N-terminal Isoleucine Research Articles

High Performance Liquid Chromatography-Quadrupole/Time of Flight–Tandem Mass Spectrometry for the Characterization of Components in Bacitracin

A selective method of high performance liquid chromateography-quadrupole/time of flight-tandem mass spectrometry is described for the characterization of related substances in bacitracin samples. The chromatographic separation of the components of bacitracin was carried out using a Diamonsil Plus C18 column, a mixture of ammonium formate buffer (50 mM, pH 4.0) and acetonitrile was used as mobile phase under gradient elution. Baseline separation of bacitracin B1 and B2 were resolved for the first time. A complete strategy for structure characterization based on the accurate molecular weight and the diagnostic fragment ions obtained by MS/MS was elucidated. Three fragmentation routes were summarized in detail from the ions of Q/TOF–MS with accurate mass values. It was worth mentioning that the characteristic ions of amino acid residues can help to determine the structure of the new components of bacitracin. About 33 components were detected and the sequences of known components like bacitracin A, B1/B2/B3, C1/C2/C3, H1/H2/H3 and F were confirmed. Bacitracin E, J1/J2/J3, isomers of the main components of bacitracin (A, B and C), dehydration product and degradation products were sequenced completely. A proposed mechanism explanation for the transformation of isomers of bacitracin A was also approached. The structure of a new and specific component (component 5) containing two methylene on the N-terminal isoleucine residue and one vinyl group on the thiazoline or one ethyl group on the thiazole was proposed and described.

Synthesis of BVD15 Peptide Analogues as Models for Radioligands in Tumour Imaging

Neuropeptide Y (NPY) Y1 receptors are overexpressed in human breast carcinomas. They also have important functional roles in breast tumour growth and metastasis. This study investigates the synthesis of 15 truncated NPY analogues as models for Y1 receptor specific radiopharmaceuticals, using competition radioreceptor binding assays from brain tissue homogenates from Y2Y4-double knockout mice. These peptides are based on the previously reported BVD15 scaffold. Different measures to improve Y1 affinity and plasma metabolic stability were investigated. Extending from the previously reported [Lys(DOTA)4]BVD15 analogue, it was found that lysine4 is capable of tolerating various modifications, including prosthetic groups and other bifunctional chelators, but also that [Lys4]BVD15 has improved Y1 affinity, relative to BVD15 itself. Substitution of lysine4 for side chain shortened analogues retains Y1 receptor affinity of the analogues. Furthermore, modifications at the N-terminal isoleucine resulted in dramatic reduction of Y1 affinity.

Peptide IC-20, encoded by skin kininogen-1 of the European yellow-bellied toad, Bombina variegata, antagonizes bradykinin-induced arterial smooth muscle relaxation

Objectives:The objectives were to determine if the skin secretion of the European yellow-bellied toad (Bombina variegata), in common with other related species, contains a bradykinin inhibitor peptide and to isolate and structurally characterize this peptide.Materials and Methods:Lyophilized skin secretion obtained from this toad was subjected to reverse phase HPLC fractionation with subsequent bioassay of fractions for antagonism of the bradykinin activity using an isolated rat tail artery smooth muscle preparation. Subsequently, the primary structure of the peptide was established by a combination of microsequencing, mass spectroscopy, and molecular cloning, following which a synthetic replicate was chemically synthesised for bioassay.Results:A single peptide of molecular mass 2300.92 Da was resolved in HPLC fractions of skin secretion and its primary structure determined as IYNAIWP-KH-NK-KPGLL-. Database interrogation with this sequence indicated that this peptide was encoded by skin kininogen-1 previously cloned from B. variegata. The blank cycles were occupied by cysteinyl (C) residues and the peptide was located toward the C-terminus of the skin kininogen, and flanked N-terminally by a classical –KR- propeptide convertase processing site. The peptide was named IC-20 in accordance (I = N-terminal isoleucine, C = C-terminal cysteine, 20 = number of residues). Like the natural peptide, its synthetic replicate displayed an antagonism of bradykinin-induced arterial smooth muscle relaxation.Conclusion:IC-20 represents a novel bradykinin antagonizing peptide from amphibian skin secretions and is the third such peptide found to be co-encoded with bradykinins within skin kininogens.

