Common evolution of waprin and kunitz-like toxin families in Australian venomous snakes.

The venoms of Australian snakes contain a myriad of pharmacologically active toxin components. This study describes the identification and comparative analysis of two distinct toxin families, the kunitztype serine protease inhibitors and waprins, and demonstrates a previously unknown evolutionary link between the two. Multiple cDNA and full-length gene isoforms were cloned and shown to be composed of three exons separated by two introns. A high degree of identity was observed solely within the first exon which coded for the propeptide sequence and its cleavage site, and indicates that each toxin family has arisen from a gene duplication event followed by diversification only within the portion of the gene coding for the functional toxin. It is proposed that while the mechanism of toxin secretion is highly conserved, diversification of mature toxin sequences allows for the existence of multiple protein isoforms in the venom to adapt to variations within the prey environment.

Crystal structure of mammalian purple acid phosphatase.

BACKGROUND: Mammalian purple acid phosphatases are highly conserved binuclear metal-containing enzymes produced by osteoclasts, the cells that resorb bone. The enzyme is a target for drug design because there is strong evidence that it is involved in bone resorption. RESULTS: The 1.55 A resolution structure of pig purple acid phosphatase has been solved by multiple isomorphous replacement. The enzyme comprises two sandwiched beta sheets flanked by alpha-helical segments. The molecule shows internal symmetry, with the metal ions bound at the interface between the two halves. CONCLUSIONS: Despite less than 15% sequence identity, the protein fold resembles that of the catalytic domain of plant purple acid phosphatase and some serine/threonine protein phosphatases. The active-site regions of the mammalian and plant purple acid phosphatases differ significantly, however. The internal symmetry suggests that the binuclear centre evolved as a result of the combination of mononuclear ancestors. The structure of the mammalian enzyme provides a basis for antiosteoporotic drug design.

The relationship between the carboxylesterase and monoacylglycerol lipase activities of chicken liver microsomes.

The carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1) and monoacylglycerol lipase (glycerol-monoester acylhydrolase, EC 3.1.1.23) activities, measured against ethyl butyrate and emulsified monooleoylglycerol respectively, were determined for chicken liver microsomes and highly purified chicken liver carboxylesterase. The activity ratio (ethyl butyrate activity/monooleoylglycerol activity) was approx. 5 for microsomes and approx. 400 for carboxylesterase. Homogenization of microsomes in 0.1 M Tris-HCl buffer (pH 7.92) released all of the ethyl butyrate activity and about half of the monooleoylglycerol activity into a soluble form. Both activities eluted from a Sephadex G-200 column with the same elution volume as that of pure carboxylesterase. This fraction (fraction B) had an activity ratio of approx. 15, an average pI of 5.01 (cf. 4.75 for carboxylesterase), and ran on polyacrylamide gel electrophoresis at pH 8.6 as a number of closely spaced esterase bands with mobilities considerably less than those of the esterase bands present in the carboxylesterase. Fraction B activities against both substrates were completely inhibited by diethyl p-nitrophenyl phosphate and completely precipitated by antibody to carboxylesterase. The remaining half of the monoacylglycerol lipase activity of microsomes was solubilized by treatment with 1.5% (w/v) Triton X-100. This solubilized monoacylglycerol lipase was completely inhibited by diethyl p-nitrophenyl phosphate, showing it to be a serine-dependent enzyme like the carboxylesterases. However, it had no detectable activity against ethyl butyrate, indicating that it is not closely related to the carboxylesterases.

Organophosphate inhibitors. The stereospecificity of hydrolysis of methyl n-butyl p-nitrophenyl phosphate by serum phosphotriesterases (EC 3.1.1.2) and by acetylcholinesterases (EC 3.1.1.7).

