Isolation and characterization of monodeamidated derivatives of bovine pancreatic Ribonuclease A

The isolation and characterization of the initial intermediates formed during the irreversible acid denaturation of enzyme Ribonuclease A are described. The products obtained when RNase A is maintained in 0.5 M HCl at 30° for periods up to 20 h have been analyzed by ion-exchange chromatography on Amberlite XE-64. Four distinct components were found to elute earlier to RNase A; these have been designated RNase Aa₂, Aa_1c, Aa_1b, and Aa_1a in order of their elution. With the exception of RNase Aa₂, the other components are nearly as active as RNase A. Polyacrylamide gel electrophoresis at near-neutral pH indicated that RNase Aa_1a, Aa_1b, and Aa_1c are monodeamidated derivatives of RNase A; RNase Aa₂ contains, in addition, a small amount of a dideamidated component. RNase Aa₂, which has 75% enzymic activity as compared to RNase A, consists of dideamidated and higher deamidated derivatives of RNase A. Except for differences in the proteolytic susceptibilities at an elevated temperature or acidic pH, the monodeamidated derivatives were found to have very nearly the same enzymic activity and the compact folded structure as the native enzyme. Fingerprint analyses of the tryptic peptides of monodeamidated derivatives have shown that the deamidations are restricted to an amide cluster in the region 67–74 of the polypeptide chain. The initial acid-catalyzed deamidation occurs in and around the 65–72 disulfide loop giving rise to at least three distinct monodeamidated derivatives of RNase A without an appreciable change in the catalytic activity and conformation of the ribonuclease molecule. Significance of this specific deamidation occurring in highly acidic conditions, and the biological implications of the physiological deamidation reactions of proteins are discussed.     

THE INFLUENCE OF ESTERIFICATION OF CARBOXYL GROUPS OF RIBONUCLEASE-A ON ITS STRUCTURE AND IMMUNOLOGICAL ACTIVITY

The effect of modification of carboxyl groups of Ribonuclease-A^a on the enzymatic activity and the antigenic structure of the protein has been studied. Modification of four of the eleven free carboxyl groups of the protein by esterification in anhydrous methanol/0.1 M hydrochloric acid resulted in nearly 80% loss in enzymatic activity but had very little influence on the antigenic structure of the protein. Further increases in the modification of the carboxyl groups caused a progressive loss in immunological activity, and the fully methylated RNase-A exhibited nearly 30% immunological activity. Concomitant with this change in the antigenic structure of the protein, the ability of the molecule to complement with RNase-S-protein increased, clearly indicating the unfolding of the peptide “tail” from the remainder of the molecule. The susceptibility to proteolysis, accessibility of methionine residues for orthobenzoquinone reaction and the loss in immunological activity of the more extensively esterified derivatives of RNase-A are suggestive of the more flexible conformation of these derivatives as compared with the compact native conformation. The fact that even the fully methylated RNase-A retains nearly 30% of its immunological activity suggested that the modified protein contained antibody recognizable residual native structure, which presumably accommodates some antigenic determinants.     

Influence of deamidation(s) in the 67–74 region of ribonuclease on its refolding

The influence of chemical mutation featuring the selective conversion of asparagine or glutamine to aspartic or glutamic acid, respectively, on the kinetics of refolding of reduced RNase has been studied. The monodeamidated derivatives of RNase A, viz. RNase Aa_1a, Aa_1b, and Aa_1c having their deamidations in the region 67–74, were found to regain nearly their original enzymatic activity. However, a marked difference in the kinetics of refolding is seen, the order of regain of enzymic activity being RNase A > Aa_1c Aa_1a > Aa_1b. The similarities in the distinct elution positions on Amberlite XE-64, gel electrophoretic mobilities, and u.v. spectra of reoxidized and native derivatives indicated that the native structures are formed. The slower rate of reappearance of enzymic activity in the case of the monodeamidated derivatives appears to result from altered interactions in the early stages of refolding. The roles of some amino acid residues of the 67–74 region in the pathway of refolding of RNase A are discussed.     

Chemical stability of peptides in polymers. 2. Discriminating between solvent and plasticizing effects of water on peptide deamidation in poly(vinylpyrrolidone).

