Biochemical activities of highly purified, catalytically active human APOBEC3G: correlation with antiviral effect.

APOBEC3G (APO3G), a cytidine deaminase with two zinc finger domains, inhibits human immunodeficiency virus type 1 replication in the absence of Vif. Here, we provide a comprehensive molecular analysis of the deaminase and nucleic acid binding activities of human APO3G using a pure system containing only one protein component, i.e., highly purified, catalytically active enzyme expressed in a baculovirus system. We demonstrate that APO3G deaminates cytosines in single-stranded DNA (ssDNA) only, whereas it binds efficiently to ssDNA and ssRNA, about half as well to a DNA/RNA hybrid, and poorly to double-stranded DNA and RNA. In addition, the base specificities for deamination and binding of ssDNA are not correlated. The minimum length required for detection of APO3G binding to an ssDNA oligonucleotide in an electrophoretic mobility shift assay is 16 nucleotides. Interestingly, if nucleocapsid protein and APO3G are present in the same reaction, we find that they do not interfere with each other's binding to RNA and a complex containing the RNA and both proteins is formed. Finally, we also identify the functional activities of each zinc finger domain. Thus, although both zinc finger domains have the ability to bind nucleic acids, the first zinc finger contributes more to binding and APO3G encapsidation into virions than finger two. In contrast, deamination is associated exclusively with the second zinc finger. Moreover, zinc finger two is more important than finger one for the antiviral effect, demonstrating a correlation between deaminase and antiviral activities.

Isolation from Nocardioides sp. strain CT16, purification, and characterization of a deoxycytidine deaminase extremely thermostable in the presence of D,L-dithiothreitol.

A deoxycytidine deaminase that was extremely thermostable in the presence of dithiothreitol was found in a mesophilic bacterium isolated from soil. The bacterium was classified as a Nocardioides sp. The enzyme was purified to a homogeneous protein by treatment at 100 degrees C, ammonium sulfate precipitation, and chromatography on DEAE-Toyopearl, hydroxyapatite, and then Sephacryl S-100. Twenty micrograms of the pure enzyme was obtained from 811 mg of the starting crude protein. After treatment at 50 degrees C for 15 min in the absence of dithiothreitol, enzyme activity was 44% of the starting activity; after treatment at 100 degrees C for 2 h in the presence of 50 mm dithiothreitol, activity was 56% of the starting activity. Dithiothreitol greatly stabilized the enzyme. Activity was maximum at 99 degrees C. The Km values for deoxycytidine, cytidine, and methyl-deoxycytidine were 55.2, 140, and 130 microM, respectively. The molecular mass was estimated to be 52 kDa by gel permeation chromatography. The enzyme molecule was dissociated into two subunits each of 18 kDa subunit when reduced with mercaptoethanol.

A rapid and versatile method for harnessing scFv antibody fragments with various biological effector functions.

A versatile expression vector is described for the rapid construction and evaluation of bispecific scFvs and scFv-based fusion proteins. An important feature of this vector is the presence of two multiple cloning sites (MCS) separated by an in frame linker sequence. The first MCS was specifically designed to contain unique SfiI and NotI restriction enzyme sites that can be used for directional and in frame insertion of scFvs (or potentially any molecule) selected from established phage-display systems. Using this new vector, a functional bs-(scFv)(2) (2C11-MOC31) was constructed for retargeted T-cell cytotoxicity towards EGP2 positive tumor cells. The vector was also used for grafting of a number of promising biological effector principles onto scFv MOC31, including the prodrug converting enzyme cytosine deaminase, the anti-angiogenic factor angiostatin, and the thrombogenic molecule tissue factor. We aimed at producing biologically active fusion proteins by directing them through the endoplasmic reticulum-based protein folding machinery of eukaryotic cells (COS-7) using a kappa light chain leader, thereby taking advantage of the associated quality control mechanisms that allow only fully folded and processed fusion proteins to be secreted into the medium. Supernatants derived from fusion protein transfected COS-7 cells, which were transiently transfected at low transfection rates, were directly assayed for the biological and/or targeting activity of the excreted fusion proteins without any prior purification steps. This procedure might help to identify those fusion proteins that have favourable characteristics like stability and biological activity in the presence of serum and at low protein concentrations. Targeted delivery of all effector principles was subsequently assessed in an in vitro model system. The method we devised is both rapid and versatile and can be useful to construct and identify series of new chimeric proteins with enhanced therapeutic potential in human cancer therapy.

