Wilms' tumor 1 splice variants have opposite effects on the tumorigenicity of adenovirus-transformed baby-rat kidney cells.

The Wilms' Tumor 1 gene (WT1) encodes a transcription factor of the zinc-finger family. As a result of alternative RNA splicing, the gene can be expressed as four polypeptides which differ in the presence or absence of two stretches of amino acids: one of 17 residues (17aa) just N-terminal of the four zinc-fingers and of three residues (K-T-S) between zinc finger 3 and 4. In this study, four human cDNA constructs encoding the Wilms' tumor 1 splice variants were stably transfected into adenovirus-transformed baby rat kidney (Ad-BRK) cells. The in vivo produced WT1 proteins that lacked the KTS residues were found to bind efficiently to both the Egr-1 consensus sequence and the recently described WTE DNA sequence, as determined by electrophoretic mobility shift assays. Our studies show distinct effects of the different WT1 isoforms. Expression of the WT1 (-/+) protein, lacking the 17aa insert, strongly suppressed the tumorigenic phenotype of the Ad-BRK cells. Intriguingly, expression of the WT1 (-/-) protein, lacking both inserts, increased the tumor growth rate. In contrast to the growth in vivo, the growth rate of the transfectants in tissue culture is not influenced by any of the WT1 isoforms. However, the suppression of tumorigenicity appears to be correlated with a reduced ability of the cells to grow in serum-free medium.

Localization of the essential histidine and carboxylate group in D-xylose isomerases.

D-Xylose isomerases from different bacterial strains were chemically modified with histidine and carboxylate-specific reagents. The active-site residues were identified by amino acid sequence analysis of peptides recognized by differential peptide mapping on ligand-protected and unprotected derivatized enzyme. Both types of modified residues were found to cluster in a region with consensus sequence: Phe-His-Asp-Xaa-Asp-Xaa-Xaa-Pro-Xaa-Gly, conserved in all D-xylose isomerases studied so far. These results are consistent with the recently published X-ray data of the enzyme active centre from Streptomyces rubiginosus showing hydrogen bond formation between Asp-57 and His-54 which locks the latter in one tautomeric form. A study of the pH-dependence of the kinetic parameters suggests the participation of a histidine group in the substrate-binding but not in the isomerization process. Comparison of the N-terminal amino acid sequences of several D-xylose isomerases further revealed a striking homology among the Actinomycetaceae enzymes and identifies them as a specific class of D-xylose isomerases.

Reaction of Woodward's reagent K with D-xylose isomerases. Modification of an active site carboxylate residue.

D-Xylose isomerases from Streptomyces violaceoruber, Streptomyces sp., Lactobacillus xylosus, Lactobacillus brevis and Bacillus coagulans were rapidly inactivated by Woodward's reagent K. Second-order rate constants in the absence of ligands, at pH 6.0 and 25 degrees C, were 41, 36, 22, 95 and 26 M-1.min-1 respectively. Spectral analysis at 340 nm revealed that inactivation was correlated with modification of five, six, two, three and six carboxylate residues per monomer respectively. In the presence of protecting ligands, modification of one carboxylate group was prevented. The results support the idea of an active site glutamate or aspartate group that may contribute to the catalytic activity of all these D-xylose isomerases.

Evidence for an essential histidine residue in D-xylose isomerases.