A chiral phosphotriester, methyl n-butyl p-nitrophenyl phosphate was used to determine the stereospecificity of hydrolysis catalysed by serum phosphotriesterases (aryl-ester hydrolases, EC 3.1.1.2) from horse, ox and rabbit. Each enzyme hydrolysed the (-)-enantiomer more quickly. The same phosphate was used to inhibit acetycholinesterase (acetycholine hydrolase, EC 3.1.1.7) from ox, rabbit and electric eel, and in each case, the (+)-enantiomer caused more rapid inhibition. Serum phosphotriesterase did not catalyse the dephosphorylation of dialkyphosphoryl-acetylcholinesterase or dialkylphosphoryl-carboxylesterase. Levels of serum phosphotriesterase in rabbits which received sub-lethal injections of the phosphotriester remained unchanged after one or several injections. In the same rabbits, the levels of blood acetylcholinesterase fell sharply following injections, but normal values were regained in 2-8 days. Serum phosphotriesterases seem incapable either of preventing acute phosphotriester poisoning or of regenerating active enzyme from phosphorylated acetylcholinesterase. However, phosphotriesterases would act in cases of chronic exposure by catalysing the hydrolysis of such organophosphate poisons as remain in the blood.

Localization of the 5-phospho-alpha-D-ribosyl-1-pyrophosphate binding site of human hypoxanthine-guanine phosphoribosyltransferase.

Human erythrocyte hypoxanthine-guanine phosphoribosyltransferase (HPRT) is inactivated by iodoacetate in the absence, but not in the presence, of the substrate, 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRib-PP). Treatment of HPRT with [14C]iodoacetate followed by tryptic digestion, peptide separation and sequencing has shown that Cys-22 reacts with iodoacetate only in the absence of PRib-PP; this strongly suggests that Cys-22 is in or near the PRib-PP binding site. In contrast, Cys-105 reacts with [14C]iodoacetate both in the presence and absence of PRib-PP. Carboxymethylation of Cys-22 resulted in an increase in the Km for PRib-PP, but no change in Vmax. Storage of HPRT also resulted in an increase in the Km for PRib-PP and a decrease in its susceptibility to inactivation by iodoacetate. Dialysis of stored enzyme against 1 mM dithiothreitol resulted in a marked decrease in Km for PRib-PP. The stoichiometry of the reaction of [14C]iodoacetate with Cys-22 in HPRT leading to inactivation (approx. 1 residue modified per tetramer) showed that, in this preparation of HPRT purified from erythrocytes, only about 25% of the Cys-22 side chains were present as free and accessible thiols. Titration of thiol groups [with 5,5'-dithiobis(2-nitrobenzoic acid)] and the effect of dithiothreitol on Km for PRib-PP indicate that oxidation of thiol groups occurs on storage of HPRT, even in the presence of 1 mM beta-mercaptoethanol.

Enzymatically active zinc, copper and mercury derivatives of the one-iron form of pig allantoic fluid acid phosphatase.

Derivatives of the violet, iron-containing acid phosphatase of pig allantoic fluid have been prepared in which one of the two iron atoms present in the native enzyme has been replaced by zinc, copper or mercury. The derivatives so formed are enzymatically active: the Zn-Fe, Cu-Fe and Hg-Fe enzymes have specific activities of about 80%, 25% and 17% respectively, of the maximum specific activity of the Fe-Fe enzyme in the standard assay at pH 4.9 with p-nitrophenyl phosphate as substrate. In contrast to the Fe-Fe enzyme, the mixed metal derivatives are not rapidly inactivated by H2O2. Visible absorption spectra of the derivatives confirm that all of the visible absorption of the Fe-Fe enzyme is due to one of the iron atoms. Attempts to prepare an active Cu-Cu enzyme were unsuccessful.

Fatty acid ethyl ester synthesis by human liver microsomes.

Fatty acid ethyl esters are a family of non-oxidative metabolites of ethanol present in many tissues after ethanol consumption. In this report we demonstrate the existence in human liver of an acyl-CoA:ethanol acyltransferase activity which may be responsible in part for the synthesis of these compounds in vivo. The effects of oleoyl-CoA and ethanol concentrations, presence or absence of bovine serum albumin and detergent, pH and enzyme concentration on this activity have been determined. Acyl-CoA:ethanol acyltransferase activity is localised in the membrane-bound fraction. Using inhibitors directed against related enzyme activities, it has been shown that the activity is not related to serine-dependent carboxylesterases or acyl-CoA:cholesterol acyltransferase, but tht it may be associated with acyl-CoA hydrolase activity. We have also compared acyl-CoA:ethanol acyltransferase activity with fatty acid ethyl ester synthase activity in microsomes and cytosol from the same liver. Our data indicate that these activities are comparable in vitro (on a unit/g liver basis), and suggest that both may be significant in vivo.