Cloning, overexpression, and purification of cytosine deaminase from Saccharomyces cerevisiae.

Cytosine deaminase is an enzyme which has been investigated for cancer chemotherapy as a result of its ability to convert the relatively nontoxic prodrug 5-fluorocytosine into the anticancer drug 5-fluorouracil. To facilitate investigations of the utility of cytosine deaminase for cancer chemotherapy, we have cloned and expressed the enzyme from Saccharomyces cerevisiae. The DNA sequence translates into a protein of 158 amino acids in length, with a predicted molecular weight of 17,563 kilodaltons. Alignment of the cytosine deaminase protein sequence from yeast with a variety of proteins defines a novel sequence motif of cytosine or cytidine binding enzymes. Recombinant expression cassettes encoding cytosine deaminase were transfected into monkey kidney COS cells, which lack endogenous cytosine deaminase, to test for production of a functional protein. Cell extracts from these transfectants contained detectable levels of enzyme activity capable of converting 5-fluorocytosine to 5-fluorouracil. Cytosine deaminase was expressed in yeast from a cDNA cassette under the control of an inducible promoter, increasing expression 250- to 300-fold relative to wild-type strains. A purification protocol has been developed which permits recovery of 60% of cytosine deaminase in active form from induced cell lysates after two purification steps. This protocol will be useful for isolating large quantities of pure enzyme which are required for the preclinical evaluation of monoclonal antibody-cytosine deaminase conjugates in combination with 5-fluorocytosine.

Cytosine deaminase. The roles of divalent metal ions in catalysis.

Cytosine deaminase (CDase, EC 3.5.4.1) isolated from Escherichia coli contains a catalytically essential divalent metal ion. Fe2+ was efficiently removed from the enzyme with o-phenanthroline to yield an apoenzyme with less than 5% of the catalytic activity of native enzyme. The time courses for inactivation and for removal of Fe2+ from the enzyme by o-phenanthroline were similar. Apoenzyme reconstituted with Fe2+, Mn2+, Co2+, or Zn2+ (M2+CDase) had kcat values of 185, 88, 50, and 32 s-1, respectively. The Km values of these M2+CDases for cytosine were similar (0.22-0.39 mM). Cytosine potently inhibited reconstitution of the apoenzyme with Fe2+. Fe2+CDase was rapidly inactivated by 1 mM H2O2 (t1/2 < 1 s), whereas Mn2+CDase, Co2+CDase, and Zn2+CDase were not inactivated by H2O2. CDase was also inhibited by excess divalent cations. Cu2+ and Zn2+ reversibly inhibited Fe2+CDase activity with inhibition constants of 1.8 and 5.8 microM, respectively. Cu2+ dissociated slowly from the secondary binding on CDase with a rate constant of 2 x 10(-3) s-1.

Generation of 5-fluorouracil from 5-fluorocytosine by monoclonal antibody-cytosine deaminase conjugates.

Cytosine deaminase (CDase) catalyzes the conversion of cytosine to uracil and is also able to convert the clinically used antifungal agent 5-fluorocytosine (5FC) into the anticancer drug 5-fluorouracil (5FU). The enzyme was purified from bakers' yeast in a six-step procedure. Studies indicated that bakers' yeast CDase had a molecular weight of approximately 32 kDa and was composed of two subunits of equal molecular weights. Monoclonal antibodies were covalently attached to CDase, forming conjugates that could bind to antigens on tumor cell surfaces. The combination of L6-CDase and 5FC was equivalent in cytotoxic activity to 5FU when tested against the H2981 human lung adenocarcinoma cell line (L6 positive, 1F5 negative). 5FC alone was noncytotoxic. The activation of 5FC was immunologically specific since 1F5-CDase did not enhance 5FC activity.