Diethyl pyrocarbonate inactivated D-xylose isomerases from Streptomyces violaceoruber, Streptomyces sp., Lactobacillus xylosus and Lactobacillus brevis with second-order rate constants of 422, 417, 99 and 92 M-1.min-1 respectively (at pH 6.0 and 25 degrees C). Activity was completely restored by the addition of neutral hydroxylamine, and total protection was afforded by the substrate analogue xylitol in the presence of either Mg2+ or Mn2+ according to the genus studied. The difference spectra of the modified enzymes revealed an absorption maximum at 237-242 nm, characteristic for N-ethoxycarbonylhistidine. In addition, the spectrum of ethoxycarbonylated D-xylose isomerase from L. xylosus showed absorption minima at both 280 and 230 nm, indicative for modification of tyrosine residues. Nitration with tetranitromethane followed by diethyl pyrocarbonate treatment eliminated the possibility that modification of tyrosine residues was responsible for inactivation, and resulted in modification of one non-essential tyrosine residue and six histidine residues. Inactivation of the other D-xylose isomerases with diethyl pyrocarbonate required the modification of one (L. brevis), two (Streptomyces sp.) and four (S. violaceoruber) histidine residues per monomer. Spectral analysis and maintenance of total enzyme activities further indicated that either xylitol Mg2+ (streptomycetes) or xylitol Mn2+ (lactobacilli) prevented the modification of one crucial histidine residue. The overall results thus provide evidence that a single active-site histidine residue is involved in the catalytic reaction mechanism of D-xylose isomerases.

Metal ion binding to D-xylose isomerase from Streptomyces violaceoruber.

The binding of two activating cations, Co2+ and Mg2+, and of one inhibitory cation, Ca2+, to D-xylose isomerase from Streptomyces violaceoruber was investigated. Equilibrium-dialysis and spectrometric studies revealed that the enzyme binds 2 mol of Co2+/mol of monomer. Difference absorption spectrometry in the u.v. and visible regions indicated that the environment of the first Co2+ ion is markedly different from that of the second Co2+ ion. The first Co2+ appears to have a six-co-ordinate. The conformational change induced by binding of Co2+ to the first site is maximum after the addition of 1 equivalent of Co2+ and yields a binding constant greater than or equal to 3.3 x 10(6) M-1. Binding of Co2+ to the second, weaker-binding, site caused a visible difference spectrum. The association constant estimated from Co2+ titrations at 585 nm agrees satisfactorily with the value of 4 x 10(4) M-1 obtained from equilibrium dialysis. Similarly, the enzyme undergoes a conformational change on binding of Mg2+ or Ca2+, the binding constants being estimated as 1 x 10(5) M-1 and 5 x 10(5) M-1 respectively. Competition between the activating Mg2+ and Co2+ and the inhibitory Ca2+ ion for both sites was further evidenced by equilibrium dialysis and by spectral displacement studies.

Effect of pH on oligomeric equilibrium and saccharide-binding properties of peanut agglutinin.

The conformation and saccharide-binding properties of peanut agglutinin (PNA) depend on pH as studied by analytical ultracentrifugation, fluorescence, circular dichroism, equilibrium dialysis, and absorption spectroscopy. PNA is tetrameric in neutral solution and dissociates reversibly into dimers below pH 5.1. Below pH 3.4, the lectin is totally dimeric. Lowering of the pH induces reversible changes in the tertiary and secondary structures of PNA. Binding of saturating amounts of lactose to tetrameric (pH 6.9) or dimeric (pH 3.2) PNA resulted in identical ultraviolet difference spectra. Fluorescence studies of PNA as a function of pH in the presence of lactose indicated that tryptophanyl residues, present at or near the saccharide binding site, are more accessible to the ligand in dimeric than in tetrameric PNA. For solutions of dimeric PNA, containing only minor amounts of tetramers (pH 3.6), equilibrium dialysis with MeUmb-beta Gal beta(1----3)GalNac showed that the binding capacity of PNA was the same as for tetrameric PNA (one binding site per protomer) but the apparent association constant was one order of magnitude lower than for tetrameric PNA. The enhancement of MeUmb-beta Gal beta(1----3)GalNac fluorescence upon binding to PNA was pH dependent: 50% at neutrality, 16% at pH 3.7, and unobservable at pH 3.0, suggesting that the microenvironment of this PNA-bound chromophore changed progressively with pH and was dependent on ionization of an acidic amino acid residue.

Transfer reactions catalyzed by beta-D-xylosidase from Penicillium wortmanni.

