Studies on the mechanism of action of the alkaline phosphatase from Dictyostelium discoideum

The Dictyostelium discoideum alkaline phosphatase was investigated kinetically in an attempt to elucidate its mechanism of action. Analysis of the hydrolysis of p-nitrophenyl phosphate by stopped-flow spectrophotometry revealed biphasic kinetics, suggesting a double displacement enzyme mechanism. Furthermore, Tris stimulated activity in an uncompetitive manner, a result that was consistent with this interpretation. The enzyme was inhibited reversibly by phosphate at low ionic strength, but the inhibition was irreversible at high ionic strength and the latter effect was enhanced at alkaline pH values. These results indicate that high ionic strength and alkaline pH conditions bring about a conformational change that renders the enzyme susceptible to irreversible inhibition by phosphate.     

Incision at nucleotide insertions/deletions and base pair mismatches by the SP nuclease of spinach

Spinach leaves contain a highly active nuclease called SP. The purified enzyme incises single-stranded DNA, RNA, and double-stranded DNA that has been destabilized by A-T-rich regions and DNA lesions [Strickland et al. (1991) Biochemistry 30, 9749-9756]. This broad range of activity has suggested that SP may be similar to a family of nucleases represented by S1, P1, and the mung bean nuclease. However, unlike these single-stranded nucleases that require acidic pH and low ionic strength conditions, SP has a neutral pH optimum and is active over a wide range of salt concentrations. We have extended these findings and showed that an outstanding substrate for SP is a mismatched DNA duplex. For base-substitution mismatches, SP incises at all mismatches except those containing a guanine residue. SP also cuts at insertion/deletions of one or more nucleotides. Where the extrahelical DNA loop contains one nucleotide, the preference of extrahelical nucleotide is A > T approximately C but undetectable at G. The inability of SP to cut at guanine residues and the favoring of A-T-rich regions distinguish SP from the CEL I family of neutral pH mismatch endonucleases recently discovered in celery and other plants [Oleykowski et al. (1998) Nucleic Acids Res. 26, 4597-4602]. SP, like CEL I, does not turn over after incision at a mismatched site in vitro. Similar to CEL I, the presence of a DNA polymerase or a DNA ligase allows SP to turn over and stimulate its activity in vitro by about 20-fold. The possibility that the SP nuclease may be a natural variant of the CEL I family of mismatch endonucleases is discussed.     

Purification and characteristics of a mitochondrial endonuclease from the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae contains a membrane-bound mitochondrial nuclease. The enzyme was purified nearly 500-fold from sphaeroplasts of the organism by differential centrifugation, differential solubilization, heparin-agarose chromatography, and gel filtration. A final specific activity of 98 mumol min-1 (mg of protein)-1 was obtained. The enzyme required further purification to achieve homogeneity. Two peaks of activity were obtained after gel filtration with apparent molecular weights of 140000 and 57000. Otherwise, these two components have nearly identical characteristics. Without detergent the enzyme is insoluble and has very low activity. Zwittergent 3-14 or Triton X-100 in the presence of KCl could be used to solubilize and activate the enzyme. A number of other detergents were much less effective in solubilizing or activating the nuclease. The enzyme requires Mg2+ for activity, and this can be replaced to some degree by Mn2+ but not by Ca2+ or Zn2+. It is most active at pH 6.5-7.0 and degrades the substrate to small oligonucleotides with 5'-phosphate ends. The relative rates of hydrolysis were 100 for poly(A), 31 for ssDNA, 19 for RNA, 2.1 for dsDNA, and less than or equal to 0.2 for poly(C). Under the assay conditions used the enzyme appears to constitute about 90% of the total nuclease activity of the cell. The enzyme is unstable, especially at neutral and alkaline pH.     

Alkaline unfolding and salt-induced folding of arginine kinase from shrimp Feneropenaeus chinensis under high pH conditions

The structural and functional properties of arginine kinase (AK) in alkaline conditions in the absence or presence of salt have been investigated. The conformational changes of AK during alkaline unfolding and salt-induced folding at alkaline pH were monitored using intrinsic fluorescence emission, binding of the fluorescence probe 1-anilino-8-naphthalenesulfonate and circular dichroism. The results for the alkaline unfolded enzyme showed that much lower pH (11.0) was required to cause the complete loss of AK activity than was required to cause an obvious conformational change of the enzyme. Compared with the completely unfolded state in 5 M urea, the high pH denatured enzyme had some residual secondary and tertiary structure even at pH 13.0. Increasing the ionic strength by adding salt at pH 12.75 resulted in the formation of a relatively compact tertiary structure and a little new secondary structure with hydrophobic surface enhancement. These results indicate that the partially folded state formed under alkaline conditions may have similarities to the molten globule state which is compact, but it has a poorly defined tertiary structure and a native-like secondary structure.     

