Biomedical subjects

A Basak

Publications and source records attributed to A Basak.

At least 37 records · Page 2Linked to original sources

PubMed1997

In vitro characterization of the novel proprotein convertase PC7.

Biochemical and enzymatic characterization of the novel proprotein convertase rat PC7 (rPC7) was carried out using vaccinia virus recombinants overexpressed in mammalian BSC40 cells. Pro-PC7 is synthesized as a glycosylated zymogen (101 kDa) and processed into mature rPC7 (89 kDa) in the endoplasmic reticulum. No endogenously produced soluble forms of this membrane-anchored protein were detected. A deletion mutant (65 kDa), truncated well beyond the expected C-terminal boundary of the P-domain, produced soluble rPC7 in the culture medium. Enzymatic activity assays of rPC7 using fluorogenic peptidyl substrates indicated that the pH optimum, Ca2+ dependence, and cleavage specificity of this enzyme are largely similar to those of furin. However, with some substrates, cleavage specificity more closely resembled that of yeast kexin, suggesting differential processing of proprotein substrates by this novel convertase. We examined the rPC7- and human furin-mediated cleavage of synthetic peptides containing the processing sites of three proteins known to colocalize in situ with rPC7. Whereas both enzymes correctly processed the pro-parathyroid hormone tridecapeptide and the pro-PC4 heptadecapeptide, neither enzyme cleaved a pro-epidermal growth factor hexadecapeptide. Thus, this study establishes that rPC7 is an enzymatically functional subtilisin/kexin-like serine proteinase with a cleavage specificity resembling that of hfurin. In addition, we have demonstrated that rPC7 can correctly process peptide precursors that contain the processing sites of at least two potential physiological substrates.

Amino Acid Sequence↗

PubMed1997

Histidine-rich human salivary peptides are inhibitors of proprotein convertases furin and PC7 but act as substrates for PC1.

A 32 amino acid peptide called histatin-3 (H3; 22% His) and its N-terminal 24 amino acid fragment histatin-5 (H5, 33% His), are found in human saliva and possess powerful antimicrobial properties. These His-rich peptides have been synthesized by Fmoc-based solid-phase chemistry. Their sequences are: DSHAKRHHGYKRKFHEKHHSHRGYRSNYLYDN (H3) and DSHAKRHHGYKRKFHEKHHSHRGY (H5). In addition, we also prepared two H5 and one H3 mutants. The H5 mutants were: DH5 (all amino acids in D configuration) and H5F (where all His are replaced by Phe at positions 3, 7, 8, 15, 18, 19, 21). The 9-24 segment of H3 with all the His at positions 15,18,19,21 replaced by Tyr was also prepared (delta 1-8 H3Y). The behavior of these five peptides was examined with three proprotein convertases (PC's) which possess cleavage specificity directed towards single and pairs of basic residues. These were: human (h)PC1, an endocrine and neural convertase, hfurin and rat (r)PC7, two widely expressed enzymes. All are serine endoproteases belonging to the kexin/subtilisin family. Our in vitro study revealed that H3 behaves as a substrate for PC1, being cleaved by this endoprotease primarily at a site carboxy terminal to the single Arg25 residue (HRGYR decrease SN). On prolonged incubation some minor cleavage was also observed C-terminal to the first LysArg6 pairs of basic amino acids namely at: HAKR decrease HH, which contains a P4 as well as P'1 and P'2 His residues. The second potential site YKRK12-FH which does not have a P4 basic residues is not cleaved, even upon incubation with excess protease. PC1 only poorly cleaves H5 at the same site mentioned above for H3, i.e., at HAKR decrease HH. As expected, neither the D-amino acid analogue (DH5), nor the Phe and Tyr mutant analogues of the long and short histatins, respectively, are cleaved at all. In contrast to the above findings for hPC1, the convertase hfurin did not cleave any of the five synthetic peptides. Instead, H3 and H5 were found to be moderately potent inhibitors of the furin-mediated cleavage of the pentapeptide pGlu-Arg-Thr-Lys-Arg-MCA fluorogenic substrate. This inhibition was reversible and competitive, with an estimated inhibition constant Ki of 1.98 microM for H3 and 2.98 microM for H5. The other analogs exhibited only a moderate to weak inhibition of furin, suggesting that substitution of all His by aromatic residues (Phe or Tyr) drastically reduces their inhibitory potency. When tested against rPC7, H3 exhibited almost identical inhibition profile with a measured Ki of 2.4 microM. The partial sequence identity of H3 to the inhibitory pro-peptide of furin and PC7 provides a rationale for our observation.