Synthesis and antibacterial activity of nisin‐containing block copolymers

Nisin, an antibacterial peptide proven to be an effective inhibitor of Gram-positive bacteria, was incorporated into novel block copolymer constructs and tested for retained antibacterial activity. Covalent coupling was achieved by chemical modification of the N-terminal isoleucine to introduce a thiol group. Thiolated-nisin derivatives were then linked to poly[ethylene oxide]-poly[propylene oxide]-poly[ethylene oxide] (PEO-PPO-PEO) triblocks that had been end-activated such that terminal hydroxyl groups of the PEO chains were replaced with pyridyl disulfide moieties. The nisin-containing block copolymers were separated from free nisin by dialysis and showed antimicrobial activity against the Gram-positive indicator strain Pediococcus pentosaceus. The contribution to antimicrobial activity from nisin that was covalently linked was not distinguished from the contribution of nisin that had associated with the PEO-PPO-PEO triblocks through noncovalent interactions. However, nisin that was covalently linked showed activity upon reduction of the disulfide bond and release from the end-activated PEO.

Peptidergic and Nitrergic Innervation of the Pineal Gland in the Domestic Pig: An Immunohistochemical Study

The presence and co-localization of vasoactive intestinal polypeptide (VIP), peptide N-terminal histidine C-terminal isoleucine (PHI), pituitary adenylate cyclase-activating peptide (PACAP), somatostatin (SOM), calcitonin gene-related peptide (CGRP), substance P (SP) and the neuronal isoform of nitric oxide synthase (NOS) were studied in neuronal structures of the pig pineal gland. Paraformaldehyde-fixed pineals of 3-month-old gilts were sliced into serial cryostat sections, which were subjected to a set of double immunofluorescence stainings. Based on the co-existence patterns of neuropeptides, five populations of nerve fibres supplying the pig pineal were distinguished: (1) PHI-positive, (2) PACAP-positive, (3) SOM-positive, (4) SP/CGRP-positive and (5) SP-positive/CGRP-negative. Only a subpopulation of PHI-positive fibres contained VIP at the level detectable by immunofluorescence. NOS was found in some intrapineal PHI- and VIP-positive fibres. PHI-, VIP- and NOS-positive nerve fibres were more numerous in the peripheral than in the central part of the pineal. PACAP-positive fibres were equally distributed within the gland. The density of SOM-positive fibres was higher in the ventro-proximal than in the dorso-distal part of the pineal. SOM was also detected in some neuronal-like cells or specialized pinealocytes situated in the central region of the gland. Two populations of fibres containing SP were found: CGRP-positive, present in the distal and central parts of the pineal as well as CGRP-negative, localized in the proximal compartment of the gland.

Exploring the ubiquitin‐proteasome protein degradation pathway in yeast

This article describes an undergraduate biochemistry laboratory investigating the ubiquitin-proteasome pathway in yeast. In this exercise, the enzyme β-galactosidase (β-gal) is expressed in yeast under the control of a stress response promoter. Following exposure to heat stress to induce β-gal expression, cycloheximide is added to halt translation, and β-gal degradation is monitored by measuring enzyme activity as a function of time. Students observe that an N-Ile-β-gal variant with an N-terminal isoleucine has a significantly lower stability than wild-type β-gal, whose N-terminal residue is methionine. This strong dependence of protein stability on the N-terminal residue is known as the "N-end rule." To corroborate the enzyme activity assay results, students perform denaturing protein electrophoresis and immunoblotting of lysates, observing that the time-dependent loss of enzyme activity is coincident with the disappearance of the β-gal protein.