EcoRI activity: Enzyme modification or activation of accompanying endonuclease?

A study has been made of the factors and mechanism leading to appearance of the so-called EcoRI activity described by Polisky et al. (1975) in the restrictase EcoRI preparations. The preparations of purified restrictase EcoRI, precipitated at 0.9 ammonium sulphate saturation, as well as that obtained using standard techniques have been found to contain an admixture of an endonuclease which at neutral pH and high ionic strength multiply cleaves those DNAs which normally have only one recognition site for EcoRI. Under the standard conditions for EcoRI digestion this activity is found only when large amounts of freshly isolated enzyme are added to the incubation mixture and it is sharply enhanced by replacement of Mg2+ with Mn2+. The number and size of DNA fragments produced under such conditions practically do not differ from those found under the so-called EcoRI conditions, that is for alkaline pH values and low ionic strength. The optimum incubation mixture for the EcoRI activity has been found to be 10 mM Tris . HCl buffer (pH 8.8) + 2 mM Mn2+. Similar activity is induced also by addition to EcoRI solution of 40--50% glycerol or a number of organic solvents (dimethylacetamide (DMA), dimethylformamide (DMF), dimethylsulphoxide (DMSO), sulphalane (SP) in concentrations from 1 to 6%. The EcoRI activity induced by 50% glycerol or at alkaline pH values and low ionic strength is suppressed or sharply inhibited by 2--3 mM parachloromercuribenzoate (PCMB), while EcoRI is not sensitive to this agent. The DNA fragments cleaved by EcoRI have cohesive termini and can be easily ligated. It is suggested that the EcoRI activity can be due not only (or largely not) to modification of the "recognizing capacity" of the EcoRI restrictase but not activation of a latent specific endonuclease which is present in the restrictase preparation as an impurity.     

Phenylethanolamine-N-methyltransferase:alcohol and the mechanism of action

Mixed-solvent systems of methanol and other alcohols and water were used to study the properties of bovine phenylethanolamine-N-methyltransferase. The presence of methanol decreased the binding affinity of the enzyme for its amine substrate but did not alter the maximum velocity. The change in binding was accompanied by an alkaline shift in the pK of an ionizable group in the active site. The well-known property of enzyme inhibition by substrate was also alleviated. Increasing the pH of the medium, in the presence or absence of methanol, increased the maximum velocity but did not alter substrate inhibition. It is proposed that substrate inhibition is due in part to the ionic state of a single unidentified ionizable group in the active site of the enzyme and to a slow release of product. Evidence that an essential, pH-dependent sulfhydryl modulates product release is presented. The properties of phenylethanolamine-N-methyl-transferase are quite responsive to changes in pH, ionic strength, and water content so that the enzyme may well be regulated at the microenvironmental level.     

Purification and properties of nuclease SP.

Single-strand-specific nucleases are a diverse and important group of enzymes that are able to cleave a variety of DNA structures present in duplex molecules. Nuclease SP, an enzyme from spinach, has been purified to apparent homogeneity, allowing for the unambiguous characterization of a number of its physical properties as well as its DNA strand cleavage specificities. The effects of ionic strength, pH, divalent metal cations, and temperature on nuclease SP activity have been examined in detail. Nuclease SP was found to be quite thermostable and could be stimulated by Co2+. In addition, the cleavage of UV-damaged and undamaged supercoiled plasmid substrates under a variety of conditions suggests that at least two types of structures are recognized and processed by nuclease SP: UV photoproduct-induced distortions and unwound "nuclease hypersensitive sites". These studies indicate that nuclease SP is functionally related to other single-strand-specific nucleases and is a potential enzymatic tool for probing and manipulating various types of DNA structures.     