Purification and characterization of S-adenosylhomocysteine deaminase from streptonigrin-producing Streptomyces flocculus.

An S-adenosylhomocysteine deaminase has been isolated and purified from streptonigrin-producing Streptomyces flocculus ATCC 13257. Deamination represents the major metabolic route of S-adenosylhomocysteine in this organism. The protein was found to be monomeric with a molecular weight of 56,100 +/- 1,600. The activity was optimal at pH 7.0 and 37 degrees C, and the deaminase was inactivated by p-chloromercuribenzoate but not by metal chelators. The Km for S-adenosylhomocysteine is 2.5 mM, and the Ki for inhibition by deoxycoformycin is 1.6 nM.

Laser photobleaching leads to a fluorescence grade adenosine deaminase.

The enzyme adenosine deaminase (adenosine aminohydrolase EC 3.5.4.4) from calf intestinal mucosa is commercially available at high purity grade yet, at the sensitivity at which fluorescence studies may be undertaken, a nonpeptidic fluorescence is detectable at lambda exmax = 350 nm and lambda emmax = 420 nm. A sevenfold decrease of this nonpeptidic fluorescence was obtained upon irradiation by the third harmonic (355 nm) of a Nd:YAG laser for 16 min, at 5 mJ/pulse, with a pulse width of 6 ns at a repetition rate of 10 Hz. The decline of fluorescence was accompanied by a negligible loss of enzymatic activity. Moreover, the integrity of the protein was ascertained by (i) its fluorescence (lambda exmax = 305 nm, lambda emmax = 335 nm) and lifetime distribution and (ii) its kinetics in the presence of the substrate adenosine and two inhibitors, all of which remained essentially unaltered. Laser photobleaching is a simple way to achieve a fluorescence grade adenosine deaminase.

The application of affinity chromatography for the separation of "high Km" and "low Km" 5'-nucleotidase and other AMP metabolizing enzymes.

AMP-sepharose 4B has been widely used as a general ligand affinity chromatography for purification of AMP deaminase, 5'-nucleotidase, adenosine kinase and other adenine nucleotide metabolizing enzymes. Since these enzymes generally differ in their kinetic properties related to the values of Km for AMP and analogous compounds, it was assumed that there may be a specific elution pattern of some of the enzymes which would enable sequential elution from the column during a single run. Using 0.5 M NaCl, 10 mM ATP and 5 mM adenosine as eluting agents, it was possible to separate on AMP-sepharose column AMP deaminase "high Km" and "low Km" 5'-nucleotidase and adenosine kinase. Adenylate kinase, adenosine deaminase and nonspecific phosphatase did not bind to the column. Using human placental extract, AMP deaminase, "high Km" and "low Km" 5'-nucleotidase and adenosine kinase were purified 2.8, 2.9, 105 and 1240 fold, respectively. AMP deaminase and "high Km" 5'-nucleotidase were further separated using phosphocellulose column chromatography and the final purification was 227 and 143 fold, respectively. The specific activities of purified enzyme preparations were 9.1, 1.0, 0.4 and 0.5 mumols/min/mg protein of AMP deaminase, "high Km" 5'-nucleotidase and adenosine kinase, respectively. This approach provides a rapid method for initial purification of these enzymes from crude soluble extracts.

Purification and partial characterization of brain adenosine deaminase: inhibition by purine compounds and by drugs.