N-Terminal fragment of hog pepsin.

One of the peptide fragments obtained in a preceding study of the cyanogen bromide hydrolysate of S-sulfo-pepsin has been investigated in more detail. The peptide was purified by ion-exchange chromatography and obtained in homogeneous form. It contains 158 amino acid residues, among their number the only histidine residue of pepsin and two half-cystine residues. Evidence was obtained that the peptide is derived from the N-terminal region of the molecule of hog pepsin. The peptide was reduced by 2-mercaptoethanol and converted into its S-(β -amino-ethyl)-cysteinyl derivative. From the tryptic digest of this derivative a 105-residue peptide containing N-terminal isoleucine was isolated which represents the N-terminal segment of the pepsin molecule.

Domain interactions between streptokinase and human plasminogen.

Plasmin (Pm), the main fibrinolytic protease in the plasma, is derived from its zymogen plasminogen (Plg) by cleavage of a peptide bond at Arg(561)-Val(562). Streptokinase (SK), a widely used thrombolytic agent, is an efficient activator of human Plg. Both are multiple-domain proteins that form a tight 1:1 complex. The Plg moiety gains catalytic activity, without peptide bond cleavage, allowing the complex to activate other Plg molecules to Pm by conventional proteolysis. We report here studies on the interactions between individual domains of the two proteins and their roles in Plg activation. Individually, all three SK domains activated native Plg. While the SK alpha domain was the most active, its activity was uniquely dependent on the presence of Pm. The SK gamma domain also induced the formation of an active site in Plg(R561A), a mutant that resists proteolytic activation. The alpha and gamma domains together yielded synergistic activity, both in Plg activation and in Plg(R561A) active site formation. However, the synergistic activity of the latter was dependent on the correct N-terminal isoleucine in the alpha domain. Binding studies using surface plasmon resonance indicated that all three domains of SK interact with the Plg catalytic domain and that the beta domain additionally interacts with Plg kringle 5. These results suggest mechanistic steps in SK-mediated Plg activation. In the case of free Plg, complex formation is initiated by the rapid and obligatory interaction between the SK beta domain and Plg kringle 5. After binding of all SK domains to the catalytic domain of Plg, the SK alpha and gamma domains cooperatively induce the formation of an active site within the Plg moiety of the activator complex. Substrate Plg is then recognized by the activator complex through interactions predominately mediated by the SK alpha domain.

Amino acid sequence of a modified β2-microglobulin in renal failure patient urine and long-term dialysis patient blood

We isolated and analyzed β2-microglobulin (BM) in urine of two renal failure patients and in ultrafiltrate from long-term dialysis patients. In both groups of patients, we found the native BM with p I 5.7 and some modified BM with p I 5.3. The amino acid sequence of the modified BM was determined and compared with that of the native BM; the N-terminal isoleucine residue was missing and the second glutamine residue was cyclized into pyroglutamic acid in the modified BM.

Beta-adrenergic and peptide N-terminal histidine and C-terminal isoleucine stimulation of N-acetyl-transferase activity and melatonin production in the cultured rat pineal gland.

The purpose of this investigation was to compare the effect of peptide N-terminal histidine and C-terminal isoleucine (PHI) with that of the beta-adrenergic agonist isoproterenol (ISO) on N-acetyltransferase (NAT) activity and melatonin production in the cultured rat pineal gland. Pineal glands were removed and placed in organ culture containing PHI, ISO, or PHI plus ISO at different dosages, and subsequent changes in NAT activity and melatonin levels were measured. PHI stimulated these parameters in both a time- and dose-dependent manner. Combined treatment with PHI (10(-6) M) and high doses of ISO (either 10(-7) or 10(-8) M) did not potentiate the effect of the peptide in terms of either NAT activity or melatonin levels in the pineal gland. However, at a lower concentration, ISO (10(-9) M) had additive effects to those of PHI in both cultured pineals and medium. The results suggest that PHI modulates melatonin synthesis in the rat pineal gland. Furthermore, stimulation of the pineal with both PHI and ISO demonstrates an additive effect rather than a synergistic action of these compounds. It is presumed that ISO and PHI stimulate pineal melatonin production via separate receptors, but they probably use the same intracellular second messenger, cAMP, to do so. This is the first study showing an effect of the peptide PHI on pineal melatonin production in any vertebrate.