Heart 6-phosphofructo-1-kinase. Subcellular distribution and binding to myofibrils

6-Phosphofructo-1-kinase (phosphofructokinase) (ATP:D-fructose-6-P 1-phosphotransferase, EC 2.7.1.11) can be identified in sheep heart homogenates in two forms, a soluble form and a form bound to the particulate fraction. Homogenates from immediately-dissected hearts have the enzyme in the soluble form, while those collected after a delay have the enzyme bound to the particulate fraction. Aldolase appears to show the same change in its location. Homogenization in a solution with concentrated macromolecular species (20% albumin) results in a greater association of phosphofructokinase and of aldolase to the particulate fraction in homogenates from immediately dissected hearts. Phosphofructokinase activity can be solubilized by two specific means: by high ionic strength, which is dependent upon specific salts; or by low ionic strength, which is dependent upon the presence of phosphofructokinase substrates or modifier ligands. These two means of solubilization are affected differently upon decreasing the pH below 6.9: the solubilization at low ionic strength is prevented, whereas phosphofructokinase is still solubilized by high ionic strength. Under the latter condition, the enzyme is in the inactive dimeric state, which can be activated at an alkaline pH. Myofibrils present in the particulate fraction can account for the binding of phosphofructokinase in heart homogenates. Purified myofibrils, when added to heart supernatant fluids, can bind phosphofructokinase at a slightly acidic pH. Conditions for phosphofructokinase binding to myofibrils, as well as its dissociation, follow what was observed with the binding of phosphofructokinase to the particulate fraction. At an acidic pH, and in the presence of a high concentration of ATP, phosphofructokinase exhibits low activity. However, if phosphofructokinase is assayed under these conditions while bound to myofibrils, the enzyme is activated.     

Endonuclease V of Escherichia coli.

A small endodeoxyribonuclease )2.3 S) that is active on single-stranded DNA has been extensively purified from Escherichia coli so as to be free of other known DNases. It has an alkaline pH optimum (9.5), requires Mg2+, and makes 3'-hydroxy and 5'-phosphate termini. The nuclease nicks duplex DNA, particularly if treated with OsO4, irradiated with ultraviolet light, or exposed to pH 5. The uracil-containing duplex DNA from the Bacillus subtilis phage PBS-2 is an especially good substrate; it is made acid-soluble by levels of the enzyme which fail to produce any acid-soluble material in other single-stranded or duplex DNAs. Neither RNA nor RNA-DNA hybrid are degraded by the enzyme. The enzyme specificity suggests that it might act at abnormal regions in DNA, so that its in vivo function could be to initiate an excision repair sequence. Its high activity on uracil-containing DNA could imply that the enzyme provides an alternative mechanism for excising uracil residues from DNA to the pathway utilizing uracil-DNA N-glycosidase. We suggest that this enzyme be designated as endonuclease V of E. coli.     

Deoxyribonucleic acid polymerases of Euglena gracilis. Primer-template utilization of and enzyme activities associated with the two deoxyribonucleic acid polymerases of high molecular weight.

The two high-molecular-weight DNA polymerases from Euglena gracilis, pol A (mol. wt. 190 000) and pol B (mol. wt. 240 000), were differentiated on the basis of associated enzymic activities and primer-template utilization. Neither enzyme had endodeoxyribonuclease activity, but pol B, like pol B of yeast and the corresponding enzyme from Tetrahymena pyriformis, exhibited at least one other nuclease activity directed against denatured DNA and the RNA of an RNA-DNA hybrid. These nuclease functions preferred an alkaline pH and Mg2+. Pol B also exhibited nucleoside diphosphokinase activity. Both enzymes were active with 'activated' DNA and poly[d(A-T)] as primer-templates and were sensitive, especially pol B, to inhibition by excess of native or heat-denatured DNA. Pol B also utilized oligo[d(T)] and poly(A) templates under certain conditions, whereas pol A exhibited only slight activity with poly[d(A)]. (U)6 was not used as a primer by either enzyme.     

Affinity chromatography on hydroxyalkyl methacrylate gels. III. Adsorption of chymotrypsin to poly(hydroxyalkyl methacrylates) with covalently bound benzyloxycarbonyl-glycyl-D-phenylalanine and -D-leucine as function of pH and ionic strength.

Chymotrypsin is specifically adsorbed at low ionic strength and alkaline pH to hydroxyalkyl methacrylate gels with N-benzyloxycarbonylglycl-D-phenylalanine or N-benzyloxycarbonylglycyl-D-leucine attached through 1,6-hexanediamine. Chymotrypsin is not adsorbed either to the unmodified gel (Spheron) or to the gel with attached, 1,6-hexanediamine (NH2-Spheron). The adsorption of chymotrypsin to Z-Gly-D-Phe-NH2-Spheron was investigated as a function of pH and ionic strength. Trypsin is not adsorbed to this gel. Chymotrypsin isolated from a crude pancreatic extract by affinity chromatography on Z-Gly-D-Phe-NH2-Spheron had the same activity as the enzyme isolated on a column of Spheron, to which the naturally-occurring trypsin inhibitor had been coupled.     