Amino Acid Sequence↗

PubMed1995

Fluorescent peptidyl substrates as an aid in studying the substrate specificity of human prohormone convertase PC1 and human furin and designing a potent irreversible inhibitor.

The substrate specificities of two human prohormone convertases, furin and PC1, were examined with a series of 7-amino-4-methylcoumarinamide (MCA) containing peptidyl substrates. Using acetyl-Arg-Ser-Lys-Arg-MCA as model, P4 Arg substitution by Lys or Orn resulted for furin in a 538- and a 280-fold lower kcat/Km value, but only in a 14- and 18-fold decrease for PC1. Substitution of P3 Ser by either Pro, Glu, or Lys does not modify significantly the kcat/Km value for PC1, whereas furin activity is seriously impaired by the Glu substitution. Elongating the peptidyl sequence up to the P8 position decreases the kcat/Km value for furin but not for PC1. In both the P3 or P5 Glu substitution, the decrease of kcat/Km was due primarily to lower kcat rather than higher Km, possibly because of the presence of a negatively charged side chain. Finally, an octapeptidyl chloromethane derivative proved to be a potent irreversible inhibitor of either PC1 and ruin. The 811-fold difference in the apparent Kapp/[I] (1.63 x 10(6) s-1 m-1), and kcat/Km determined with the corresponding peptidyl MCA substrate (2.01 x 10(3) s-1 m-1), supports the proposal that cleavage of the acylenzyme represents the rate-limiting step for PC1 and furin.

Amino Acid Sequence↗

PubMed1995

An internally quenched fluorogenic substrate of prohormone convertase 1 and furin leads to a potent prohormone convertase inhibitor.

Based upon the observed cleavage of various peptidyl substrates by the recombinant prohormone convertases PC1 and furin, an intramolecularly quenched fluorogenic peptidyl substrate, (o-aminobenzoyl)-Lys-Glu-Arg-Ser-Lys-Arg-Ser-Ala-Leu-Arg-Asp-(3-nitro)Ty r-Ala, was synthesized. In spite of the distance (approx. 33 A) separating the fluorescent donor/acceptor pair, the highly fluorescent o-aminobenzoyl group is efficiently quenched by long-range resonance energy transfer to the (3-nitro)Tyr moiety. Both recombinant human PC1 and human furin recognize and cleave specifically this substrate at the expected Arg-Ser site in a sensitive manner. The Km values for human PC1 and human furin were 17 microM and 30 microM respectively, with Vmax. values of 6.4 microM/h and 18 microM/h. These values differ significantly from those obtained when using a 7-amino-4-methylcoumarin-containing pentapeptidyl substrate where, for similar Km values, the Vmax. values were much lower. The peptide sequence was used to synthesize another peptide incorporating a ketomethylene arginyl pseudopeptide bond. This compound proved to be a potent competitive inhibitor of both human PC1 and human furin, displaying Ki values of 7.2 microM and 2.4 microM respectively.

Amino Acid Sequence↗

PubMed1994

The convertases furin and PC1 can both cleave the human immunodeficiency virus (HIV)-1 envelope glycoprotein gp160 into gp120 (HIV-1 SU) and gp41 (HIV-I TM).

Intracellular proteolytic processing of human immunodeficiency virus envelope glycoprotein precursor (gp160) is an essential step for virus infectivity. Northern blot analysis provided evidence that furin and PC1, but not PC2, are expressed in the CD4+ human lymphoblastoid H9 cell line, suggesting the possible participation of these convertases in human immunodeficiency virus (HIV) gp160 proteolytic processing. Purified PC1 and furin cleaved specifically in vitro gp160 into gp120 (HIV-I SU) and gp41 (HIV-I TM). NH2-terminal sequence analysis of the produced gp41 (HIV-I TM) demonstrated that the cleavage occurred within the sequence Arg-Glu-Lys-Arg decreases Ala-Val-Gly-Ile, which is identical to the bond cleaved in vivo. Transition state analog peptides were designed and tested in vitro for their ability to inhibit the PC1- or furin-mediated gp160 cleavage. The best inhibitor was decanoyl-Arg-Lys-Arg-Arg-psi [CH2NH]-Phe-Leu-Gly-Phe-NH2.