Organophosphate inhibitors: the reactions of bis(p-nitrophenyl) methyl phosphate with liver carboxylesterases and alpha-chymotrypsin.

Bis(p-nitrophenyl) methyl phosphate (BNMP) has been tested as a spectrophotometric titrant for a group of serine hydrolases. Bis(p-nitrophenyl) methyl phosphate reacts rapidly with liver carboxylesterases from chicken, sheep, and horse, and more slowly with alpha-chymotrypsin, releasing 2 mol of p-nitrophenol per active site titrated, and producing a phosphorylated enzyme very stable to dephosphorylation. However, pig liver carboxylesterase produces 2.2 mol of p-nitrophenol per active site titratedmreaction of pig and chicken liver carboxylesterases with bis(p-nitrophenyl) [3H]methyl [32P]phosphate clarified this differencemone molecule of the chicken enzyme reacts with one molecule of bis(p-nitrophenyl) methyl phosphate, releasing both p-nitrophenol residues, and resulting in an inhibited enzyme with one phosphorus atom and one methyl group covalently bound. Pig enzyme reacts rapidly, forming (presumably) methyl p-nitrophenyl phosphoryl-carboxylesterasemthis further reacts, concurrently producing methyl phosphoryl-carboxylesterase plus p-nitrophenol, or free enzyme plus methyl p-nitrophenyl phosphate, in the ratio of about 5 : 1 at pH 7.55. The free enzyme produced undergoes further reaction with bis(p-nitrophenyl) methyl phosphate until all the carboxylesterase is inhibited.

Mössbauer and EPR study of the binuclear iron centre in purple acid phosphatase.

Mössbauer spectra have been determined on 57Fe-enriched samples of both pink (reduced) and purple (oxidized) forms of pig allantoic acid phosphatase (EC 3.1.3.2), and EPR spectra on corresponding unenriched samples. The spectra show unambiguously that both forms of the enzyme contain two distinct, antiferromagnetically coupled, high-spin iron atoms: a ferrous-ferric ion pair in the pink, reduced form, and a pair of ferric ions in the purple, oxidized form.

Expression of active human hypoxanthine-guanine phosphoribosyltransferase in Escherichia coli and characterisation of the recombinant enzyme.

A plasmid, pRG1, has been constructed by incorporating the coding sequence of human hypoxanthine-guanine phosphoribosyltransferase (HPRT) into the expression vector pT7-7. Expression of human HPRT has been achieved in HPRT- Escherichia coli cells transformed with pRG1 and pGP1-2, as shown by: (1) exclusive labelling with [35S]methionine of a polypeptide with the same mobility as purified human HPRT on SDS-PAGE; and (2) measurement of HPRT activity after cell lysis. Although the majority of the recombinant HPRT was present in the particulate fraction after cell lysis and centrifugation, sufficient HPRT activity was present in the supernatant fraction to allow comparison with the HPRT purified from human erythrocytes and the activity in human haemolysates and lymphoblast lysates. Small differences in electrophoretic mobility on native gels were found between HPRT activity from these sources. The Km values of recombinant HPRT for the substrates 5-phospho-alpha-D-ribosyl-1-pyrophosphate and guanine were compared with those of lymphoblast and erythrocyte HPRT.

Structures of free and complexed forms of Escherichia coli xanthine-guanine phosphoribosyltransferase.