The mechanistic role of water in the deamidation of a model asparagine-containing hexapeptide (Val-Tyr-Pro-Asn-Gly-Ala) in lyophilized formulations containing poly(vinylpyrrolidone) (PVP) and glycerol was investigated. Glycerol was used as a plasticizer to vary formulation glass transition temperature (T(g)) without significantly changing water content or activity. Increases in moisture and glycerol contents increased the rate of peptide deamidation. This increase was strongly correlated with T(g) at constant water content and activity, suggesting that increased matrix mobility facilitates deamidation. In rubbery systems (T > T(g)), deamidation rates appeared to be independent of water content and activity in formulations with similar T(g)s. However, in glassy formulations with similar T(g)s, deamidation increased with water content, suggesting a solvent/medium effect of water on reactivity in this regime. An increase in water content also affected the degradation product distribution; less of the cyclic imide intermediate and more of the hydrolytic products, isoAsp- and Asp-hexapeptides, were observed as water content increased. Thus, residual water appears to facilitate deamidation in these solid PVP formulations both by enhancing molecular mobility and by solvent/medium effects, and also participates as a chemical reactant in the subsequent breakdown of the cyclic imide.     

Acid-induced COX-2 expression and prostaglandin E<Subscript>2</Subscript> production via activation of ERK1/2 and p38 MAPK in cultured feline esophageal smooth muscle cells

Cyclooxygenase (COX)-2 is known to play an important role in inflammatory conditions such as reflux esophagitis resulting from acid reflux. In this study, we tested whether an acidic medium (pH 4.0) induces an increase in COX-2 expression or PGE₂ production, and explored the implication of mitogen-activated protein kinases (MAPKs) activation in these responses in cultured cat esophageal smooth muscle cells. Acidic cytotoxicity was assessed and expression changes in COXs or phosphorylated MAPKs were analyzed by Western blotting. PGE₂ production was measured by immunoassay. No significant decrease in cell viability was observed for 6 h exposure to acidic medium. COX-2 expression and PGE₂ production significantly increased to maximal levels at 6 h exposure to acidic medium. The cells also exhibited significant activation of ERK1/2 and p38 MAPK, but not JNK within 10 min under acidic medium. The increments of COX-2 expression and PGE₂ production by acidic medium were decreased by pretreatment with PD98059 or SB202190, respectively. These results suggest that acidic environments may enhance the COX-2 expression and PGE₂ production through activation of ERK1/2 and p38 MAPK in the cultured cat esophageal smooth muscle cells.     

Enzymatic methyl esterification of a deamidated form of mouse epidermal growth factor

The enzyme S-adenosylmethionine:protein carboxyl-O-methyl-transferase, type II (EC2.1.1.77; PCMT) from eukaryotes methyl esterifies peptides containing isoAsp residues, which can arise from spontaneous deamidation of labile Asn residues. We report here a study on in vitro methyl esterification of mouse EGF by bovine brain PCMT. This peptide contains two Asn in the sequences Asn¹-Ser² and Asn¹⁶-Gly¹⁷. It is known from the literature that the presence of a small residue on the carboxyl side of asparaginyl makes this residue susceptible to deamidation through the spontaneous formation of a succinimide intermediate. Therefore EGF was incubated under deamidating conditions (pH9.0, 37° for 48 h) and the extent of deamidation monitored by enzymatically measuring the NH₃ produced during the alkali treatment: a release of 0.80 mol NH₃/mol EGF was calculated. The alkali-treated EGF, analyzed by anion-exchange chromatography, shows two major components identified as native EGF (nEGF) and its deamidated form (dEGF). When incubated in the presence of purified PCMT neither nEGF nor dEGF showed any methyl accepting capability. Since it is known that the three-dimensional structure of a protein may hinder the methyl esterification of a potential ethyl accepting site, dEGF was unfolded by reducing and alkylating the intrachain disulfide bridges. Only a slight increase in the methyl accepting capability could be observed. Conversely, when EGF was deamidated after its unfolding, the resulting protein was stoichiometrically methylated by PCMT, presumably at level of isoAsp¹⁶. Our findings strongly suggest that the three-dimensional structure of a protein is a major specificity determinant for both deamidation and methyl esterification processes.     

