in the nucleotide binding domain

Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the nucleotide binding site by the substrate photoaffinity analogue [gamma-32P]-8-azido-dATP. The alpha and beta polypeptides of photolabeled terminal transferase were resolved by high-performance liquid chromatography. The beta polypeptide was digested with trypsin and fractionated by reverse-phase chromatography. Two 32P-containing fractions were isolated and subjected to amino acid sequence analysis. Peptides were identified as Ile209-Lys232 (B26) and Val233-Lys239 (B27). Peptide B26 was further resolved into two overlapping species; one contained an additional lysine residue at the N-terminus which resulted from tryptic cleavage between Lys207 and Lys208. In order to ensure that the sequenced peptides corresponded to the photolabeled species, we devised an anion-exchange procedure to isolate photolabeled peptides from the mixture. Analysis of photolabeled peptides from terminal transferase alpha beta using DEAE-cellulose chromatography followed by reverse-phase HPLC confirmed that the photolabeled species were peptides B26 and B27. Peptide B26, the major photolabeled species, contained a conserved octapeptide region found in several eucaryotic DNA polymerases. In addition, peptide B27 was flanked by a sequence that has been implicated in triphosphate binding in other proteins. Structure predictions, based on sequence data, place the two peptides identified by photolabeling in spatial proximity consistent with the participation of both in the nucleotide binding domain.     

Primary structure of Dioclea glabra trypsin inhibitor, DgTI, a Bowman-Birk inhibitor.

A novel serine proteinase inhibitor, DgTI, was purified from Dioclea glabra seeds by acetone precipitation, and ion-exchange and reverse phase chromatography. The inhibitor belongs to the Bowman-Birk family, and its primary sequence, determined by Edman degradation and mass spectrometry, of 67 amino acids is: SSGPCCDRCRCTKSEPPQCQCQDVRLNSCHSACEACVCSHSMPGLCSCLDITHFCHEPCKSSGDDED++ +. Although two reactive sites were determined by susceptibility to trypsin (Lys(13) and His(40)), the inhibitory function was assigned only to the first site. The inhibitor forms a 1:1 complex with trypsin, and Ki is 0.5 x 10(-9) M. Elastase, chymotrypsin, kallikreins, factor Xa, thrombin, and plasmin were not inhibited. By its properties, DgTI is a Bowman-Birk inhibitor with structural and inhibitory properties between the class of Bowman-Birk type I (with a fully active second reactive site), and Bowman-Birk type II (devoid of second reactive site).     

Characterization of a Kunitz trypsin inhibitor with a single disulfide bridge from seeds of Inga laurina (SW.) Willd

Inga laurina is a tree that belongs to the Mimosoideae sub-family of the Leguminosae. A protein inhibitor of trypsin (ILTI) was isolated from its seeds by ammonium sulphate precipitation, ion-exchange chromatography and rechromatography on an HiTrap Q ion-exchange column. By SDS-PAGE, ILTI yielded a single band with a Mr of 20 kDa with or without reduction. ILTI was found to be a single polypeptide chain containing 180 amino acids, the sequence of which was clearly homologous to the Kunitz family of serine protease plant protein inhibitors, and it also showed significant similarity to the seed storage proteins, sporamin and miraculin. However, ILTI displayed major differences to most other Kunitz inhibitors in that it contained only one disulfide bridge, and did not have two polypeptide chains as for the majority of other Kunitz inhibitors purified from Mimosoideae species. ILTI inhibited bovine trypsin with an equilibrium dissociation constant (K(i)) of 6 x 10(-9)M, but did not inhibit chymotrypsin, papain and alpha-amylase. Its amino acid sequence contained a Lys residue at the putative reactive site (position 64). ILTI was stable over a wide range of temperature and pH and in the presence of DTT.     

Leucaena leucocephala serine proteinase inhibitor: primary structure and action on blood coagulation, kinin release and rat paw edema

A serine proteinase inhibitor isolated from Leucaena leucocephala seeds (LlTI) was purified to homogeneity by acetone fractionation, ion exchange chromatography, gel filtration and reverse phase chromatography (HPLC). SDS-PAGE indicated a protein with M(r) 20000 and two polypeptide chains (alpha-chain, M(r) 15000, and beta-chain, M(r) 5000), the sequence being determined by automatic Edman degradation and by mass spectroscopy. LlTI is a 174 amino acid residue protein which shows high homology to plant Kunitz inhibitors, especially those double chain proteins purified from the Mimosoideae subfamily. LlTI inhibits plasmin (K(i) 3.2 x 10(-10) M), human plasma kallikrein (K(i) 6.3 x 10(-9) M), trypsin (K(i) 2.5 x 10(-8) M) and chymotrypsin (K(i) 1.4 x 10(-8) M). Factor XIIa activity is inhibited but K(i) was not determined, and factor Xa, tissue kallikrein and thrombin are not inhibited by LlTI. The action of LlTI on enzymes that participate in the blood clotting extrinsic pathway is confirmed by the prolongation of activated partial thromboplastin time, used as clotting time assay. The inhibition of the fibrinolytic activity of plasmin was confirmed on the hydrolysis of fibrin plates. LlTI inhibits kinin release from high molecular weight kininogen by human plasma kallikrein in vitro and, administered intravenously, causes a decrease in paw edema induced by carrageenin or heat in male Wistar rats. In addition, lower concentrations of bradykinin were found in limb perfusion fluids of LlTI-treated rats.     

The effect of N-terminal acetylation and the inhibition activity of acetylated enkephalins on the aminopeptidase M-catalyzed hydrolysis of enkephalins.

