Purification and characterization of an acid proteinase from mesophilic Mucor sp. solid-state cultures.

The fourth-day extract of a solid-state culture of the mesophilic Mucor sp. (M-105) strain showed a high milk-clotting activity and a clotting/proteolytic activity ratio similar to that of commercial preparations from microbial origin used in cheese manufacture. After ultrafiltration of the crude extract, the milk-clotting proteinase was purified in two steps: ion-exchange followed by size-exclusion chromatography. Enzyme homogeneity was assessed by HPLC, SDS-PAGE and N-terminal residue determination. A pI value of 4.21 was obtained and a molecular weight of 33 kDa was calculated from size-exclusion chromatography and SDS-PAGE data. The optimum pH for proteolytic activity towards dimethylcasein was in the 3.0-3.5 range. The proteinase retained 26 and 13% of its proteolytic activity after a 30-min incubation period, at pH 5.0 and 50 and 60 degrees C, respectively. This evidenced a lower heat stability than that of the thermophilic enzymes currently used in the cheese industry and also than that of bovine chymosin. The enzyme was fully inhibited by pepstatin A and no effect was observed with PMSF, p-CMPS or EDTA. The N-terminal amino acid sequence: GTGTVPVTDDGNLNEYYXTVTVGXP was compared with those from other fungal enzymes.

Circulating antibodies against nicotinic acetylcholine receptors in chagasic patients.

Human and experimental Chagas' disease causes peripheral nervous system damage involving neuromuscular transmission alterations at the neuromuscular junction. Additionally, autoantibodies directed to peripheral nerves and sarcolemmal proteins of skeletal muscle have been described. In this work, we analyse the ability of serum immunoglobulin factors associated with human chagasic infection to bind the affinity-purified nicotinic acetylcholine receptor (nAChR) from electric organs of Discopyge tschudii and to identify the receptor subunits involved in the interaction. The frequency of serum anti-nAChR reactivity assayed by dot-blot was higher in seropositive chagasic patients than in uninfected subjects. Purified IgG obtained from chagasic patients immunoprecipitated a significantly higher fraction of the solubilized nAChR than normal IgG. Furthermore, immunoblotting assays indicated that alpha and beta are the main subunits involved in the interaction. Chagasic IgG was able to inhibit the binding of alpha-bungarotoxin to the receptor in a concentration-dependent manner, confirming the contribution of the alpha-subunit in the autoantibody-receptor interaction. The presence of anti-nAChR antibodies was detected in 73% of chagasic patients with impairment of neuromuscular transmission in conventional electromyographical studies, indicating a strong association between seropositive reactivity against nAChR and electromyographical abnormalities in chagasic patients. The chronic binding of these autoantibodies to the nAChR could induce a decrease in the population of functional nAChRs at the neuromuscular junction and consequently contribute to the electrophysiological neuromuscular alterations described in the course of chronic Chagas' disease.

Involvement of histidine 134 in the binding of alpha-bungarotoxin to the nicotinic acetylcholine receptor.

Peptides corresponding to the sequence alpha 124-147 of the Torpedo californica and Homo sapiens nicotinic cholinergic receptors were synthesized. The His residue at position 134 was ethoxyformylated or substituted by Ala. Effects of such modifications were studied by: (a) a toxin blot assay and (b) a competition assay between each peptide and the Discopyge Ischudii receptor for 125I alpha-bungarotoxin, in solution. Apparent Kd values were 0.1 and 0.8 microM for Torpedo californica and Homo sapiens native peptides, respectively, and no binding was observed when the His residue was modified or substituted by Ala. ic50 values for the Torpedo californica and Homo sapiens fragments were 1.0 and 0.8 microM, respectively, and no significant displacement occurred when His 134 was ethoxyformylated or substituted by Ala. Hydroxylamine treatment restored 80-100% of their binding ability. Results strongly support the involvement of His 134 in the binding of alpha-bungarotoxin either to the Torpedo californica or the Homo sapiens receptor.

Further characterization and kinetic parameter determination of a milk-clotting protease from Mucor bacilliformis.