Influence of calcium ions on the structure and stability of recombinant human deoxyribonuclease I in the aqueous and lyophilized states

The effect of calcium ions on the structure and stability of recombinant human DNase I (rhDNase) in the aqueous and solid (lyophilized) states was investigated. Fourier transform infrared (FTIR) spectroscopy was used to examine the overall secondary structure, while chemical stability was monitored in terms of deamidation and soluble aggregate formation at 40 degrees C. The exogenous calcium was removed by EGTA. This process can remove all but approximately one calcium ion per protein molecule. Analysis of the FTIR spectra in the amide III region in either the aqueous or lyophilized state demonstrated that removal of exogenous Ca2+ by EGTA-treatment had little effect on the secondary structure (and lyophilization-induced rearrangement thereof). For the aqueous solution, circular dichroism was used as an independent technique and confirmed that there was no large overall change in the secondary or tertiary structure upon the removal of calcium. The primary degradation route for the aqueous protein was deamidation. For the EGTA-treated protein, there was also severe covalent aggregation, e.g., formation of intermolecular disulfides facilitated by the cleavage of Cys173-Cys209. The aggregates exhibited a markedly different secondary structure compared to the native protein. For instance, the beta-sheet band observed at ca. 1620 cm-1 wavenumber in the amide I second derivative spectra was increased. Enzymatic activity was completely lost upon aggregation, consistent with the cleavage of the aforementioned native disulfide. For the protein lyophilized in the presence of Ca2+, there was no increase in deamidated species during solid-state storage; however, some aggregation was observed. For the lyophilized EGTA-treated protein, aggregation was even more pronounced, and there was some loss in enzymatic activity upon reconstitution. Thus, the removal of calcium ions by EGTA-treatment decreased the stability of rhDNase in both the aqueous and solid states even though no large overall calcium-induced structural changes could be observed by the techniques used in this study.     

Anomalous mobility of sulfitolysed proteins in SDS-PAGE. Analysis and applications

Some cysteine-containing proteins upon sulfitolysis have been found to show anomalously retarded SDS-PAGE mobilities in non-reducing gels. These proteins include bovine serum albumin, ovalbumin, aldolase, ribonuclease and a recombinant fusion protein (XA) consisting of a portion of γ-interferon linked to the A chain of human insulin. This mobility shift has been employed to determine the stability of the sulfonated products and to study the kinetics of the sulfitolysis reaction. Partially sulfonated products of intermediate shifts were observed at 0.01%β-ME, while 0.05%β-ME gave a shift characteristic of the completely reduced protein. The undiluted sulfitolysis reagent reacted with XA to give within 1 min a gel shift characteristic of the fully sulfitolysed protein. Its transition stages could be visualized at 15, 30 and 60 min when the reagent was diluted four-fold. In the presence of 8 M urea, the sulfitolysis of BSA was nearly complete at 30 min when the sulfitolysis reagent was used at a dilution of 1:5. However, under the same conditions BSA was predominantly unsulfitolysed in the absence of urea. In order to elucidate the mechanism of sulfonation shift. several derivatives of XA, e.g. performic acid oxidized, alkylated with (a) iodoacetamide and (b) iodoacetate, have been prepared. While the mobility of XASSO^?₃ was sensitive to the presence of β-ME, all other derivatives moved in a β-ME-insensitive fashion. Furthermore, while the nonreducing mobilities of the acidic derivatives (-SSO_-3, -SO_-3 and -SCH_-2CO_-2) were anomalously retarded and identical, the mobility of the iodoacetamide derivative was intermediate between the retarded acidic derivatives above and XA below. These studies have suggested a role of the extended conformation of the A chain of insulin in causing a mobility shift of the acidic derivatives in this series. Similar results were observed in an analogous series of derivatives prepared from BSA. Non-denaturing gel filtration analyses of native vs. sulfitolysed samples of serum albumin. ovalbumin and ribonuclease have indicated that the sulfitolysed proteins elute earlier than their native counterparts and appear to be significantly larger than their true molecular weights. Circular dichroism analysis has indicated significant loss in helicity of sulfitolysed BSA. This suggests that the retarded mobility of sulfitolysed proteins seen on SDS-PAGE is likely to be due to an expansion in the hydrodynamic volumes of these proteins, a phenomenon triggered by cleavage of disulfide bonds and further accentuated by the introduction of strongly negatively charged sulfonates. © Munksgaard 1996.     