The beta-D-xylosidase from Penicillium wortmanni catalyses (i) the hydrolysis of beta-D-xylopyranosides and alpha-L-arabinopyranosides, (ii) the transfer of the corresponding glycosyl residue to alcohols, and (iii) the dismutation of aryl beta-D-xylopyranoside substrates. These reactions all occur with retention of configuration and can be rationalized by a symmetrical reaction scheme. The key intermediate is an enzyme-glycosyl complex with a lifetime that is sufficient for the diffusion away of the leaving (aglycon) group and the binding of an acceptor group before water reacts with the intermediate. The exact nature of this complex is unknown, but it must contain an aglycon site which binds preferentially alcohols and sugar molecules having the D-xylose structure.

The pH dependence and group modification of beta-D-xylosidase from Bacillus pumilus: evidence for sulfhydryl and histidyl groups.

The pH dependence of the kinetic parameters of beta-D-xylosidase (EC. 3.2.1.37) from Bacillus pumilus reveals that an acidic functional group with pK 8.0 is involved in the catalysis. The fast inactivation of the dimeric enzyme by near equivalent amounts of methylmethanethiolsulfonate indicates that one thiol group per monomer is essential for catalysis, consistent with previously reported results. From the reactivity of the thiol groups with respect to 5,5'-dithiobis(2-nitrobenzoic acid), the absence of subunit cooperativity was indicated. The present study also reports on the inactivation of the enzyme by diethylpyrocarbonate, and provides evidence of the importance of a histidine residue. A mechanism of catalysis is presented, in which the thiol group interacts with the substrate via partial proton transfer. The mode of participation of the histidine group is difficult to specify, but may be associated with the maintenance of the active conformation of the enzyme.

Effects of alcohols on hydrolysis catalyzed by beta-D-glucosidase from Stachybotrys atra.

The interaction of alcohols in the hydrolysis of aryl beta-D-glucopyranosides and aryl beta-D-xylopyranosides by beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) from Stachybotrys atra has been investigated. The results constitute support for the presence of a glycosyl-enzyme intermediate, formed during the first step (glycosylation) of the proposed two-step mechanism. Transfer of the glycosyl group to an alcohol, with the formation of an alkyl glycopyranoside, can take place in parallel to the transfer to a water molecule (second or deglycosylation step). The alcohol binds to the free enzyme and to the glycosyl-enzyme intermediate. The glycosyl-enzyme-alcohol complex undergoes hydrolysis in addition to the alcoholysis. For aryl beta-D-glucopyranosides the deglycosylation step is rate-limiting. For aryl beta-D-xylopyranosides two kinds of substrate behaviour can be observed. Depending on the substituent group on the phenyl ring, either both steps are rate-controlling or the first step is rate-limiting. Electron-withdrawing substituents increase the rate at which the substrate aglycon group is released.

Naturally occurring leukemia viruses in H-2 congenic C57BL mice. I. High lymphoma incidence following milk-borne transmission of virus.

Two modes of transmission of ecotropic type C viruses occur naturally in C57BL mice: maternal (i.e., through milk) and genetic. By selection of virus-positive and virus-negative B10.ASgSn [B10.A (H-2a)] mothers and foster-nursing of C57BL/10ScSn [B10 (H-2b)] newborns, four sublines of C57BL mice were obtained: B10.A V+, B10.A V-, B10 V+, and B10 V-. (V+ denotes positive for milk-transmitted B-tropic virus; V- denotes negative for milk-transmitted B-tropic virus). Milk transmission of naturally prevalent B-tropic virus (V+ sublines) led to persistent infection of all offspring over at least 8 generations. Milk transmission of virus was associated with a very high incidence of lymphomas. The H-2 complex influenced the titers of virus after milk transmission, which were higher in B10.A V+ mice than in B10 V+ mice. H-2 control of virus titers, as measured by serum p30 assay, was confirmed in (B10.A V+ X B10 V+)F2 mice. Resistance to the virus was dominant, because serum p30 levels in F1 and H-2a/b F2 animals were similar to those in the B10 V+ subline and lower than those in the B10.A V+ subline. The H-2 complex also influenced the incidence of lymphomas (78 and 42%, respectively, in the B10.A V+ and B10 V+ sublines). Most B10.A V+ lymphomas were of T-cell origin, whereas most B10 V+ lymphomas were classified as non-T/non-B cells. Genetic transmission of virus (V- sublines) led to heterogeneous expression of both N- and B-tropic viruses, which thereby established the mottled trait for expression of genetically transmitted type C viruses in C57BL mice. Genetic transmission was associated with a low incidence of lymphomas that occurred in senescence.