Further characterization of an aspartyl protease from Mucor bacilliformis with milk-clotting activity was performed. An extinction coefficient, epsilon 278 cm = 1.61 mL/mg/cm, a molecular mass of 35,400 Da and a pI of 5.2 were determined. Proteolytic activity and kinetic parameters were evaluated by using the hexapeptide Leu-Ser-pNO2-Phe-Nle-Ala-Leu-OMe as the substrate. The effect of pH and temperature on peptide cleavage, as well as protease heat stability, was determined. Such properties, taken as a whole, indicate that the M. bacilliformis protease can be considered a potential substitute for bovine chymosin in cheese manufacture.

Localization of histidine residues relevant for the binding of alpha-bungarotoxin to the acetylcholine receptor alpha-subunit in V8-proteolytic fragments.

Histidine residues have been shown to be critical for alpha-BgTx binding to the acetylcholine receptor (Lacorazza et al., 1992; Bouzat et al., 1993; Lacorazza et al., 1995). Receptor subunits from Discopyge tschudii were modified with diethylpyrocarbonate (DEP). DEP treatment produces a concentration-dependent decrease of [125I] alpha-BgTx binding to the alpha-subunit. The neurotoxin binding capacity was fully restored by adding the nucleophile hydroxylamine. By proteolytic mapping of the alpha-subunit with V8-protease, we determined that the binding capacity to the fragment alpha V8-19 decreased 80% by DEP treatment. In addition, the [125I] alpha-BgTx binding to the same fragment decreased by 70% when the subunits were reduced and affinity-alkylated. We report the N-terminal sequence of both subunits and V8-fragments (alpha V8-10, alpha V8-13, and alpha V8-18), which constitute a first contribution to the knowledge of the primary structure of the Discopyge tschudii receptor. We propose that the fragment alpha V8-19 contains one or more of the histidine residues involved in the alpha-BgTx binding and probably includes the Cys alpha 192-193 disulfide bond. Only two histidine residues are present in the extracellular sequence of Torpedo californica for such fragments: His alpha 186 and alpha 204.

Key histidine residues in the nicotinic acetylcholine receptor.

Reactivity of histidine residues of the Discopyge tschudii nicotinic acetylcholine receptor was studied by reaction with DEP and the influence of their modification on functional properties of the receptor was evaluated. Determination of two kinetically distinguishable classes was achieved. The fast-reacting class is composed of 7 histidine residues with an apparent velocity constant k1 = 0.0248 +/- 0.0031 min-1. The second includes--at least--21 histidine residues with a velocity constant k2 = 0.0016 +/- 0.0009 min-1. The circular dichroism spectra of the native receptor and the most DEP-derivative indicate no significant modifications in the alpha-helix content, and fourth derivative spectroscopy analyses show that the environment around the aromatic amino acids remains unchanged. DEP treatment of the receptor results in a time- and reagent concentration-dependent loss of its alpha-bungarotoxin binding ability; these results agree with those obtained with the membrane-bound receptor. The decrease in the neurotoxin binding capacity was correlated with the DEP-reaction extent of the slow groups. Incorporation of 1.93 +/- 0.23 mol of DEP accounted for the maximal binding capacity drop, thus indicating the involvement of two histidine residues per alpha-bungarotoxin binding site. Neither amino groups nor tyrosine residues were modified during the reaction with DEP, indicating that the derivatization of histidine residues is responsible for the observed effect. Faster-reacting residues appear to be involved in agonist-induced ion flux through the nAChR channel. These results strongly support the connection between histidine residues and the receptor functional activity and lead us to infer that the changes observed in alpha-bungarotoxin binding and ionic channel capacity are the consequence of independent events induced by reaction with DEP.

Oxidation of methionine residues in equine growth hormone by Chloramine-T.