Rat brain adenosine deaminase (E.C. 3.5.4.4.) was purified 667-fold from the supernatant fraction by the following techniques: heat treatment (60 degrees C), fractionation with ammonium sulfate, column chromatography on DEAE-Sepharose, and preparative gel electrophoresis. The purified enzyme was homogeneous by the criterion of polyacrylamide disc gel electrophoresis and isoelectric focusing. Amino acid composition is given. The isoelectric point of the enzyme (5.2) was determined by isoelectric focusing on agarose. The apparent molecular weight was estimated to be 39,000 (Stokes Radius [Rs] = 27.3 A) using a calibrated Sephacryl S-300 column. The study of the influence of the temperature on the initial reaction rates allowed calculation of Ea (8.9 Kcal/mole) and delta H (5.0 Kcal/mole) values. The variation of V and Km with pH suggests the existence of a sulfhydryl group and an imidazole group in the enzyme-substrate complex. The enzyme had a Km (adenosine) of 4.5 X 10(-5) M and was inhibited by inosine, guanosine, adenine, and hypoxanthine but not by other intermediates of purine metabolism. None of the inhibitors were active as substrates. The enzyme was also inhibited by dimethyl sulfoxide and ethanol. Inhibition by ethanol can account partially for the CNS depressant effects of levels 3 and 4 of alcohol intoxication. A number of drugs having therapeutic uses such as sedative, anxiolytic, analgesic, and relaxant are modulators of the enzyme. Among these, lidoflazine, phenylbutazone, and chlordiazepoxide are the most potent as inhibitors (Ki 30, 54, and 83 microM, respectively), whereas medazepam is the most potent as activator (Ka 0.32 mM). Thus, it is concluded that some drugs that inhibit adenosine uptake also modulate adenosine deaminase activity. Besides, since the enzyme is located extracellularly [Franco et al, 1986], these drugs can modulate the physiological effects exerted by extracellular adenosine.

Implantable enzyme capsules for cancer chemotherapy from bakers' yeast cytosine deaminase immobilized on epoxy-acrylic resin and urethane prepolymer.

For trial use in the local chemotherapy of cancer by a combination of cytosine deaminase (EC 3.5.4.1) and 5-fluorocytosine (J. Biotechnol., (1985), 2, 13-21), 40 U of partially purified cytosine deaminase was obtained from 500 g of commercial compressed bakers' yeast. The enzyme, which is unstable, was immobilized to stabilize it by the use of commercial epoxy-acrylic beads (Eupergit C). The immobilized enzyme was made into enzyme capsules with cellulose tubing for dialysis to encapsulate it or urethane polymer to entrap it, which materials are biocompatible. The activity of the intact cellulose capsules thus made was 0.4% that of the immobilized enzyme inside. The enzyme capsules also were stable. Ten days after the cellulose capsules were implanted in rats, 25% of the starting activity remained. When the polyurethane capsules were tested in vitro for 9 mo for thermostability at 37 degrees C, the activity decreased rapidly (with a half-life of 28 d) during the first 4 mo, and then slowly (half-life, about 100 d) during the next 5 mo. A calculation to transform the biphasic decline into a sum of the exponential decline of two components of enzymic activities with different strengths and half-lives showed that the larger half-life was 5 mo.

Purification of cytidine deaminase from chicken liver.

Cytidine deaminase (cytidine aminohydrolase, EC 3.5.4.5) from chicken liver has been obtained in pure form through a rapid procedure consisting of organic solvent precipitation, heat treatment, anionic-exchange chromatography, hydrophobic interaction chromatography followed by two rapid chromatographies using an FPLC system. The final preparation is pure but shows microheterogeneity as judged by a single band obtained by SDS-gel electrophoresis and a series of superimposed active bands obtained on native gel electrophoresis and gel isoelectrofocusing. The native protein molecular weight determined by gel filtration is 50,000. SDS-gel electrophoresis experiments conducted in the presence and in the absence of reducing agents, suggest an oligomeric structure of four apparently identical subunits of 12,000 molecular weight. The absorption spectrum of the native protein reveals a maximum at 278 nm and a minimum at 261 nm with a small shoulder at 285 nm. The isoelectric point has an average value around pH 4.45.