Amino Acid Sequence↗

PubMed1994

Immunodiagnosis of childhood pulmonary and extrapulmonary tuberculosis using Mycobacterium tuberculosis ES antigen by penicillinase ELISA.

The diagnostic potential for detection of IgG to Mycobacterium tuberculosis excretory secretory (ES) antigen in childhood pulmonary and extrapulmonary tuberculosis was explored. IgG antibody to M. tuberculosis ES antigen was detected by indirect penicillinase ELISA. Twenty (80%) out of 25 pulmonary tuberculosis cases (clinically diagnosed and/or AFB-positive), five of nine tuberculous pleural effusion cases and only six of 69 cases in the control group were positive for IgG antibody to M. tuberculosis ES antigen. All CSF and sera were positive for IgG antibody in 12 cases of clinically diagnosed tuberculous meningitis (TBM). Out of 35 cases in the control group for TBM, all five cases of pyogenic meningitis but none of the 13 cases of viral encephalitis, five cases of enteric encephalopathy and 12 cases with no CNS infection were positive for anti-tubercular IgG antibody in CSF samples. Only two of them, i.e. one case of pyogenic meningitis and the other with no CNS infection, were positive for antibody in sera. The study demonstrated the potential of this assay in the diagnosis of tuberculosis in children where bacteriological confirmation is very difficult.

Antibodies, Bacterial↗

PubMed1994

Design and synthesis of novel inhibitors of prohormone convertases.

Prohormone convertase-1 (PC1) and furin are subtilisin-like endopeptidases involved in the biosynthesis of peptide hormones. Five decapeptides representing the junction between the pro-region and the catalytic region of PC1 were prepared. The core sequence corresponded to D-Tyr-Arg-Ser-Lys-Arg- Xaa-Val-Gln-Lys-Asp where D-Tyr replaces the native Glu residue and Xaa, representing the P1' position, corresponds to L-Ser, L-Leu or the unnatural amino acids, D-Ser, beta-Ala, gamma-Abu, beta-Cha or gamma-Hyp. Another analog incorporating an Orn residue in place of the Arg at the P1 site was also prepared. These peptides, synthesized by solid-phase Fmoc chemistry, were fully characterized by FAB-MS, 1H-NMR and amino acid composition. Except for Orn, gamma-Hyp, L/D-Ser and L-Leu containing analogs, the others were found to be moderate to potent competitive inhibitors of hPC1 activity in the following order: gamma-Abu > beta-Cha > beta-Ala, with Ki values ranging from 1 to 8.6 microM. Both L-Ser and L-Leu analogs were correctly cleaved at the acyl carbon COOH-terminal to the Lys-Arg pair by human PC1, whereas beta-Cha, gamma-Abu, beta-Ala and D-Ser analogs proved to be very poor substrates. The Orn and gamma-Hyp derivatives were not cleaved by the enzyme at all. The three analogs containing beta-Cha, gamma-Abu and beta-Ala also proved to be potent inhibitors of the human furin activity in the following order: beta-Ala > beta-Cha > gamma-Abu, with Ki ranging from 0.8 to 2.2 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Enzymic characterization of murine and human prohormone convertase-1 (mPC1 and hPC1) expressed in mammalian GH4C1 cells.

Prohormone convertase-1 (PC1), an endopeptidase that is structurally related to the yeast subtilisin-like Kex2 gene product, has been proposed to be involved in mammalian tissue-specific prohormone processing at pairs of basic residues. To better study this enzyme, a rat somatomammotroph cell line, GH4C1, was infected with vaccinia virus recombinants of murine PC1 (mPC1) and human PC1 (hPC1). An enzymically active form of each protein was secreted into the cell medium and partially purified by anion-exchange chromatography. The 80-85 kDa enzyme was shown to be Ca(2+)-dependent and exhibited a pH optimum of 6.0 when assayed against a synthetic fluorogenic substrate, acetyl-Arg-Ser-Lys-Arg-4-methylcoumaryl-1-amide. mPC1 and hPC1 displayed identical cleavage selectivity towards a number of fluorogenic substrates, and those incorporating an Arg at the P4 site were most favoured. Synthetic peptides, encompassing the junction between the putative pro-region and the active enzyme, and between the pro-region and the biologically active parathyroid hormone, were shown to be recognized and cleaved specifically at the pair of basic residues by both enzymes. Group-specific proteinase inhibitors such as metal ion chelators and p-hydroxymercuribenzoate, but not phenylmethanesulphonyl fluoride and pepstatin, strongly inhibit the PC1-associated activity. In addition, it is shown that an enzyme activity displaying identical properties is present in the cell medium of uninfected corticotroph AtT-20 cells and that its level is increased following stimulation of secretion by the secretagogue 8-bromo cyclic AMP.