1. Reactivity of methionine residues towards Chloramine-T was studied in the equine growth hormone. 2. With a 20.0-fold molar excess of reagent over methionine, full oxidation of the four residues of the protein is achieved. 3. Methionine 4 is the most reactive group, followed by methionines 72 and 178--methionine 123 being the less reactive residue. 4. As judged by circular dichroism spectra and binding assays, protein conformation and binding capacity to specific receptors remains unchanged even after full oxidation of all four methionine residues. 5. Results agree with data previously obtained with bovine growth hormone.

Effect of chemical modification of extracellular histidyl residues on the channel properties of the nicotinic acetylcholine receptor.

We have examined the effect of chemical modification with diethyl pyrocarbonate (DEP) on the properties of acetylcholine (ACh)-activated channels in the cloned muscle-cell line BC3H-1. After protein modification, patch-clamp recordings showed alterations in the kinetics of the nicotinic acetylcholine receptor (AChR) channel. The major effect was observed in the channel mean open time, which was reduced up to about 12-fold at 466 microM DEP. The specificity of the effect was first established through comparison with both untreated cells and cells treated with inactivated DEP. Consistent with an increase in the number of unprotonated histidine residues (pKa = 6.0), this effect increased concomitantly with the pH of the reaction medium, being faster at pH 8 than at pH 6. The changes were dependent on time and DEP concentration, with an apparent EC50 = 114 microM. Modified channels also showed an increase in the number of events per burst of openings together with a decrease in burst durations. The amplitude of the channel-closed time component of about 1 ms increased with respect to the longest-duration-closed component. The number of alpha-bungarotoxin sites was slightly reduced after the modification, without affecting ligand binding affinity. The results suggest that DEP affects extracellular histidine residues involved in the ion translocation function of the AChR, but not its toxin-recognition ability. DEP could, therefore, induce a dissociation between toxin and agonist binding, as is often observed in neuronal AChR.

Involvement of a disulfide bond in the binding of flunitrazepam to the benzodiazepine receptor from bovine cerebral cortex.

The effects of chemical modification of a disulfide bond(s) (-SS-) or sulfhydryl group(s) (-SH) on the [3H]-flunitrazepam ([3H]FNZ) binding to membrane-bound or immunoprecipitated benzodiazepine (BZD) receptors (BZD-R) from bovine cerebral cortex were examined. Reduction of -SS- with dithiothreitol (DTT) brought about a reversible, time- and dose-dependent inhibition of [3H]FNZ binding to the membrane-bound BZD-R. Alkylation of the membranes with the -SH-modifying reagent iodoacetamide (IAA) or 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) produced a slight inhibition of [3H]FNZ binding in a dose-dependent manner. Scatchard analysis of saturation curves of [3H]FNZ binding in the presence and absence of 5 mM DTT revealed changes in affinity without modification in the maximal binding capacity, thus indicating a competitive mode of interaction. DTT pretreatment of both the membrane-bound and the immunoprecipitated BZD-R led to [3H]FNZ binding inhibition. Consistent with the modification of a binding site is the observation that reduction of -SS- does not bear on the binding affinity, but rather reduces the number of sites. Complete protection from DTT inhibition of [3H]FNZ binding by FNZ (an agonist) or by Ro 15-1788 (an antagonist) suggests the presence of -SS- at, or very close to, the BZD recognition binding site. No protection against IAA or DTNB inhibition was provided by FNZ. Photoaffinity labeling experiments with [3H]FNZ revealed a clear-cut band of 50 kDa in native and alkylated membranes but an extremely weak label in 5 mM DTT/IAA-treated membranes. The present results provide evidence for the participation of a disulfide bond in the recognition binding site of the bovine cerebral cortex BZD-R.

Role of histidine residues in the alpha-bungarotoxin binding site of the nicotinic acetylcholine receptor.