Beta-adrenergic and peptide N-terminal histidine and C-terminal isoleucine stimulation of N-acetyl-transferase activity and melatonin production in the cultured rat pineal gland

Beta-adrenergic and peptide N-terminal histidine and C-terminal isoleucine stimulation of N-acetyl-transferase activity and melatonin production in the cultured rat pineal gland

Localisation and characterisation of functional vasoactive intestinal peptide receptors in feline kidney

Specific 125I-labelled vasoactive intestinal peptide (VIP) binding was determined in feline renal cortical and medullary plasma membranes. For the cortex, Scatchard analysis of the data resulted in a curvilinear plot with a high-affinity site K0.5 of 8.4 +/- 2.6 nmol l-1 (SE, n = 6) and a second low-affinity site K0.5 204 +/- 16 nmol l-1 with binding site concentrations (Bmax) of 385 +/- 44.5 and 2710 +/- 181.3 fmol mg protein-1 respectively. Conversely a similar analysis of the results obtained for outer medullary membranes gave a single site with a K0.5 of 1.2 +/- 0.2 nmol l-1 (SE, n = 4) and Bmax of 157.8 +/- 24.7 fmol mg-1. Inner medullary membrane binding data. Gave a single site of lower affinity (K0.5 = 62.5 +/- 21.6 nmol l-1; n = 3). Structurally related peptides, glucagon and secretin, were ineffective (up to 1 mumol l-1) in displacing VIP from specific sites in both cortex and medulla. Porcine PHI 1-27 (a peptide having N-terminal histidine and C-terminal isoleucine) and a VIP antagonist [4-Cl-D-Phe6Leu17]VIP both displaced 125I-VIP from cortical and medullary membrane binding sites with IC50 values of 43.0 nmol l-1 and 1.3 mumol l-1 (cortex) and 132.0 nmol l-1 and 1.5 mumol l-1 (medulla) respectively. The localisation of specific VIP binding sites in feline kidney was investigated further by in vitro autoradiography.(ABSTRACT TRUNCATED AT 250 WORDS)

PH dependence of the inhibition of chymotrypsin by a peptidyl trifluoromethyl ketone.

The effects of pH on the kinetics of association and dissociation of chymotrypsin and the dipeptidyl trifluoromethyl ketone (TFK) N-acetyl-L-leucyl-L-phenylalanyltrifluoromethane (1) were examined through the pH range 4-9.5. The pH dependence of the association rate (kon) is similar to that of kcat/Km for ester and peptide substrates and is dependent on two pK's at 7.0 and 8.9. We assign these pK's to the active site His and to the amino group of the N-terminal isoleucine residue. Ki for the complex of 1 and chymotrypsin has a pH dependence very similar to that of kon, and we conclude that the same ionizable groups which determine the pH dependence of kon are involved. The dissociation constant of the enzyme-inhibitor complex (koff) shows no pH dependence between pH 4 and pH 9.5. The data indicate that the inhibitor reacts with a form of the enzyme in which His 57 is unprotonated, and the resulting complex contains no groups which ionize between pH 4 and pH 9.5. This is consistent with conclusions previously reached from NMR data (Liang & Abeles, 1987). These experiments led to the conclusion that 1 reacts with chymotrypsin to form a tetrahedral complex in which His 57 is protonated (pK greater than 9.5) and the OH group of serine 195 has added to the carbonyl group of 1 to form an ionized hemiketal (pK less than 4.9). The pK of His 57 is increased by greater than 3 units over that in the free enzyme, and the pK of the hemiketal decreased by greater than 4 units compared to the pK in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasoactive intestinal peptide stimulation of renal adenylate cyclase and antagonism by (4Cl-D-Phe6Leu17)VIP.