Glycosaminoglycans modulate activation, activity, and stability of tripeptidyl-peptidase I in vitro and in vivo

Tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal exopeptidase that sequentially removes tripeptides from the N termini of polypeptides and shows a minor endoprotease activity. Mutations in TPP I lead to classic late-infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disease. TPP I proenzyme is converted in lysosomes into a mature enzyme with the assistance of another protease and is able to autoactivate in acidic pH in vitro via a unimolecular mechanism. Because autoactivation in vitro at the pH values reported for lysosomes generated inactive enzyme, we intended to determine whether physiologically relevant factors can modify this process to also make it plausible in vivo. Here, we report that high ionic strength and glycosaminoglycans (GAGs) increase yields (ionic strength) or yields and rates (GAGs) of activation, enhance degradation of liberated TPP I prosegment fragments, and switch effective autoactivation of TPP I proenzyme toward less acidic pH values (up to pH 6.0). Although ionic strength and GAGs also inhibited TPP I activity in vitro and in living cells, the degree of inhibition (from 20 to 60%) appears to be of rather limited functional significance. Importantly, binding to GAGs improved thermal stability of TPP I and protected the enzyme against alkaline pH-induced denaturation in vitro (t((1/2)) of mature enzyme at pH 7.4 increased by approximately 8-fold in the presence of heparin) and in vivo ( approximately 2-fold higher loss of TPP I in cells deficient in GAGs than in control cells after bafilomycin A1 treatment). These findings elucidate a potent physiologically relevant mechanism of TPP I regulation by GAGs and suggest that generation of the active enzyme via autoactivation can be accomplished not only in vitro but in vivo as well.     

The role of arginine 143 in the electrostatics and mechanism of Cu,Zn superoxide dismutase: Computational and experimental evaluation by mutational analysis

Cu, Zn superoxide dismutase protects cells from oxidative damage by removing superoxide radicals in one of the fastest enzyme reactions known. The redox reaction at the active-site Cu ion is rate-limited by diffusion and enhanced by electrostatic guidance. To quantitatively define the electrostatic and mechanistic contributions of sequence-invariant Arg-143 in human Cu, Zn superoxide dismutase, single-site mutants at this position were investigated experimentally and computationally. Rate constants for several Arg-143 mutants were determined at different pH and ionic strength conditions using pulse radiolytic methods and compared to results from Brownian dynamics simulations. At physiological pH, substitution of Arg-143 by Lys caused a 2-fold drop in rate, neutral substitutions (Ile, Ala) reduced the rate about 10-fold, while charge-reversing substitutions (Asp, Glu) caused a 100-fold decrease. Position 143 mutants showed pH dependencies not seen in other mutants. At low pH, the acidic residue mutations exhibited pro-tonation/deprotonation effects. At high pH, all enzymes showed typical decreases in rate except the Lys mutant in which the rate dropped off at an unusually low pH. Increasing ionic strength at acidic pH decreased the rates of the wild-type enzyme and Lys mutant, while the rate of the Glu mutant was unaffected. Increasing ionic strength at higher pH (>10) increased the rates of the Lys and Glu mutants while the rate of the wild-type enzyme was unaffected. Reaction simulations with Brownian dynamics incorporating electrostatic effects tested computational predictability of ionic strength dependencies of the wild-type enzyme and the Lys, Ile, and Glu mutants. The calculated and experimental ionic strength profiles gave similar slopes in all but the Glu mutant, indicating that the electrostatic attraction of the substrate is accurately modeled. Differences between the calculated and experimental rates for the Glu and Lys mutants reflect the mechanistic contribution of Arg-143. Results from this joint analysis establish that, aside from the Cu ligands, Arg-143 is the single most important residue in Cu, Zn superoxide dismutase both electrostatically and mechanistically, and provide an explanation for the evolutionary selection of arginine at position 143. © 1994 Wiley-Liss, Inc.     

Kinesin undergoes a 9 S to 6 S conformational transition.