Structures of free, substrate-bound and product-bound forms of Escherichia coli xanthine-guanine phosphoribosyltransferase (XGPRT) have been determined by X-ray crystallography. These are compared with the previously determined structure of magnesium and sulphate-bound XPRT. The structure of free XGPRT at 2.25 A resolution confirms the flexibility of residues in and around a mobile loop identified in other PRTases and shows that the cis-peptide conformation of Arg37 at the active site is maintained in the absence of bound ligands. The structures of XGPRT complexed with the purine base substrates guanine or xanthine in combination with cPRib-PP, an analog of the second substrate PRib-PP, have been solved to 2.0 A resolution. In these two structures the disordered phosphate-binding loop of uncomplexed XGPRT becomes ordered through interactions with the 5'-phosphate group of cPRib-PP. The cyclopentane ring of cPRib-PP has the C3 exo pucker conformation, stabilised by the cPRib-PP-bound Mg2+. The purine base specificity of XGPRT appears to be due to water-mediated interactions between the 2-exocyclic groups of guanine or xanthine and side-chains of Glu136 and Asp140, as well as the main-chain oxygen atom of Ile135. Asp92, together with Lys115, could help stabilise the N7-protonated tautomer of the incoming base and could act as a general base to remove the proton from N7 when the nucleotide product is formed. The 2.6 A resolution structure of XGPRT complexed with product GMP is similar to the substrate-bound complexes. However, the ribose ring of GMP is rotated by approximately 24 degrees compared with the equivalent ring in cPRib-PP. This rotation results in the loss of all interactions between the ribosyl group and the enzyme in the product complex.

Identification of mammalian-like purple acid phosphatases in a wide range of plants.

Purple acid phosphatases (PAPs) comprise a family of binuclear metal-containing hydrolases, members of which have been isolated from plants, mammals and fungi. Polypeptide chains differ in size (animal approximately 35kDa, plant approximately 55kDa) and exhibit low sequence homology between kingdoms but all residues involved in co-ordination of the metal ions are invariant. A search of genomic databases was undertaken using a sequence pattern which includes the conserved residues. Several novel potential PAP sequences were detected, including the first known examples from bacterial sources. Ten plant ESTs were also identified which, although possessing the conserved sequence pattern, were not homologous throughout their sequences to previously known plant PAPs. Based on these EST sequences, novel cDNAs from sweet potato, soybean, red kidney bean and Arabidopsis thaliana were cloned and sequenced. These sequences are more closely related to mammalian PAP than to previously characterized plant enzymes. Their predicted secondary structure is similar to that of the mammalian enzyme. A model of the sweet potato enzyme was generated based on the coordinates of pig PAP. These observations strongly suggest that the cloned cDNA sequences represent a second group of plant PAPs with properties more similar to the mammalian enzymes than to the high molecular weight plant enzymes.

Purification and characterization of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase and comparison with the human enzyme.

The human malaria parasite Plasmodium falciparum is auxotrophic for purines and relies on the purine salvage pathway for the synthesis of its purine nucleotides. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) is a key purine salvage enzyme in P. falciparum, making it a potential target for chemotherapy. Previous attempts to purify this enzyme have been unsuccessful because of the difficulty in obtaining cultured parasite material and because of the inherent instability of the enzyme during purification and storage. Other groups have tried to express recombinant P. falciparum HGXPRT but only small amounts of activity were obtained. The successful expression of recombinant P. falciparum HGXPRT in Escherichia coli has now been achieved and the enzyme purified to homogeneity in mg quantities. The measured molecular mass of 26 229+/-2 Da is in excellent agreement with the calculated value of 26232 Da. A method to stabilise the activity and to reactivate inactive samples has been developed. The subunit structure of P. Jilciparum HGXPRT has been determined by ultracentrifugation in the absence (tetramer) and presence (dimer) of KC1. Kinetic constants were determined for 5-phospho-alpha-D-ribosyl-1-pyrophosphate, for the three naturally-occurring 6-oxopurine bases guanine, hypoxanthine, and xanthine and for the base analogue, allopurinol. Differences in specificity between the purified P. falciparum HGXPRT and human hypoxanthine guanine phosphoribosyltransferase enzymes were detected which may be able to be exploited in rational drug design.

Inflammatory cells, microglia and MHC class II antigen-positive cells in the spinal cord of Lewis rats with acute and chronic relapsing experimental autoimmune encephalomyelitis.

Chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE) was induced in Lewis rats by inoculation with guinea pig spinal cord and adjuvants and treatment with low dose cyclosporin A (CsA). Acute EAE was induced by the same method without CsA treatment. Immunocytochemistry and flow cytometry were used to assess inflammatory cells and MHC class II (Ia) antigen expression in the central nervous system of these rats. The inflammatory infiltrate was composed mainly of CD4+ T cells and macrophages, and alpha beta T cells constituted about 65% of the CD2+ T cells. After recovery from acute EAE and during the first remission of CR-EAE, the number of T cells was significantly less than in the preceding episodes. The number of T cells was higher in the second episode of CR-EAE than in the first remission. Throughout the course of CR-EAE, the majority of the CD2+ T cells were CD45RC-. The ratio of IL-2R+ cells to CD2+ cells ranged from 10.5 to 24.0%. The ratio of CD4+ T cells to B cells was lower in the later episodes of CR-EAE than in the first episode. Ia antigen was expressed on infiltrating round cells at all stages of CR-EAE and on microglial cells (identified by dendritic morphology) with increasing intensity throughout the course of CR-EAE. With flow cytometry, the number of Ia+ cells obtained from the spinal cord rose throughout the course of CR-EAE. The number of FSClowOX1low cells, which we consider represent microglia, also increased during the course of CR-EAE.

Studies on the reactivity of acyl glucuronides--VIII. Generation of an antiserum for the detection of diflunisal-modified proteins in diflunisal-dosed rats.

Acyl glucuronide metabolites of carboxylic drugs such as the salicylate derivative diflunisal (DF) have been shown to react with proteins to produce covalent adducts. To aid in the study of the formation and distribution of these adducts in both humans and rats, we raised an antiserum against human serum albumin modified by covalent attachment of DF via an amide bond, using a carbodiimide reagent. This antiserum had wide reactivity, reacting with all types of DF-modified proteins tested and with free DF (albeit at a lower affinity). It did not cross-react with other salicylates or other non-steroidal anti-inflammatory drugs. The antiserum has been used in immunoblotting to detect proteins covalently modified by DF in the plasma and livers of rats treated with the drug for 7 days. Although some cross-reactivity was apparent on the blots, a series of DF-modified proteins was found in cytosolic, mitochondrial and mixed membrane fractions of hepatocytes, with molecular weights ranging from 28 to 130 kDa.

Studies on the reactivity of acyl glucuronides--III. Glucuronide-derived adducts of valproic acid and plasma protein and anti-adduct antibodies in humans.

The major metabolite of the anti-epileptic agent valproic acid (VPA) is its acyl glucuronide conjugate (VPA-G), which undergoes non-enzymic, pH-dependent rearrangement via acyl migration to a mixture of beta-glucuronidase-resistant forms (collectively VPA-G-R). We have compared the reactivity of VPA-G and VPA-G-R towards covalent VPA-protein adduct formation by incubation in buffer, human serum albumin (HSA) and fresh human plasma at pH 7.4 and 37 degrees. In all three media, the predominant reaction of VPA-G over 30 hr was rearrangement to VPA-G-R (ca. 24%). Hydrolysis was quite minor (ca. 2%) and covalent adduct formation negligible (when protein was present). On the other hand, both hydrolysis (ca. 27%) and adduct formation (ca. 7%) were extensive when VPA-G-R was incubated with HSA or plasma. These data do not support a transacylation mechanism for VPA-protein adduct formation, since this pathway should be much more highly favoured by VPA-G (an acyl-substituted acetal) than VPA-G-R (simple esters). VPA-protein adducts were found in the plasma of epileptic patients taking VPA chronically (mean 0.77 +/- SD 0.63 microgram VPA equivalents/mL, N = 17). An enzyme linked immunosorbent assay was developed, using HSA modified by incubation with VPA-G-R, to test the immunoreactivity of the patients' plasma. Of 57 patients tested, nine showed measurable levels of antibodies to these adducts, but the titres were very low, with no difference in response to modified and unmodified protein detectable at plasma dilutions of 1:16 or greater. These results suggest that the VPA-protein adducts have little immunogenicity, and are in agreement with clinical observations that drug hypersensitivity responses have not been associated with VPA therapy. Thus, although the in vitro data show that VPA-G is an example of a relatively unreactive acyl glucuronide, covalent VPA-plasma protein adducts and anti-adduct antibodies are nonetheless formed in vivo, at least in some patients on chronic therapy with the drug.