Influence of neurophysin residues 1–8 on the optical activity of neurophysin-peptide complexes Direct evidence that the 1–8 sequence alters the environment of bound peptide

Circular dichroism was used to compare the environment of peptides bound to native and des 1-8 neurophysin in order to further elucidate the role of the neurophysin 1-8 sequence in peptide-binding. A very large positive ellipticity (～6000 degcm²dmol^?1), shown earlier to be induced in tyrosine at position 2 of peptides bound to the native protein, was determined by the present study to be paralleled by similar induced changes in tyrosine at peptide position 1. Deletion of the neurophysin 1-8 sequence led to loss of half of the induced optical activity at peptide positions 1 and 2 and changes in binding-induced optical activity in the protein, the latter partially assignable to protein disulfides. In the rnononitrated native and des 1-8 proteins, the optical activity of neurophysin Tyr-49, a residue at the peptide-binding site, was reduced by 80% in complexes of the des 1-8 protein relative to those of the native protein. The results suggest a role for neurophysin Arg-8 in modulating the optical activity at the binding site by directly placing a charge proximal to the binding site and/or by altering binding site conformation. The data provide the first unambiguous evidence of a difference in the environment of bound peptide between the native and des 1-8 proteins.     

Antibacterial activity of some derivatives of 7-aminocephalosporanic acid against Staphylococcus aureus and synergism between these and other antibiotics

The N-phenylacetyl derivative of 7-aminocephalosporanic acid (cephaloram) had roughly the same activity as benzylpenicillin against a number of Gram-positive organisms and about one-eighth of the activity of benzylpenicillin against penicillinsensitive strains of Staphylococcus aureus. This derivative and the N-α-phenoxypropionyl derivative of 7-aminocephalosporanic acid were 4 to 8 and 4 to 16 times as active as methicillin against penicillinase- and nonpenicillinase-producing staphylococcal strains, respectively. Neither the presence of horse serum nor changes in inoculum size appreciably affected the activities of any of the derivatives of 7-aminocephalosporanic acid which were tested. After forty-eight subcultures in the presence of antibiotic the increase in minimum inhibitory concentration against the staphylococcus was about four-times as great for cephaloram as for cephalosporin C. The resistant penicillinase-producing strains remained stable after six subcultures in antibiotic-free medium, and all the strains retained coagulase activity. Some degree of cross-resistance was found between the derivatives of 7-aminocephalosporanic acid and those of 6-aminopenicillanic acid. Synergism was observed in vitro between certain derivatives of 7-aminocephalosporanic acid and 6-aminopenicillanic acid when they were tested together or with fusidic acid or cephalosporin P₁ against a weak penicillinase-producing strain of Staphylococcus aureus. Cephalosporin C and cephalosporin C (pyridine), each in combination with benzylpenicillin, showed a significant degree of synergism in protection experiments in mice infected with a strong penicillinase-producing strain of Staphylococcus aureus.     

Serum-induced sensitization of cyclic AMP accumulation in 1321N1 human astrocytoma cells.

Exposure of 1321N1 human astrocytoma cells to fresh medium containing fetal bovine serum induced a marked increase in the subsequent ability of isoproterenol and forskolin to stimulate cAMP accumulation in intact cells, compared with cells exposed to fresh medium without serum. This "sensitization" of cAMP accumulation by serum was dose dependent, occurred rapidly, was maintained in the continuing presence of serum, and reversed rapidly upon removal of serum. Preliminary characterization of the sensitizing factor(s) in serum has been performed, but the factor(s) remain to be identified. Sensitization appeared to result from an increase in maximal response and not from changes in the potency of isoproterenol or forskolin. The protein kinase C inhibitor staurosporine inhibited serum-induced sensitization. Furthermore, down-regulation of protein kinase C almost completely eliminated the subsequent ability of serum to induce sensitization, indicating involvement of protein kinase C in the serum effect. Pretreatment of cells with pertussis toxin also markedly reduced subsequent sensitization induced by serum, suggesting involvement of a pertussis toxin-sensitive guanine nucleotide-binding protein in the pathway for serum-induced sensitization. The rate of cAMP degradation was not changed in sensitized cells, but some increase in adenylyl cyclase activity was retained in broken cell preparations from sensitized cells, suggesting increased synthesis of cAMP by adenylyl cyclase as the mechanism for sensitization.     