Naturally occurring leukemia viruses in H-2 congenic C57BL mice. II. Antibody response to viral envelope antigens.

In C57BL mice milk-borne infection with B-tropic murine leukemia virus (V+ denoting positive for milk-transmitted B-tropic virus and V- denoting negative for milk-transmitted B-tropic virus) was accompanied by an antibody response against viral envelope antigens (VEA). Milk transmission of virus led to higher virus titers and lymphoma incidence in B10.A (H-2a) mice than in B10 (H-2b) mice, and the latter strain produced higher titers of anti-VEA antibodies than did the former. H-2 control of the antivirus-antibody response was confirmed in the (B10.A V+ X B10 V+)F2 cross. B10 V+ mice produced both IgM and IgG antibodies, whereas in the sera of B10.A V+ mice only IgM antibodies were demonstrable. The production of IgG and high-titer IgM antibodies to VEA was dominant in (B10.A V+ X B10 V+)F1 animals. The failure of B10.A V+ mice to produce IgG antibodies against VEA was not due to an intrinsic defect of helper T-cell function because these mice produced IgG antivirus antibodies after sc immunization with killed viral vaccine. Furthermore, in B10.A mice without milk-transmitted virus (B10.A V- subline), expression of genetically transmitted virus upon aging was associated with the production of IgG antibodies to VEA. The combined data were compatible with the existence of an H-2-linked dominant immune-response gene regulating the antibody response to milk-transmitted murine leukemia virus.

Binding of alkyl beta-D-xylopyranosides, containing branched-chain, cyclic, and substituted aglycon groups, to beta-D-xylosidase from Bacillus pumilus PRL B12.

For a number of alkyl beta-D-xylopyranosides having branched-chain, cyclic, and substituted aglycon groups, and binding to beta-D-xylosidase from B. pumilus PRL B12, the binding constant Ki and (for some of them) the thermodynamic equilibrium parameters delta H0, delta S0, and delta G0 have been determined. Although the aglycon is bound through hydrophobic forces, no simple relationships between the binding parameters and the relative hydrophobicity of the alkyl beta-D-xylopyranosides could be demonstrated. All of the available evidence suggests that the aglycon sub-site has a highly specific structure which forces the atoms of the aglycon group to occupy well-defined positions. The supplementary energy-requirements resulting from the imposed restrictions seem to be the main reason for the irregular way in which the binding parameters depend on the aglycon structure.

Binding of manno-oligosaccharides to concanavalin A. Substitution titration with a fluorescent-indicator ligand.

The association constants for binding of methyl alpha-D-mannopyranoside (I), mannobiose (II) and mannotriose (III) to concanavalin A were determined in the temperature range 285-313 K by a substitution titration, using 4-methylumbelliferyl alpha-D-mannopyranoside as a carbohydrate-specific and fluorescent indicator. All binding equilibria are simple, but establish extremely slowly with II and III. At 298.3 K, K increases moderately from I to III: (6.4 +/- 0.5) x 10(3), (1.2 +/- 0.1) x 10(4) and (1.10 +/- 0.05) x 10(5) M-1. For binding of I, II and III, the - delta H degree values are constant (36 +/- 2 kJ mol-1) and equal to the average value (36.1 +/- 0.6 kJ mol-1) obtained for the three corresponding 4-methylumbelliferyl alpha-D-manno-oligosaccharides [Van Landschoot, A., Loontiens, F. G., and De Bruyne, C. K (1978) Eur. J. Biochem. 83, 277-285]. The data are interpreted as arising from specific binding to a single mannopyranosyl residue in (alpha 1 leads to 2)-linked manno-oligosaccharides.