This paper studies the effect of histidine chemical modification of the membrane-bound acetylcholine receptor from Discopyge tschudii on its specific alpha-bungarotoxin binding. The acylating reagent ethoxyformic anhydride (diethyl pyrocarbonate, DEP), was used. DEP-treatment induces a loss of binding capacity, time and DEP-concentration dependent. After a 30 min period of derivatization with 2 mM final DEP-concentration, at pH 7.4, the decrease reaches 70%; the loss of binding capacity is faster at pH 7.4 than at pH 6.0, as expected, since the amount of unprotonated species is higher under the first condition. Moreover, when ethoxyformylation is carried out at different pH values, the most important neurotoxin binding decrease occurs between pH 6.0 and 8.0. Furthermore, ethoxyformylation reversion restores such capacity. Consistent with the modification of a binding site, the ethoxyformylation does not bear on the affinity but reduces the number of receptors. Ethoxyformylation in the presence of carbamylcholine shows some ligand protective effect. These results, as a whole, strongly indicate a relevant role for histidine residues at the alpha-bungarotoxin binding site of the nicotinic acetylcholine receptor.

An equine growth hormone molecular species lacking the 76-92 peptidic fragment.

A new eGH molecular species was isolated and purified by reverse phase HPLC. SDS-polyacrylamide gel electrophoresis, amino acid composition, and C- and N-terminal determinations support a primary structure identical to that described by Zakin et al. (1976), except for the lack of the 76-92 peptidic fragment and the maintaining of 30% of its biological activity.

Role of histidine residues in the binding mechanism of the growth hormone family.

1. Reactivity of the hGH histidine residues were studied by reaction with ethoxyformic anhydride. Localization in the molecule of three kinetically distinguishable classes, each including only one residue, was achieved. 2. The first was composed of residue 151, with an apparent velocity constant k = 0.735/min, (similar to that of histidines 19 and 21 in bGH and eGH). The second histidine, 18, with a velocity constant k = 0.135/min, (similar to that of histidine 169 in the above hormones), and a third, histidine 21, which does not react at all. 3. Neither histidine 151 nor 18 seem to be involved, at least not directly, in bGH binding to specific rat liver sites, since the decrease in this capacity was only 47% after modification of the former by 77 and 65% after total modification of the latter. 4. These results, and those previously obtained with bGH and eGH, suggest that either histidine 21 is the only indispensable histidine for the binding of growth hormones to specific rat liver sites, or that histidine 21 and/or 18 (19 in bGH and eGH), are located within the growth hormone binding site interaction area.

Conformational comparison in the growth hormone family.

1. The method of Kubota et al. [Biochim. biophys. Acta 701, 242-252 (1982)] was applied to several members of the growth hormone family in order to examine their conformational homology. 2. The method neither detects differences between rat, cow, sheep, horse and alpaca hormones, nor between monkey and human hormones. 3. Lack of homology between primate and non-primate growth hormones was found in segments 42-49 and 184-191. The first fragment could be linked to species-specificity.

Antigenicity of basement membrane fractions obtained from rat seminiferous tubules.

A noncollagenous fraction of basement membrane (D-STBM) obtained from rat testes was submitted to sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis (SDS-PAGE), and eight well-defined bands were detected. A cross-reaction with an antiserum against laminin was revealed by immunoblotting in five bands, with molecular weights ranging from 54 to 64 kDa. No further resolution of these components could be obtained by size exclusion and ionic exchange chromatography. Fifty-two percent of the rats immunized with D-STBM and adjuvants developed a mild multifocal damage of the testis. The lesions were characterized by foci of seminiferous tubules with different degrees of sloughing and/or atrophy of the germinal epithelium. Giant multinucleated cells were frequently seen, and mild interstitial mononuclear cell infiltrates were also detected. By immunofluorescence, deposits of rat IgG with a faint discontinuous linear pattern were observed along the walls of the seminiferous tubules. Circulating antibodies to D-STBM were detected by ELISA in 100% of the rats, whereas in a cross-reaction with laminin antibodies were detected in only 63%. All rats studied revealed a positive delayed type of hypersensitivity (DTH) response to D-STBM. None of the control rats injected with saline and adjuvants presented circulating antibodies to D-STBM or laminin or a positive DTH reaction to D-STBM. Some control group rats (10%) revealed few isolated seminiferous tubules with some degree of sloughing of the germinal epithelium.

An approach to the three-dimensional structure of bovine growth hormone based on chemical modification and secondary structure prediction.