The effect of vasoactive intestinal peptide (VIP) and related peptides [glucagon, secretin, PHI 1-27 (peptide with N-terminal histidine and C-terminal isoleucine)] on renal adenylate cyclase (AC) has been determined in several species. The largest stimulation (4.1 +/- 0.5-fold basal) of AC by 1 mumol.l-1 VIP was observed in feline cortical plasma membranes. In rabbit and guinea-pig, VIP increased AC activity 1.5 +/- 0.3- and 1.8 +/- 0.3-fold respectively but glucagon had no such action. Conversely in the rat glucagon stimulated AC some 3-fold over basal activity whereas VIP had little effect. In dog, cat and mouse both peptides were effective in increasing AC activity. For cat, half-maximal stimulation of cortical plasma membrane AC by VIP was seen at 27.0 +/- 9.0 nmol.l-1 (SE N = 9 animals). VIP also increased AC activity in both outer (red) and inner (white) medulla. In feline cortical membranes VIP and PTH (parathyroid hormone) when added in combination were fully additive. However for VIP and glucagon in combination there was no cumulative increase in AC activity, indeed the resultant activity was less than that attained by VIP alone. The VIP analogue (4Cl-D-Phe6Leu17)VIP at 10 mumol.l-1 produced a right shift in the VIP-dose response curve and increased the EC50 from 17.2 +/- 5.8 nmol.l-1 to 132.0 +/- 22.2 nmol..-1 VIP (SE N = 4). There was no reduction in the maximum response elicited by VIP consistent with a competitive type of antagonism by this analogue. PHI-stimulated AC was also reduced by (4Cl-D-Phe6Leu17)VIP resulting in a similar right shift in the dose response curve.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of vasoactive intestinal polypeptide on gastric motility in the lamb.

1. Intra-arterial infusions of vasoactive intestinal polypeptide (VIP) were made in anaesthetized lambs in which activity of the reticulo-omasal orifice (ROO) was recorded manometrically and in conscious lambs in which activity of the reticulum, ROO and abomasum were recorded by electromyographic (EMG) techniques. 2. Spontaneous rhythmic opening and closing movements of the ROO occurred in anaesthetized lambs at 3-5 min-1. Infusions of VIP into the left gastric artery at rates of 0.5-3.0 nmol min-1 produced changes in activity of the ROO. Within 120 s of commencement of the infusions there was an increase in frequency and magnitude of the movements of the ROO for up to 120 s. This was followed with infusion of VIP at the lower levels (0.5-1.0 nmol min-1), by a marked reduction and sometimes complete loss of the rhythmic movements. There was always complete cessation of activity of the ROO with infusion of VIP at 1.5-3.0 nmol min-1. 3. In conscious lambs the frequency of the diphasic reticular EMG bursts which recur at intervals of ca. 1 min was not affected by infusions of VIP at 3.0 nmol min-1 for 10 min. 4. Between each diphasic reticular EMG burst in the conscious lamb there was normally phasic activity of the ROO consisting of EMG bursts of long (ca. 4 s) and short (ca. 1 s) duration. Within 90 s of commencement of infusion of VIP at 3.0 nmol min-1 short-burst EMG activity disappeared with the remaining long bursts being of greater duration (5.4 +/- 1.2 s) than before infusion. After a series of four to fifteen such more prolonged long bursts there was quiescence of the EMG of the ROO. After infusion of VIP EMG activity recommenced first as a series of eight to fourteen long bursts which was followed by the reappearance also of short-burst activity. Infusions of VIP at 8-10 nmol min-1 produced a more prompt cessation of EMG activity of the ROO. Of other peptides which were infused only PHI (a peptide with N-terminal histidine and C-terminal isoleucine amide) produced cessation of the EMG activity of the ROO. However, on a molar basis VIP was 2-3 times more potent than PHI in causing cessation of activity of the ROO. 5. Infusion of VIP at 3.0 nmol min-1 produced a cessation or diminution of EMG activity of the body, antrum and pylorus of the abomasum.(ABSTRACT TRUNCATED AT 400 WORDS)