Addition of NaCl or KCl in the presence of 50 nM ATP induces a shift in the sedimentation coefficient (apparent S20,w) of kinesin from 9.4 S at low ionic strength to 6.5 S at high ionic strength. The midpoint for the transition occurs at ionic strength values of 0.39, 0.25, and 0.18 for pH values of 6.3, 6.9, and 8.3, respectively. Gel filtration experiments indicate that the transition to the 6.5 S species is accompanied by a decrease in the diffusion coefficient. Under all conditions which were tested, the 64-kDa beta subunits comigrate with the 120-kDa alpha subunits without any evidence for dissociation of the alpha 2 beta 2 complex. These results are consistent with the change in sedimentation coefficient being due to a conformational transition between a folded form at low ionic strength and an extended form at high ionic strength. This conformational transition is not significantly affected by the nature of the nucleotide bound at the active site since similar results are obtained both in the presence of excess EDTA, which removes the bound ADP, and after replacement of the bound ADP with adenosine 5'-(beta,gamma-imino)triphosphate. The alpha 2 form of kinesin, which lacks the beta subunits, undergoes a similar transition between a 6.7 S form at low ionic strength and a 5.1 S form at high ionic strength with a midpoint for the transition at an ionic strength of 0.5 at pH 6.9. Electron microscopic observation also indicates a transition between a folded conformation at low ionic strength and an extended conformation at high ionic strength for both the alpha 2 beta 2 and alpha 2 species.     

Characterization of the Bacillus subtilis bacteriophage PBS2-induced DNA polymerase and its associated exonuclease activity.

The DNA polymerase induced by Bacillus subtilis bacteriophage PBS2 has a Stokes radius of 7.2 in buffers of high ioninc strength, suggesting a molecular weight in the range 145,000 to 195,000. The polypeptide bands observed on gel electrophoresis in dodecyl sulfate have apparent molecular weights of 78,000 and 69,000 (and possibly another 27,000) in equimolar amounts. In buffers of low ionic strength, the enzyme appears to form large aggregates and even precipitates, with about 90% loss of activity. A nuclease activity co-purifies with the PBS2 DNA polymerase and shows similar responses to changes in pH, MgCl2, N-ethylmaleimide, temperature, and dextran sulfate levels. The nuclease produces deoxyribonucleoside 5'monophosphates from denatured DNA containing thymine or uracil. No endonuclease activity is detectable on supercoiled DNA. The inhibition of nuclease activity by added deoxyribonucleoside triphosphates, the DNA-dependent turnover of triphosphates, to free monophosphates during DNA polymerization, the inhibition of nuclease activity by 3'-phosphates on the DNA template-primer, and the pattern of digestion of 5'-[32P]phosphate-labeled DNA all indicate that the PBS2 DNA polymerase-associated hydrolytic activity is a 3' leads to 5'-exonuclease.     

Properties of mammalian nuclear-envelope nucleoside triphosphatase.

The nucleoside triphosphatase activities of the nuclear envelopes from rat liver, pig liver and simian-virus-40-transformed mouse-embryo 3T3 cells were shown to exhibit similar parperties. All three preparations hydrolyse ATP, 2'-dATP, 3'-dATP, GTP, CTP and UTP in the presence of Mg2+, Ca2+, Mn2+ and Co2+ with a pH optimum of 8.0, are sensitive to inhibition by mercurials, arsenicals, quercetin, proflavin and adenosine 5'-[gamma-thio]triphosphate and are partially inactivated by exposure to high ionic strength. The kinetic behaviour is similar for all substrates irrespective of the source of material. The typical Eadie-Hofstee plot, which is concave upwards at pH 8.0 when the ionic strength is 20mM, becomes linear when the pH is increased to 8.5 or the ionic strength to 160mM. The overall evidence, particularly the labelling of only one polypeptide by [gamma-32P]ATP, suggests that under the conditions of preparation and assay used only one class of nucleoside triphosphatase active sites is detectable in nuclear envelopes. The importance of these results for an understanding of the role of the enzyme in vivo is discussed.     

Phosphate binding to Escherichia coli alkaline phosphatase. Evidence for site homogeneity.

The reversible, noncovalent binding of inorganic phosphate to Escherichia coli alkaline phosphatase at pH 8 has been examined by equilibrium dialysis at two temperatures and two ionic strengths. Binding occurs with a stoichiometry of two phosphate ions per dimeric enzyme molecule and a single dissociation constant that is not very sensitive to temperature or ionic strength. These results contradict published evidence for anti-cooperative binding of inorganic phosphate to alkaline phosphatase. Reasons are presented for believing that the apparent anti-cooperativity reported by other workers is artifactual.     