Amino Acid Sequence↗

PubMed1993

Synthesis of the segment (11-23) located in the first tandem repeat of plasma kallikrein: comparative binding studies of this and another segment (328-343) to high-molecular-mass kininogen.

The synthesis of porcine plasma kallikrein (pPK) segment (11-23), of sequence Phe-Phe-Arg-Gly-Gly-Asp-Val-Ser-Ala-Met-Tyr-Thr-Pro, present in the first tandem repeat sequence of the regulatory chain of PK, has been accomplished following the peptide fragments (5 + 4 + 4) condensation strategy in solution, as well as by fluorenylmethoxycarbonyl solid-phase chemistry. This and another synthetic PK segment of residues (328-343) present in the fourth tandem repeat sequence [Cys(ACM)-Ser-Leu-Arg-Leu-Ser-Thr-Asp-Gly-Ser-Pro-Thr-Arg-Ile-Thr-Tyr] and synthesized by a solid-phase method, were fully characterized by 1H nuclear magnetic resonance, fast atom bombardment mass spectrometry, amino acid composition and reversed-phase high-performance liquid chromatography. Proteolysis of these peptides by either rat PK (rPK) or trypsin resulted in cleavages between Arg decreases Gly for pPK (11-23) and between Arg decreases Leu and Arg decreases Ile for rPK (328-343). Kinetic studies revealed that for peptide pPK (11-23), the catalytic efficiency (kcat/Km) of rPK is congruent to 9-fold higher than that of trypsin, but for the other peptide, rPK (328-343), kcat/Km of trypsin is congruent to 49-fold higher than that of rPK. The facile cleavage of pPK (11-23) by rPK confirms the Arg13 decreases Gly14 position as the site of autolytic degradation of PK and also explains its special preference for Phe-Phe-Arg sequence.

Amino Acid Sequence↗

PubMed1993

Radiolabeled biotinyl peptides as useful reagents for the study of proteolytic enzymes.

A radiometric assay for studying the proteolytic activity of endopeptidases using a radiolabeled biotinyl peptide substrate is described. The method relies on the use of a peptidyl substrate incorporating susceptible bonds located between a biotinyl group at one end and a radioiodinated group at the other end. Two tyrosine-containing peptidyl substrates, a fragment of rat plasma kallikrein and a derivative of Leu-enkephalin, were coupled to biotin by reacting with N-hydroxysuccinimidyl-6-(biotinamido) hexanoate. The enzymatic activity is measured by the release into solution of the radiolabeled peptide fragment following selective retrieval, using immobilized avidin, of the biotinyl undigested substrate and the unlabeled biotinyl peptide fragment. This study illustrates that retrieval can be done either before incubation with the enzyme by immobilizing the labeled substrate onto avidin-agarose or, alternatively, after incubation by treating the resulting digest with immobilized avidin. The identity of the labeled released peptides and hence the sites of cleavage can be obtained following separation by RP-HPLC. This assay, in addition to allowing proteolysis to occur with the substrate either in solution or immobilized, is rapid, sensitive (less than 1 pg/ml of trypsin), reproducible, and applicable to the detection of members of all endopeptidase classes. Furthermore, incubation of the radiolabeled biotinyl peptide substrates with enzymes immobilized within polyacrylamide gel slices can be used to detect proteolytic activity following electrophoretic separations under denaturing or nondenaturing conditions.

Amino Acid Sequence↗

PubMed1993

Synthesis and reaction of potential alternate substrates and mechanism-based inhibitors of clavaminate synthase.