Dog mastocytoma tryptase: Affinity purification, characterization, and amino-terminal sequence

A tryptic protease with the characteristics of a mast cell tryptase was purified from dog mastocytoma cells propagated in nude mice. Partial amino acid sequence of the mastocytoma tryptase revealed unexpected differences in comparison with other mast cell and leukocyte granule protease sequences. Extraction from mastocytoma homogenates at high ionic strength, followed by gel filtration and benzamidine affinity chromatography yielded a product with several closely spaced bands ( M r 30,000–32,000) on gel electrophoresis and a single N-terminal sequence. Nondenaturing analytical gel filtration revealed an apparent M r of 132,000, suggesting noncovalent association as a tetramer. Studies with peptide p-nitroanilides indicated pronounced substrate preferences, with P1 arginine preferred to lysine. Benzoyl- l-Lys-Gly-Arg- p-nitroanilide was the best of the substrates screened. Inhibition by diisopropyl fluorophosphate and tosyllysine chloromethyl ketone indicated that the enzyme is a serine protease. Like the tryptases of human mast cells, mastocytoma tryptic protease was inhibited by NaCl, resistant to inactivation by α 1-proteinase inhibitor and plasma, and stabilized by heparin. Comparison of the N-terminal 24 residues of mastocytoma tryptase revealed 80% identity with the more limited sequence reported for human lung tryptase, and surprisingly, closer homology to serine proteases of digestion and clotting than to other leukocyte granule proteases sequenced to date, including mast cell chymase. The N-terminal isoleucine is the homolog of trypsinogen Ile-16 which becomes the new N-terminus upon cleavage of the activation peptide. Thus, the tryptase N-terminus is related to the catalytic domain of activated serine proteases, and lacks the N-terminal regulatory domains found in most clotting and complement serine proteases. These findings provide further evidence that tryptases are unique serine proteases and that they may be less closely related in evolution and function than are other leukocyte granule proteases described to date.

Vasoactive intestinal polypeptide regulation of rabbit renal adenylate cyclase activity in vitro.

1. The effect of vasoactive intestinal polypeptide (VIP) upon adenylate cyclase activity was determined in purified cortical basolateral membranes and in glomeruli and tubular elements obtained from rabbit kidney. 2. In purified basolateral membranes prepared from cortex, 1 microM-VIP consistently stimulated adenylate cyclase activity above basal levels (1.55 +/- 0.09-fold (mean +/- S.E. of mean), n = 10 animals). Half-maximal stimulation was observed at 17 +/- 11 nM-VIP (S.D., n = 9). 3. Related peptides, e.g. secretin, glucagon, gastric inhibitory peptide, human pancreatic growth hormone releasing factor, and peptide having N-terminal histidine and C-terminal isoleucine amide (PHI), were without effect or gave lower stimulations of adenylate cyclase activity when tested at 1 microM. 4. Significant VIP degradation was observed under the assay conditions used but this did not substantially alter the response or selectivity to VIP. 5. In separate preparations of isolated glomeruli and proximal tubules addition of 1 microM-VIP resulted in a 3.3 +/- 1.1-fold (S.D., n = 3) and 2.2 +/- 1.0-fold (S.D., n = 3) stimulation (respectively) of adenylate cyclase activity. 6. In isolated medullary tubule suspensions, isolated by collagenase-hyaluronidase digestion of outer (red) medulla, and in thick ascending-limb-enriched preparations prepared by Percoll density gradient fractionation, 1 microM-VIP significantly increased adenylate cyclase activity by 2.4 +/- 0.6-fold (S.D., n = 3) and 2.1 +/- 0.7-fold (S.D., n = 3) respectively. 7. A possible role for VIP in the regulation of renal function in the rabbit is discussed in relation to the occurrence of VIP stimulation of adenylate cyclase activity in several renal cellular elements.