Redox properties of the [2Fe-2S] center in the 24 kDa (NQO2) subunit of NADH:ubiquinone oxidoreductase (complex I)

The redox properties of the [2Fe-2S] cluster in the 24 kDa subunit of bovine heart mitochondrial NADH:ubiquinone oxidoreductase (complex I) and three of its homologues have been defined using protein-film voltammetry. The clusters in all four examples display characteristic, pH-dependent redox transitions, which, unusually, can be masked by high ionic strength conditions. At low ionic strength (10 mM NaCl) the reduction potential varies by approximately 100 mV between high and low pH limits (pH 5 and 9); thus the redox process is not strongly coupled and is unlikely to form part of the mechanism of energy transduction in complex I. The pH dependence was shown to result from pH-linked changes in protein charge, due to nonspecific protonation events, rather than from the coupling of a specific ionizable residue, and the ionic strength dependence at high and low pH was modeled using extended Debye-Hückel theory. The low potential of the 24 kDa subunit [2Fe-2S] cluster, out of line with the potentials of the other iron-sulfur clusters in complex I, is suggested to play a role in coupling reducing equivalents at the catalytic active site. Finally, the validity of using the [2Fe-2S] cluster in an isolated subunit, as a mechanistic basis for coupled proton-electron transfer in intact complex I, is evaluated.     

Affinities of various nucleases to DNA-Sepharose under non-digestive conditions: survey for productive affinity chromatography

1. It has been reported that DNase I can be highly purified from pancreas extract by affinity chromatography on a dDNA-Sepharose column under non-digestive conditions. In the present study, the adsorption-elution of other nucleases on the column under non-digestive conditions was studied. 2. All the seven kinds of nucleases tested were adsorbed when applied on a dDNA-Sepharose column under conditions which did not allow the enzymes to hydrolyze the DNA. The non-digestive conditions were as follows. i) For DNase II (pI=10.2), pH 3.0 in the presence of 50 mM sodium sulfate (inhibitor), ii) for micrococcal nuclease (pI=9.6), pH 4.0 in the absence of Ca2+ (activator), iii) for restriction endonucleases Eco RI (pI=5+1), Hind III (pI=5+1), and Bam HI (pI=5+1), pH 4.0 in the presence of 20% glycerol and 0.1% Neopeptone (stabilizers), and iv) for nucleases S1 (pI=5+1) and nuclease P1 (pI=4.5), pH 7.0. At the respective pH's, the enzymes other than nucleases S1 and P1 were cationic so as to exhibit electrostatic attraction to the anionic dDNA-Sepharose. Although S1 and P1 were anionic, they still adsorbed to the column. 3. All the adsorbed nucleases described above were eluted by a concentration gradient of KCl without changing pH. The ionic strengths required for elution were 0.19 for DNase II, 0.53 for micrococcal nuclease, 0.73 for Eco RI, 0.72 for Hind III, 0.37 for Bam HI, 0.17 for P1, and 0.13 for S1. The fact that the ionic strength required for the elution of DNase I (pI=5.0) was 0.39 at pH 4.0 indicates that the former five enzymes except DNase II can be chromatographed with almost the same or higher efficiency than DNase I, because the proteins adsorbed with no-specific affinity could be mostly eluted at lower ionic strength. On the other hand, the fact that nucleases P1 and S1 were adsorbed in spite of electrostatic repulsion suggests that these two enzymes can also be effectively chromatographed, especially when other cationic proteins are previously removed by an appropriate method such as adsorption to a typical cation exchanger.     

Human alpha-L-iduronidase. 1. Purification, monoclonal antibody production, native and subunit molecular mass

Human alpha-L-iduronidase from liver was purified about 20 000-fold with a new rapid three-step, five-column procedure which consisted of a Concanavalin-A-Sepharose/Blue-A-Agarose coupled step, a CM-Sepharose/Bio-Gel HT coupled step followed by a cupric-ion-chelating Sepharose 6B step. The behaviour of alpha-L-iduronidase on gel permeation chromatography was dependent upon both pH and ionic strength of the eluting buffer. The formation of species with enzyme activity which behaved as large-molecular-mass aggregates was favoured under conditions of low ionic strength and neutral pH. The amount of high-Mr species diminished as the pH decreased or the ionic strength increased to favour a single active species of Mr 65 000. A specific monoclonal antibody was generated against liver alpha-L-iduronidase. The antibody specifically immunoprecipitated enzyme activity from both crude and purified sources. The subunit Mr of liver alpha-L-iduronidase was estimated to be 65 000 using SDS-PAGE. Monoclonal antibody immunoprecipitation of radiolabelled enzyme was used to provide definitive confirmation of this subunit size.