The formation and stability of imidazolidinone adducts from acetaldehyde and model peptides. A kinetic study with implications for protein modification in alcohol abuse.

The kinetics of the reaction of acetaldehyde (AcH) with the alpha-amino group of several di- and tripeptides to form 2-methylimidazolidin-4-one adducts were determined at pH 7, 4, 37 degrees C, using reverse phase HPLC to separate peptides from adducts. The imidazolidin-4-one structure of the adducts was confirmed by 13C NMR spectroscopy. The reaction of val-gly-gly with AcH was shown to follow second-order kinetics over a wide range of concentrations of both reactants, with k2 = 0.734 +/- 0.032 M(-1) min(-1). Under conditions similar to those in the liver of an alcoholic during chronic ethanol oxidation ([Ach]o = 50-910 microm; [free peptide alpha-amino groups]o = 1.5 mM), the reaction proceeded until effectively all of the AcH had been consumed. The side chain of the N-terminal amino acid was shown not to have a marked effect on the rate of imidazolidinone formation. The decomposition of the imidazolidinone adduct of val-gly-gly and AcH was observed at 60-100 degrees C. Extrapolation of an Arrhenius plot to 37 degrees C provided an estimate of K(obs) of 0.002 h-1 (t1/2 approximately 14 days). Based on these kinetic studies, it is concluded that imidazolidinone adducts of AcH with proteins may be present in the liver and, possibly, in the blood of alcoholics.

A novel thermostable dextranase from a Thermoanaerobacter species cultured from the geothermal waters of the Great Artesian Basin of Australia.

A Gram-negative sporulating thermophilic anaerobe, designated AB11Ad, was isolated from the heated waters of the Great Artesian Basin of Australia. It grew on a variety of carbohydrates including glucose, starch, and dextran and produced a thermostable and thermoactive extracellular endo-dextranase. The enzyme was produced more actively under pH controlled continuous culture conditions than under batch conditions. Ammonium sulfate precipitated crude dextranase exhibited a temperature optimum of 70 degrees C and a pH optimum between 5 and 6. The half life was approximately 6.5 h at 75 degrees C and 2 h at 80 degrees C at pH 5.0 and in the absence of added dextran. 16S rRNA sequence analysis indicated that isolate AB11Ad was a member of the genus Thermoanaerobacter.

Effects of acetaldehyde on polymerization of microtubule proteins.

The in vitro effects of ethanol and acetaldehyde on polymerization of calf brain microtubular proteins (MTP) were examined. While ethanol up to 100 mM had no effect on the polymerization of MTP, acetaldehyde above 0.5 mM had an inhibitory effect. This effect was not dependent on the presence of microtubule-associated proteins (MAPs), since acetaldehyde had a similar effect on the polymerization of highly purified tubulin. Electron microscopy revealed that the number and the length of microtubules at equilibrium was reduced by the presence of acetaldehyde. Acetaldehyde raised the critical concentration for tubulin assembly and caused greater inhibition at lower tubulin concentrations. Acetaldehyde augmented the depolymerizing effects of Ca2+ on preassembled microtubules. In addition, acetaldehyde itself caused depolymerization of microtubules but only in the absence of MAPs. Long-term (19.5 h) incubation of MTP with acetaldehyde led to significant loss of polymerization ability which could not be reversed by removal of acetaldehyde. This loss of activity was apparently independent of the observed formation of reducible adducts between acetaldehyde and MTP.

The reaction of acetaldehyde with brain microtubular proteins: formation of stable adducts and inhibition of polymerization.

Stable adducts were formed by treatment of bovine brain microtubular proteins (MTP) with acetaldehyde, followed by gel filtration to remove excess acetaldehyde. The extent of stable adduct formation was determined using [14C]acetaldehyde and was correlated with acetaldehyde concentration and reaction time. Significant inhibition of MTP polymerization was observed at adduct concentrations of 0.6 mol acetaldehyde/mol tubulin dimer. The data suggest that repeated exposure of MTP to low concentrations of acetaldehyde, as would occur in the brain and other tissues of alcoholics, may inhibit MTP polymerization with neurological consequences.