Clavaminate synthase is an FeII/alpha-ketoglutarate-dependent enzyme central to the biosynthesis of the beta-lactamase inhibitor clavulanic acid. In the presence of dioxygen it catalyzes the oxidative cyclization/desaturation of proclavaminic acid to clavaminic acid in a two-step process. Samples of (4'R)- and (4'S)-D,L-[4'-2H]proclavaminic acid have been prepared and used to demonstrate that oxazolidine ring formation occurs with retention of configuration. The stereochemical course of oxygen insertion from substrate that takes place in this oxidative cyclization is the same as that observed from molecular oxygen in several hydroxylation reactions catalyzed by other FeII/alpha-ketoglutarate-dependent enzymes. The ferryl (FeIV = O) species thought to be transiently involved in each of these processes was investigated in the present work with clavaminate synthase and three structural analogues of proclavaminic acid bearing vinyl or ethynyl groups at C-4' or a cyclopropyl at C-4. In the synthesis of the former two derivatives and proclavaminic acid stereoselectively labeled with deuterium at C-4', introduction of the unsaturated substituents in a stereochemically defined manner at C-4' relied upon ready access to (4R)-4-thiophenyl-2-azetidinone. Trimethylsilyl substitution could be easily achieved at C-3 of the optically pure starting material to give the readily separable cis and trans diastereomers. In radical chain reactions in which the thiophenyl was replaced by deuterium or in anionic reactions in which the thiophenyl was eliminated as its sulfone and replaced by addition of carbanions, the steric bulk of the trimethylsilyl group at C-3 governed the approach of incoming reagents to give the trans product. The enzymatic fate, however, of these derivatives was disappointing, yielding neither detectable reaction nor hoped-for inactivation of clavaminate synthase. Finally, as mixed competitive/noncompetitive inhibitors of catalysis, they gave unexceptional inhibition constants in the range 2-10 mM.

Aza Compounds↗

PubMed1992

Affinity purification of proteinases by a combination of immobilized peptidyl aldehyde and semicarbazone.

D-Phe-Phe-argininal semicarbazone and Tyr-Gly-Gly-Phe-Leu-Arg-argininal semicarbazone were prepared using the solution phase synthesis method and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. The tripeptide and heptapeptide semicarbazones were individually immobilized on affi-Gel 15 resulting in two affinity columns called S3 and S7, respectively. A third affinity column was obtained by hydrolysing the semicarbazone moiety in column S3 to aldehyde (column A3). Serine proteinases such as trypsin or rat plasma kallikrein almost quantitatively bind to either S3 or A3 affinity columns. Under optimized conditions, more than 97% of trypsin bound to both columns S3 and A3. At a lower ionic strength and higher pH, 80-85% of rat plasma kallikrein bound to the same columns. Elution of both enzymes was achieved using mild conditions at near neutral pH and in the presence of a small amount of denaturant. Both proteinases were identified and characterized by high-performance liquid chromatography, sodium dodecylsulphate polyacrylamide gel electrophoresis and by their substrate specificity and inhibition profiles. A single purification (six-to seven-fold) step using either column S3 or A3 allowed the preparation of pure trypsin from commercial sources. Starting from rat plasma partially purified by a phenyl boronate column, fractionation on the S3 column allowed approximately an 87-fold purification of rat plasma kallikrein. However, serial purification of rat plasma kallikrein on column S7 followed by column A3 resulted in a purification factor of about 455.

Aldehydes↗

PubMed1991

Structure of a psoralen derivative of a monosubstituted 18-crown-6 ether.

N-[(2,5,9-Trimethyl-7-oxo-7H-furo[3,2-g]-[1] benzopyran-3-yl)methyl]-1,4,7,10,13,16-hexaoxacyclooctadecane- 2,3-dicarboximide, C29H35NO11, Mr = 573.60, monoclinic, P21, alpha = 18.877 (9), b = 6.888 (6), c = 24.488 (10) angstrom, beta = 119.90 (4) degrees, V = 2760.2 angstrom3, Z = 4, Dx = 1.380 Mg m-3, lambda (Cu K a) = 1.54178 angstrom, mu = 0.85 mm-1, F(000) = 1216, T = 170 K, R = 0.065, wR = 0.057 for 3720 observed reflections. There are two molecules (I and II) in the asymmetric unit and the 18-crown-6 part of molecule II is disordered. The psoralen moieties of the two molecules are nearly centrosymmetrically related.

Bleomycin↗

PubMed1991

Detection of endopeptidase activity and analysis of cleavage specificity using a radiometric solid-phase enzymatic assay.

A radiometric procedure to detect the presence of proteolytic enzymes and analyze their substrate specificity is described. The enzymatic activity is first measured by the release into solution of a radiolabeled reporter group from an immobilized peptidyl substrate. Two peptidyl substrates encompassing multiple cleavage sites, a derivative of Leu-enkephalin and a peptide related to the bait region of human alpha 2-macroglobulin, are prepared and linked via a spacer molecule to an insoluble support. The labeled peptides released are then separated by high-performance liquid chromatography. The position of the released peptides upon chromatography allows direct identification of the sites of cleavage. The assay, using a radioactive iodinated tyrosine residue as reporter group, is extremely sensitive (less than 0.02 pg/ml of trypsin), reproducible, and easy to perform while yielding unambiguous identification of the sites of cleavage. This assay can be used to detect the presence of enzymatic activities and/or of enzyme inhibitors. Furthermore, it can be easily adapted to detect from a variety of sources all four classes of enzymes known by using appropriate peptidyl substrate sequences, buffer, pH, and incubation conditions.