Separation of human from mouse and monkey adenosine deaminase by ion-exchange chromatography following retroviral-mediated gene transfer.

A method for the chromatographic separation of human adenosine deaminase (ADA) from murine and monkey ADA is described. This procedure was developed in order to detect the expression of low or moderate levels of human ADA following retroviral-mediated gene transfer of cloned human ADA gene sequences into both mouse and monkey cells. Protein separation was achieved on a Mono Q (HR 5/5) anion-exchange column using the Pharmacia fast protein liquid chromatography system and was found to be a highly reproducible method yielding enzymatically active protein. An increasing linear gradient extending from 0.05 to 0.5 M potassium chloride (pH 7.5) was used to elute the enzyme. Under these conditions, most human ADA does not bind to the column and elutes in the low-salt buffer (0.05 M KCl), while murine ADA elutes at 0.12 M KCl and monkey ADA at 0.15 M KCl. The column fractions were assayed for ADA activity, and the characteristic isozyme banding patterns for human, mouse, and monkey ADA were confirmed by starch gel electrophoresis. This procedure allows the rapid and reproducible separation of human ADA from that of other species and yields partially purified enzymatically active protein.

Immunoaffinity purification and fluorescence studies of human adenosine deaminase.

Human thymus adenosine deaminase was isolated by using a monoclonal antibody affinity column. The highly purified enzyme produced by this rapid, efficient procedure had a molecular weight of 44,000. Quenching of the intrinsic protein fluorescence by small molecules was used to probe the accessibility of tryptophan residues in the enzyme and enzyme-inhibitor complexes. The fluorescence emission spectrum of human adenosine deaminase at 295-nm excitation had a maximum at about 335 nm and a quantum yield of 0.03. Addition of polar fluorescence quenchers, iodide and acrylamide, shifted the peak to the blue, and the hydrophobic quencher trichloroethanol shifted the peak to the red, indicating that the emission spectrum is heterogeneous. The fluorescence quenching parameters obtained for these quenchers reveal that the tryptophan environments in the protein are relatively hydrophobic. Binding of both ground-state and transition-state analogue inhibitors caused decreases in the fluorescence intensity of the enzyme, suggesting that one or more tryptophans may be near the active site. The kinetics of the fluorescence decrease were consistent with a slow conformational alteration in the transition-state inhibitor complexes. Fluorescence quenching experiments using polar and nonpolar quenchers were also carried out for the enzyme-inhibitor complexes. The quenching parameters for all enzyme-inhibitor complexes differed from those for the uncomplexed enzyme, suggesting that inhibitor binding causes changes in the conformation of adenosine deaminase. For comparison, parallel quenching studies were performed for calf adenosine deaminase in the absence and presence of inhibitors. While significant structural differences between adenosine deaminase from the two sources were evident, our data indicate that both enzymes undergo conformational changes on binding ground-state and transition-state inhibitors.

Adenosine deaminase from Saccharomyces cerevisiae: purification and characterization.

Adenosine deaminase from Saccharomyces Cerevisiae was purified about 1600 fold by salt fractionation, ion exchange and affinity chromatography. Some physico-chemical properties have been determined: the molecular weight of the enzyme by gel filtration is 85,000 daltons; one -SH is readily titrated by paramercuribenzoate per 78,000 mol. weight; optimum pH is 7; Km for adenosine is 40.7 microM; 2'-deoxyadenosine is not a substrate. Deazaadenosine analogues are good inhibitors, while erythro-9-(2-hydroxy-3-nonyl) adenine binds with low affinity. These properties are compared with those of other adenosine deaminases.

Affinity difference of adenosine deaminases for the purine riboside-epoxyactivated Sepharose 6B column.