Effect of carboxylate group modification on enzymatic and cardiotoxic properties of snake venom phospholipases A2

By treating Naja nigricollis and Naja naja atra phospholipase A₂ with carbodiimide and semicarbazide, we obtained derivatives having varied numbers of modified carboxylate groups. When tested on artificial and natural substrates, derivatives of both enzymes with a modified carboxylate group at the active site (Asp-49) retained little enzymatic activity ($\text{1}\text{4}$1 to 10%). However, the derivatives of N. nigricollis also lost most of their lethal potency (5% of native), while those of N. n. atra retained considerable lethality (29%). Caboxyl modification with protection of Asp-49 in N. n. atra enzyme resulted in a derivative with lethal potency equal to or greater than the native enzyme and enzymatic activity which was low on all substrates (12 – 17% of native). Similar protection of Asp-49 at the active site in N. nigricollis enzyme produced a derivative with decreased enzymatic activity on artificial substrate (22% of native) and decreased lethality (17 – 33% of native), but with full enzymatic activity on natural substrates. When tested on electrical and mechanical properties of the isolated perfused heart and the isolated ventricle muscle wall, the derivatives of both enzymes retained considerably more of the cardiotoxic activity than would have been expected based on their residual enzymatic activity. The one exception occurred with the least modified N. nigricollis derivative which had an unaltered Asp-49, this enzyme retained both cardiotoxic activity and full enzymatic activity on natural substrates. The extent of phospholipid hydrolysis following treatment was measured in the isolated heart preparation and in hearts removed from mice following i.v. injection of the phospholipases. Very low levels of phospholipid hydrolysis were observed and no correlation could be made between the extent of hydrolysis and the pharmacological potencies of these enzymes. Modification of the enzymatic active site, whether of Asp-49 in this study of His-48 in prior studies, leads to a large decrease in both enzymatic activity and lethal potency. Asp and Gluresidues outside of the enzymatic site contribute significantly to the lethal potency of the N. nigricollis enzyme and to the enzymatic activity of the N. n. atra enzyme. Based on these and previous data we conclude that changes in isoelectric points are not responsible for altered lethal potencies following chemical modification and that some pharmacological effects of snake venom phospholipases A₂ are due to a non-enzymatic action, suggesting two distinct but perhaps overlapping active sites.     

Conformational changes of bovine plasma albumin prior to the salting-out of the protein in concentrated salt solution

By working at very low protein concentration (ca. 0.003%), it is possible to measure tryptophyl fluorescence intensity at 350nm (F₃₅₀) of bovine plasma albumin (BPA) as a function of pH under precipitating conditions (acidic concentrated salt solutions). Under such conditions, distinct changes in F₃₅₀ were seen before the starting of precipitation of BPA and no further changes in F₃₅₀ over the precipitating pH range. Comparison of pH-profiles monitored by F₃₅₀ with those by solubility in the presence of various salts at various concentrations indicated that the change of solubility is observed after definite changes in conformation of the protein.     

ENOLASE FROM BACILLUS STEAROTHERMOPHILUS

Enolase from Bacillus stearothermophilus was homogeneous in the ultracentrifuge and had the hydrodynamic properties of a globular protein with a molecular weight of 3.51 times 105 (sedimentation-diffusion) and 3.60 times 105 (Archibald approach to equilibrium). Electrophoretic and gel filtration measurements in presence of dissociating agents indicated that the enzyme consists of eight apparently identical polypeptide chains of molecular weight of 47 000. The denaturation of enolase has been studied as a function of urea concentration. A broad transition curve was observed, the enzyme in 8 M urea being inactive and dissociated into monomers. The far-ultraviolet circular dichroism spectrum of enolase in 8 M urea solution indicates extensive unfolding of the globular structure. Accordingly, fluorescence emission measurements indicate that the tryptophanyl residues of the denatured enolase are largely exposed to the aqueous solvent medium. The enzyme can be reversibly denatured by 8 M urea and the enzymatic activity then recovered by dilution 1:100 of the denatured enzyme into buffer or by removing urea by dialysis. Under suitable conditions and in the presence of Mg2+-ions, 90–100% of original activity was recovered and the renatured enzyme was indistinguishable from the native one, as judged from several enzymological and physicochemical criteria, including molecular weight. The far-ultraviolet circular dichroism spectrum and the fluorescence emission properties of the native enzyme were regained very rapidly (less than 1 min), while enzymatic activity was recovered much more slowly. Octameric enzyme species were obtained on refolding the denatured enzyme at temperatures between 4 and 55.     