Co-existence and co-secretion of the structurally related peptides VIP and PHI

Using regional specific antisera the concentrations of vasoactive intestinal polypeptide (VIP) and the peptide with N-terminal histidine and C-terminal isoleucine (PHI) in various peripheral tissues and VIP producing tumours were compared with their immunohistochemical localization. In normal tissue the VIP levels were in general higher than the PHI levels, while the VIP/PHI ratio in tumour tissue varied considerably more than in normal tissue. By immunohistochemistry it was found that VIP and PHI immunoreactivity occurred in the same autonomic neurons. Gel chromatography revealed that VIP and PHI immunoreactivity in both normal and tumour tissue consisted of two larger molecular forms in addition to "authentic" peptides. These larger molecular forms which had overlapping elution positions probably represent VIP/PHI precursors. In tumour tissue the larger molecular forms constituted a larger proportion of the total immunoreactivity. Neurally induced relaxation of smooth muscle caused a simultaneous release of VIP and PHI which in combination with the observed relaxatory effect of the peptide suggest a role in the control of smooth muscle activity. Similarly VIP and PHI were co-secreted from tumour tissues as evidenced from elevated plasma levels in patients with VIP producing tumours. In conclusion VIP and PHI seem to co-exist and be co-secreted. Differences in posttranslational processing may create variable content and release of the two peptides.

Isolation and characterization of hirudin isoinhibitors and sequence analysis of hirudin PA.

A five-step isolation procedure has been developed for the purification of isoforms of hirudin (isohirudins) from whole leeches. The final purification of two thrombin-inhibiting preparations by reversed-phase high-performance liquid chromatography yielded several isohirudins with either N-terminal valine or isoleucine but with identical inhibition characteristics, i.e. specific thrombin inhibiting activities of 680-720 IU/mg and dissociation constants Ki of the thrombin-inhibitor complexes close to 3 X 10(-11) mol/l. The inhibitor with N-terminal isoleucine was designated hirudin PA. This inhibitor contains 66 amino-acid residues and has a molecular mass of 7,087 Da. The complete amino-acid sequence of hirudin PA was established by automated solid-phase Edman degradation of the native and oxidized inhibitor and two of its tryptic fragments. On the basis of the primary structures two types of thrombin inhibitors from the leech can be distinguished, designated hirudin and hirudin PA. The degree of structural homology of both isoinhibitors is approximately 82%; both have a tyrosine-O-sulfate residue near the C-terminus.

Binding of a curarimimetic toxin from cobra venom to the nicotinic acetylcholine receptor. Interactions of six biotinyltoxin derivatives with receptor and avidin.

We have reacted N-hydroxysuccinimidyl biotin with the principal curarimimetic toxin in Naja naja siamensis venom, biotinylating each of the five lysine residues and the N-terminal isoleucine. The six monobiotinyl-toxins were isolated by ion-exchange chromatography, and the residue modified in each was identified by peptide mapping and amino acid analysis. We evaluated the role of each lysine in the binding of toxin to the acetylcholine receptor by measuring the affinity of each biotinyltoxin for receptor and by determining which biotinyltoxins could bind receptor and avidin simultaneously. The effect of biotinylation of each residue decreased the affinity of toxin for receptor in the order Lys 23 greater than Lys 49 greater than Lys 35 greater than Lys 69 congruent to Lys 12 greater than Ile 1. Biotinyltoxin modified either at Lys 12 or at Lys 69 is effective in cross-linking avidin to receptor, while biotinyltoxin modified at Lys 49 can form a low-affinity avidin-biotinyltoxin-receptor complex. Taken together, these results help define the surface of toxin that binds to receptor.