The small-size adenosine deaminase (Mr = 35,000 and 43,000) in calf intestinal mucosa, frog liver and scallop adductor muscle and the large-size deaminase (Mr = 100,000) in frog liver probably formed by adding a conversion protein to the small enzyme, were tightly bound to the purine riboside affinity column. Binding of the other large-size enzymes (Mr = 140,000) in the midgut gland of scallop and mussel to the column was not successful. It seems that the binding difference does not depend on a change in affinity between active site and ligand but rather on the stereospecific positioning of active site in the enzyme molecules.

Purification, stability and kinetic properties of highly purified adenosine deaminase from Bacillus cereus NCIB 8122.

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) from Bacillus cereus NCIB 8122 has been purified to electrophoretic homogeneity by ammonium sulfate precipitation, gel filtration through Sephadex G-100, DEAE-Sephadex A-50 chromatography and ion-exchange HPLC on DEAE-Polyol. The enzyme activity is stabilized (at temperatures from 0 degrees C to 40 degrees C) by 50 mM NH4+ or K+, while it is irreversibly lost in the absence of these or a few other monovalent cations. Glycerol (24% by volume) helps the cation in stabilizing the enzyme activity above 40 degrees C, but also exerts per se a noticeable protecting effect at room temperature. B. cereus adenosine deaminase displays the following properties: Mr on Sephadex G-200, 68,000; Mr in SDS-polyacrylamide gel electrophoresis, 53,700; optimal pH-stability (in the presence of 50 mM KCl) over the range 8-11 at 4 degrees C, and maximal catalytic activity at 30 degrees C between pH 7 and 10; Km for adenosine around 50 microM over the same pH range and Km for 2'-deoxyadenosine around 400 microM.

Isolation of mutant adenosine deaminase by coformycin affinity chromatography.

Adenosine deaminase is a purine salvage enzyme that catalyzes the deamination of adenosine and deoxyadenosine. Deficiency of the enzyme activity is associated with T-cell and B-cell dysfunction. Mutant adenosine deaminase has been isolated from heterozygous and homozygous deficient lymphoblast cell lines with the aid of an affinity matrix consisting of coformycin (a potent inhibitor of the enzyme) as the affinity ligand, bound to 3,3'-iminobispropylamine-derivatized Sepharose. Routinely, 80-90% of adenosine deaminase in crude cell homogenates could be bound to the material. Adenosine deaminase was specifically eluted by enzyme inhibitors or less efficiently by high substrate concentrations. Protein preparations isolated from several different deficient cell lines were highly purified and exhibited molecular weights identical to wild-type adenosine deaminase. This method produces a protein that is suitable for structural studies.

Cytidine deaminase from Escherichia coli B. Purification and enzymatic and molecular properties.

Cytidine deaminase (cytidine aminohydrolase, EC 3.5.4.5) from Escherichia coli has been purified to homogeneity through a rapid and efficient two-step procedure consisting of anion-exchange chromatography followed by preparative electrophoresis. The final preparation is homogeneous, as judged by a single band obtained by disc gel electrophoresis performed in the absence and presence of denaturing agents. The native protein molecular weight determined by gel filtration is 56 000. Sodium dodecyl sulfate disc gel electrophoresis experiments conducted upon previous incubation of the enzyme with dimethyl suberimidate suggest an oligomeric structure of two identical subunits of 33 000 molecular weight. The absorption spectrum of the protein reveals a maximum at 277 nm and a minimum at 255 nm. The isoelectric point is at pH 4.35. Amino acid analysis indicates an excess of acidic amino acid residues as well as six half-cystine residues. No interchain disulfide groups have been evidenced. According to Cleland's nomenclature, kinetic analysis shows a rapid-equilibrium random Uni-Bi mechanism. Cytidine deaminase is competitively inhibited by various nucleosides. Km values for cytidine, deoxycytidine, and 5-methylcytidine are 1.8 X 10(-4), 0.9 X 10(-4), and 12.5 X 10(-4) M, respectively.