PROTEOLYTIC SUSCEPTIBILITY AND METHIONINE MODIFICATION OF MONODEAMIDATED RIBONUCLEASE A

The susceptibility of a monodeamidated RNAaseA (RNAaseAa₁) towards carboxypeptidaseA, α-chymotrypsin and pepsin has been studied. Similar to RNAaseA, the C-terminal of RNAaseAa₁ is not available for carboxypeptidase A hydrolysis. The thermal stability of RNAaseAa₁ as probed through chymotryptic digestion is found to be less than that of RNAaseA. Preliminary chromatographic analysis of the digested material, however, suggests that the nature of thermal transition might be the same in the two proteins. Pepsin inactivates RNAaseAa₁ more slowly than does RNAaseA. Accordingly, less peptide bonds, almost half that of RNAaseA, are cleaved by pepsin in RNAaseAa_1. The accumulation of RNAase-P type intermediates is not evident during peptic digestion of RNAaseAa_1. Reaction with O-benzoquinone at low pH shows that methionines of the deamidated protein seem to have higher reactivities. These observations indicate a different structure for RNAaseAa₁ at elevated temperature and low pH.     

Effects of solution polarity and viscosity on peptide deamidation

Abstract: Deamidation kinetics were measured for a model hexapeptide (l ‐Val‐l ‐Tyr‐l ‐Pro‐l ‐Asn‐Gly‐l ‐Ala, 0.02 mg/mL) in aqueous solutions containing glycerol (0–50% w/w) and poly(vinyl pyrrolidone) (PVP, 0–20% w/w) at 37 °C and pH 10 to determine the effects of solution polarity and viscosity on reactivity. The observed pseudo‐first order deamidation rate constants, k_obs, decreased markedly when the viscosity increased from 0.7 to 13 cp, but showed no significant change at viscosities > 13 cp. Values of k_obs also increased with increasing dielectric constant and decreasing refractive index. Molecular dynamics simulations indicated that the free energy associated with Asn side‐chain motion is insensitive to changes in dielectric constant, suggesting that the observed dielectric constant dependence is instead related primarily to the height of the transition state energy barrier. An empirical model was proposed to describe the effects of the viscosity, refractive index and dielectric constant on k_obs. Analysis of the regression coefficients suggested that both permanent and induced dipoles of the medium affect the deamidation rate constant, but that solution viscosity is relatively unimportant in the range studied.     

Biological activities of a major protein secreted from the rat seminal vesicles after structural modification catalyzed by transglutaminase in vitro

The immunosuppressive, anti-inflammatory and anti-thrombotic properties of SV-IV, a major protein secreted from the epithelium of rat seminal vesicles, were investigated after transglutaminase-catalyzed covalent incorporation of two molecules of spermidine (Spd) into the protein at the level of Gln-9 and Gln-86. The modified molecular form of the protein (Spd₂-SV-IV) showed a more marked inhibitory activity on Con A-induced lymphocyte blastogenesis in comparison with the native protein, whereas no differences in the ability to inhibit the mixed lymphocyte reaction and to decrease the rat epididymal sperm immunogenicity were found between modified and native SV-IV. Spd₂-SV-IV was also less effective than native SV-IV to inhibit platelet aggregation induced in vivo by different thrombogenic agents. In contrast, superimposable inhibitory tracings were observed in the in vitro platelet aggregation experiments performed with the two different molecular forms on the protein. Finally, Spd₂-SV-IV was shown to retain unchanged the anti-inflammatory activity of native SV-IV.     