Amino Acid Sequence↗

PubMed1990

Syntheses of argininal semicarbazone containing peptides and their applications in the affinity chromatography of serine proteinases.

Eight argininal semicarbazone containing peptides prepared by liquid phase synthesis were all found to be reversible inhibitors of model serine proteinases including trypsin and plasma kallikrein (PK). Among the peptides tested, those having a Lys residue at position P2 displayed the maximum binding potency towards PK. One of the peptides, Leu-enkephalin-argininal semicarbazone, a comparatively weak inhibitor, was chosen in order to develop an affinity-based purification protocol for PK. The affinity column was prepared by covalent attachment of the NH2-terminal moiety of the peptidyl semicarbazone to a solid-phase matrix bearing a spacer group. For efficient binding of PK, it was found necessary to optimize parameters like the concentration of inhibitor linked to the solid matrix, the ionic strength of the buffer used, the temperature and the pH. The majority of the bound enzyme could be recovered following elution with guanidine hydrochloride or benzamidine hydrochloride in a high salt buffer at pH 6.0. The usefulness of the affinity procedure towards the purification of other serine proteinases is also discussed.

Amino Acid Sequence↗

PubMed1985

Dopaminergic activity of four analogs of butaclamol.

The displacing potency of four analogs of the neuroleptic drug butaclamol were evaluated using dog striatal tissue and [3H]-Spiroperidol as ligand. Although significantly less powerful than the parent compound, two of them (N-isobutyl butaclamol equatorial; N-methyl butaclamol equatorial) could be used for dopaminergic receptor studies.

Animals↗

PubMed1979

Impact of cardiopulmonary resuscitation training on resuscitation.

Restoration of adequate spontaneous circulation after "arrest" and cardiopulmonary resuscitation (CPR) of 546 patients before and 460 patients after initiation of a CPR training course in a 500-bed city hospital is reported. Between January 1972 and June 1976, adequate circulation after CPR was present in 38.6% of patients before and 50.4% after training ICU nurses and house physicians in modern resuscitation techniques. Factors crucial to resumption of adequate circulation are: (1) CPR training of all hospital personnel so that effective CPR can be started immediately after recognition of an arrest situation, (2) production of a palpable pulse with closed chest cardiac massage, and (3) prompt effective therapy so that the time interval between arrest and resumption of adequate spontaneous circulation is short.

Blood Circulation↗

PubMedDate not supplied

Application of the multiple antigenic peptides (MAP) strategy to the production of prohormone convertases antibodies: synthesis, characterization and use of 8-branched immunogenic peptides.

Antiserum against an N-terminal sequence of murine prohormone convertase-1 (mPC1) incorporating the sequence immediately following the junction between the putative pro-region and the active enzyme was obtained. This was accomplished using the multiple antigenic peptide (MAP) approach whereupon an 8-branched polylysine core to which are grafted multiple copies of a 16 amino acid peptide representing the N-terminal sequence of mPC1 (positions 84-99) was synthesized by solid-phase Fmoc chemistry. The ensuing peptide was purified and fully characterized by RP-HPLC, 1H-NMR, amino acid composition, peptide sequencing and ion-spray mass spectrometry. The immunological properties of the resulting antibodies in detecting recombinant PC1 in both crude and purified preparations were compared with antibodies raised against a similar N-terminal segment of PC1 but using the conventional method of peptide-carrier protein conjugation and also developed against a C-terminal fusion protein of PC1. Our data indicate that the MAP antibody was as efficient as both the amino and carboxy-terminal antibodies in qualitative as well as quantitative analysis of PC1 encoded protein by radioimmunoassay. Following an identical approach, antibodies against other prohormone convertases like furin, PC5/6 and PACE4 were also developed and subsequently applied to a number of biochemical and immunological studies. In each case, the case of preparation and high immunogenicity of the MAP approach were confirmed and reside in the simplicity and rapidity with which a potent and useful antiserum is obtained.

Amino Acid Sequence↗