Three different methods have been applied to the prediction of secondary structure. The prediction that better fitted the chemical data was chosen. Two regions of the bovine growth hormone molecule (111-117 and 166-174) appear to be exposed to the solvent, according to hydropathic analysis but have several charged residues not reactive towards their specific reagents. Two molecular domains are postulated, each one bearing a region with charged residues on its surface and interacting with the other in the molecule by means of saline bridges. The hydrophobic core of the molecule is formed by the ensemble of the hydrophobic region predicted between residues 81 and 108, and the hydrophobic faces of the amphiphilic helices 109-127 and 9-33.

Ethoxyformylation of histidine residues in equine growth hormone.

Reactivity of histidine residues in equine growth hormone to ethoxyformic anhydride was studied. The existence of two kinetically different sets was demonstrated: one of them including only the slow reacting histidine 169 (k = 0.164 min-1) and the other containing fast reacting histidines 19 and 21 (k = 0.892 min-1). A correlation between the decrease in the capacity to compete with 125I-labeled hormone for rat liver binding sites and the degree of ethoxyformylation of the fast group was found. Circular dichroism studies indicated no significant conformational changes in the protein with all three residues modified. These results fully agree with those obtained for bovine growth hormone which is further evidence supporting the vinculation of histidines 19 and/or 21 with the binding site of these hormones to their specific receptors.

Hydrophobicity modulation as a tool for the purification of histidyl peptides.

A methodology is described for purification of histidyl peptides based on the changes in hydrophobicity induced by the specific and reversible modification of histidine residues by ethoxyformic anhydride. The mixture of modified peptides is subjected to HPLC; peptides containing modified histidines are detected at 242 nm, recovered, deethoxyformylated, and rechromatographed under the same conditions, then being detected at 220 nm. This procedure allows their isolation free from contaminants.

Molecular characterization of a protein, insoluble at low temperature, produced by Clostridium botulinum type G.

A preliminary study of a low-toxicity protein, called cryoprotein, produced by Clostridium botulinum type G, led to a better characterization of this substance and to discriminate its relationship with type G botulinum toxin. This sparingly soluble protein has been characterized as an aggregated form of a soluble precursor with an Mr of 170,000. This phenomenon is temperature-dependent. The monomeric protein is usually contaminated with a lower Mr form (150,000) quite probably originated by a limited proteolytic process. The amino acid composition of this protein is relatively analogous to that of the botulinum toxins A and B, the only notable exception being the absence of cysteine. The N-terminal amino acid is alanine and the C-terminal sequence is Val-Ala-Leu-OH. The low toxicity which is usually demonstrable in samples of this protein disappears after a reductive treatment, strongly suggesting that it is not an intrinsic property. Taking into account that some of its physiochemical properties are similar to those of the known botulinal toxins, it is quite probable that this substance accompanies G toxin preparations currently obtained by routine methods, increasing its non-toxic antigenic mass. This fact could be critical to the sensitivity and specificity of G toxin immunological detection methods.

Amino acid sequences around the cystine residues in alpaca growth hormone.

Half-cystine peptides were isolated from the enzymatic digest of reduced and carbamidomethylated alpaca growth hormone. From their amino acid composition and determination of the end terminal residues we propose the amino acid sequence around the two disulphide bridges, partially based on homology with other mammalian growth hormones. The proposed sequence is identical to that of equine growth hormone and very similar to those of rat, bovine and ovine hormones.

Basic and acidic hydrophilic residues involved in the interaction between protomers of the bovine growth hormone dimer.

Reactivity of histidine and arginine residues--as well as of carboxyl-groups--in the covalently stabilized dimer of bovine growth hormone, was studied in comparison with their reactivity in the monomeric form. Results obtained by reaction of histidine and arginine residues with ethoxyformic anhydride and cyclohexanedione, respectively, were similar for both proteins. The reactivity of two carboxyl-groups towards a soluble carbodiimide becomes impaired in the dimer, thus suggesting that their location is within the protomer interaction area.