Organophosphate neuropathy due to methamidophos: biochemical and neurophysiological markers

Neuropathy target esterase (NTE), the putative target enzyme for organophosphate induced delayed polyneuropathy (OPIDP), can be measured in lymphocytes but has rarely been assessed in acute human poisoning. Serum autoantibodies to nervous system proteins develop in hens poisoned with neuropathic insecticides and also have not been studied after human poisoning. Serial lymphocyte NTE (LNTE) was measured in a 16-year-old boy after acute poisoning with methamidophos for evaluation as a predictor of subsequent neuropathy. The profiles of serum autoantibodies to neurofilament triplet proteins, myelin basic protein, and glial fibrillary acidic protein were measured in order to characterize changes occurring as a result of OPIDP. Clinical neuropathy characterized by steppage gate and profound lower extremity weakness, decreased grip and pinch strength, and decreased ulnar and absent tibial compound muscle action potentials developed 2 weeks following poisoning. Sensory examination and nerve conduction studies were normal. On day 3 following poisoning LNTE was depressed (77% compared with subsequent baseline enzyme activity). Marked increases in serum immunoglobulin G (IgG) autoantibodies to glial fibrillary acidic protein and to neurofilament 200 were observed after the development of OPIDP. We conclude that inhibition of lymphocyte NTE is predictive of subsequent OPIDP. Serum autoantibody titers to nervous system proteins may be useful markers of neuropathy. Received: 16 March 1999 / Accepted: 27 April 1999     

Rapid Prediction of Deamidation Rates of Proteins to Assess Their Long-Term Stability Using Hydrogen Exchange–Mass Spectrometry

Deamidation is an important degradation pathway for proteins. Estimating deamidation propensities is essential for predicting their long-term stability. However, predicting deamidation rates in folded proteins is challenging because higher-order structure has a significant and unpredictable effect on deamidation. Here, we investigated the correlation between amide hydrogen exchange (HX) and deamidation to assess the potential of using hydrogen exchange–mass spectrometry (HX-MS) to rapidly predict deamidation propensity. Maltose-binding protein and a structurally less stable mutant, W169G, were stored in the dark at pH 7.0 at 23 ± 2°C for 1 year. Deamidation at each asparagine site was measured using liquid chromatography–mass spectrometry after trypsin digestion. Deamidation rates at each deamidation site were determined based on first-order kinetics. HX rates at the deamidation sites were determined before storage using the shortest peptic peptide containing each site using conventional bottom-up HX-MS at pD 7.0 at 25°C. We observed a power law correlation between deamidation half-life and HX half-life for the NG sites with measurable kinetics. For NA sites, slow deamidation was only observed at 2 sites located in rapidly exchanging regions. Our findings demonstrate that HX-MS can be used to reliably and rapidly rank deamidation propensity in folded proteins.     

Toxicological effects of a sub-lethal concentration of inorganic Mercury on the fresh water fish,Tilapia mossambica,Peters

Tilapia mossambica, Peters were exposed to a sub-lethal concentration of 0.5 mg · 1^–1 of mercury as mercuric nitrate [Hg(NO₃)₂] in aquaria under controlled laboratory conditions. Main clinical symptoms such as inappetence and ataxia appeared after 2 days of exposure. Blindness was noticed in 60% of the fish after a 10 day exposure. The whole-animal oxygen uptake decreased to 27% after an exposure period of 7 days, and remained more or less at this level during the remainder of the 35-day exposure period. When the test fish were transferred to mercury-free, oxygen-saturated water, the oxygen uptake rate recovered to 74% of the initial value.The effects exhibited by the exposed fish were a decrease in protein content, haemoglobin percentage, RBC count and microhaematocrit percentage. A gradual decrease in haematocrit percentage after 1 week, followed by an increase after 21 days of exposure, was accompanied by an initial enlargement of the red blood cells, while the subsequent decrease in haematocrit percentage was due to haemolysis of red blood cells. Mercury accumulated more in liver than in brain and muscle, and was depleted more rapidly from the liver than from the brain when the test fish were transferred to mercury-free, oxygen-saturated water. The physiological and biochemical changes and active metabolic rates were found to be directly related to the mercury concentrations in the tissues and in the medium.