High performance liquid chromatography and high performance liquid chromatography/electrospray ionization-mass spectrometry were used to study the effect of N-terminal acetylation and the inhibition activity of acetylated enkephalins on the aminopeptidase M (EC 3.4.11.2)-catalyzed hydrolysis of methionine (Met-enk) and leucine enkephalins (Leu-enk). Acetylation imparts a significant enhancement in the proteolytic stability of these two peptides. After 30 min of the reaction, < 10% of both acetylated enkephalins was hydrolyzed. In an 8-h incubation period, only a maximum of 54% acetylated (Ac)-Met-enk and 38% Ac-Leu-enk was hydrolyzed. Vmax and Km [infil] for the degradation of Ac-Met-enk were 1.4 nmol/min/50 ng and 2.2 mM, respectively. The corresponding values for the reaction of Ac-Leu-enk were 0.5 nmol/min/50 ng and 0.9 mM. Also, the aminopeptidase M activity on Met-enk can be inhibited in the presence of Ac-Met-enk, which acts as a mixed-type inhibitor with the inhibition constant (K(i)) of I x 10(-3) M.     

Purification, characterization, and cDNA cloning of a Bowman-Birk type trypsin inhibitor from Apios americana Medikus tubers

An Apios americana trypsin inhibitor, AATI, was purified from Apios tubers by chromatography on DEAE Cellulofine A-500 and Sephadex G-50. The molecular mass of AATI was determined to be 6,437 Da by matrix-assisted laser desorption and ionization time-of-flight mass spectrometer (MALDI-TOF-MS). It showed strong inhibitory activity toward serine proteases, and the inhibition constants toward trypsin and chymotrypsin were 3.0 x 10(-9) M and 1.0 x 10(-6) M respectively. The inhibitory activity was not affected by heating at 80 degrees C for 2 h or by incubation at a wide range of pH values, suggesting that AATI has remarkable heat-stability and pH-stability. AATI cDNA consists of 552 nucleotides, and includes an open reading frame encoding a protein of 116 amino acids. The results of N-terminal amino acid sequencing of AATI and MALDI-TOF-MS analysis suggested that the deduced amino acid sequence had 50 and seven extra amino acids at the N- and C-termini respectively. Thus the mature AATI protein consists of 59 amino acid residues. Comparison of the amino acid sequence with those of the trypsin inhibitors from plants suggests that AATI belongs to the Bowman-Birk family and that it contains two possible reactive sites toward trypsin at Lys62 and Arg88.     

Degradation of thymic humoral factor gamma2 by human plasma: involvement of angiotensin converting enzyme

The degradation of thymic humoral factor-gamma2 (THF-gamma2), an immunoregulatory octapeptide important for T-lymphocyte regulation, by enzymes present in human plasma, was investigated. THF-gamma2 was metabolized through two steps that involved the detaching of N-terminal amino acid leucine followed by hydrolysis of the Lys(6)-Phe(7) bond. The THF-gamma2 cleavages were sensitive to aminopeptidase and metalloproteinase inhibitors. The degradation was completely blocked by amastatin and specific inhibitors of angiotensin converting enzyme (ACE). The cleavages occurred independently, with two different kinetics, faster for the N-terminal hydrolysis than for that of the Lys(6)-Phe(7) bond. Purified human plasma ACE was used to characterize the hydrolysis of Lys(6)-Phe(7) bond. The K(m) and K(cat) values for THF-gamma2 hydrolysis were 0.273 mM and 107 s(-1), respectively. The optimum of chloride concentration was 300 mM, while that of pH was 7.6. The presence of ACE in circulating mononuclear cells raises the possibility that it may play a role in modulating the THF-gamma2 activity.     

Tryptic hydrolysis of hGH-RH(1-29)-NH2 analogues containing Lys or Orn in positions 12 and 21.

Two analogues of the 29 amino acid sequence of human growth hormone-releasing hormone, namely [Nle27]hGH-RH(1-29)-NH2 and [Orn(12,21),Nle27]hGH-RH(1-29)-NH2, have been synthesized and subjected to digestion by trypsin. The course of degradation was followed using RP-HPLC and ESI-MS. Several intermediates and final products of degradation were identified and conclusions regarding the rate of cleavages at different positions occupied by Lys and Arg residues were drawn. The analogue containing ornithine was found to be less susceptible to hydrolysis by trypsin: the 12-13 and 21-22 peptide bonds were completely resistant to the cleavage. The results show that by replacing Lys with Orn, a possibility exists to design new peptides, which could be more stable in biological fluids.     

A new protein inhibitor of trypsin and activated Hageman factor from pumpkin (Cucurbita maxima) seeds

A protein inhibitor (CMTI-V; Mr 7106) of trypsin and activated Hageman factor (Factor XIIa), a serine protease involved in blood coagulation, has been isolated for the first time from pumpkin (Cucurbita maxima) seeds by means of trypsin-affinity chromatography and reverse phase high performance liquid chromatography (HPLC). The dissociation constants of the inhibitor complexes with trypsin and Factor XIIa have been determined to be 1.6 x 10(-8) and 4.1 x 10(-8) M, respectively. The primary structure of CMTI-V is reported. The protein has 68 amino acid residues and one disulfide bridge and shows a high level of sequence homology to the Potato I inhibitor family. Furthermore, its amino terminus consists of an N-acetylates Ser. The reactive site has been established to be the peptide bond between Lys44-Asp45. The modified inhibitor which has the reactive site peptide bond hydrolyzed inhibits trypsin but not the Hageman factor.