Preparation and properties of insolubilized restriction endonucleases.

Type II restriction endonucleases Bam HI and Eco RI were covalently coupled to Sepharose. These insolubilized enzymes generated fragment patterns for several viral DNAs identical to those produced by the respective free enzymes. Conditions for optimal activity were similar for both bound and unbound forms of the enzymes. Insolubilization improved thermal stability of Bam HI and Eco RI. The bound enzyme can be recovered from reaction mixtures and reused several times. Upon storage at 4 degrees C, coupled endonucleases remained stable for several months.     

Alpha-D-Mannosidase. Preparation and properties of free and insolubilized enzyme.

Alpha-D-Mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.24) has been purified to homogeneity as demonstrated by polyacrylamide gel electrophoresis and ultracentrifugation. The molecular weight of the enzyme is approx. 200000; the protein appears to contain 4 subunits, with molecular weights of 66000 and 44000. The enzyme was immobilized on Sepharose and the properties of the coupled and free enzyme were compared. Both were stable up to 70 degrees C with rapid loss of activity between 75-80 degrees C; both retained 25-30% activity in 6 M urea and 65% of the original activity could be restored in the coupled preparation by removal of the urea. The pH maximum of each form was approximately the same, with the maximum of the immobilized enzyme shifted slightly to a lower pH. The coupled alpha-D-mannosidase presented in this report offers the possibility of digesting high molecular weight substrates, such as glycoproteins, with the advantages of (1) recovering large quantities of digested substrate; (2) recovery of the active glycosidase; and (3) digestion at high temperatures and under conditions that denature many proteins.     

Factors affecting the properties of insolubilized antibodies

IgG separated from an antiserum to estradiol was coupled under various experimental conditions to Sepharose activated either with CNBr or by conversion into a long-armed derivative (the N-hydroxysuccinimide ester). The conjugates were characterized by measurement of the binding parameters, in order to evaluate separately the loss of sites and the loss of affinity. The cross-reactivity with estriol and estrone was measured to obtain information on the occurrence of structural alterations of the antibody site.The results show that the loss of immunoreactivity varies in extent (from 95% to less than 10%) and in nature (loss of sites or of affinity or a combination of both effects) depending on the coupling conditions.The use of a hydrocarbon extension to keep the protein distant from the matrix does not prevent the loss of active sites but is effective in safeguarding the affinity of the residual sites. The loss of sites can be substantially reduced by coupling at a pH value around neutrality and by keeping the protein/matrix mass ratio low. At a coupling pH of 6.4 and at a mass ratio of 0.1–0.2 nmol IgG/mg of Sepharose, the antibodies were insolubilized with a negligible loss of sites and affinity; on increasing the mass ratio (up to 10 nmol IgG/mg Sepharose) there is a progressive loss of sites accompanied by a substantial lowering of the affinity of the residual sites.On the basis of the above-mentioned findings, the nature of the effects occurring when antibodies are transferred from solution onto a solid matrix is discussed.     

Factors affecting the properties of insolubilized anitbodies.

IgG separated from an antiserum to estradiol was coupled under various experimental conditions to Sepharose activated either with CNBr or by conversion into a long-armed derivative (the N-hydroxysuccinimide ester). The conjugates were characterized by measurement of the binding parameters, in order to evaluate separately the loss of sites and the loss of affinity. The cross-reactivity with estriol and estrone was measured to obtain information on the occurrence of structural alterations of the antibody site. The results show that the loss of immunoreactivity varies in extent (from 95% to less than 10%) and in nature (loss of sites or of affinity or a combination of both effects) depending on the coupling conditions. The use of a hydrocarbon extension to keep the protein distant from the matrix does not prevent the loss of active sites but is effective in safeguarding the affinity of the residual sites. The loss of sites can be substantially reduced by coupling at a pH value around neutrality and by keeping the protein/matrix mass ratio low. At a coupling pH of 6.4 and at a mass ratio of 0.1-0.2 nmol IgG/mg of Sepharose, the antibodies were insolubilized with a negligible loss of sites and affinity; on increasing the mass ratio (up to 10 nmol IgG/mg Sepharose) there is a progressive loss of sites accompanied by a substantial lowering of the affinity of the residual sites. On the basis of the above-mentioned findings, the nature of the effects occurring when antibodies are transferred from solution onto a solid matrix is discussed.     

Preparation and use of insolubilized amyloglucosidase for the production of sweet glucose liquors

Amyloglucosidase (EC. 3.2.1.3), partially purified from an Aspergillus species, was chemically attached to DEAE cellulose using the bifunctional reagent 2-amino-4,6-dichloro-s-triazine. The action of the insolubilized enzyme derivative on dilute maltose and dextrin solutions was studied in a packed bed. A second and deeper bed was used to demonstrate the possibility of a continuous process for raising the dextrose; equivalents of “glucose” liquors of high concentration formed by acid hydrolysis of maize starch.     

Kinetic properties of a nucleoside phosphotransferase of chick embryo

1. A nonspecific nucleoside phosphotransferase (nucleotide : 3'-deoxynucleotide 5'-phosphotransferase, EC 2.7.1.77), purified from chick embryos, catalyzes the transfer of phosphate ester from a nucleotide donor to a nucleoside acceptor. 2. The enzyme exhibits sigmoidal kinetics with respect to nucleoside monophosphate donors, but with respect to nucleoside di- or triphosphate donors and nucleoside acceptors hyperbolic kinetics were obtained. 3. The nucleoside phosphotransferase of chick embryo is unstable to heat and is protected from inactivation by a large number of nucleosides. 4. Nucleoside di- and triphosphates lower both the concentration of nucleoside monophosphates required for half-maximal velocity and the kinetic order of reaction measured with these phosphate donors. On the contrary, nucleoside di- or triphosphate do not modify the kinetic parameters evaluated for nucleoside acceptors. 5. We suggest that the nucleoside phosphotransferase contains both substrate and regulatory sites. It seems that the free apoenzyme is converted, by means of cooperative interactions between regulatory sites, into an enzyme-nucleotide complex, which is particularly stable at 37 degrees C.     

Mitochondrial GTP-AMP phosphotransferase. 1. Purification and properties.

GTP-AMP phosphotransferase has been purified 116-fold with a yield of 24% from beef heart mitochondria using freeze-thawing, alkali and acid treatment and successive column chromatography on phosphocellulose, Sephadex G-100 and blue-dextran--Sepharose. It has crystallized from poly-(ethylene glycol) and is essential homogeneous by sodium dodecylsulfate electrophoresis and isoelectrofocusing. The specific activity of the crystalline preparation was 290 U/mg. The molecular weight was found to be 26000 and the isoelectric point to be 9.8. Amino acid analysis showed 21 aspartic acid or asparagine, 19 threonine, 12 serine, 26 glutamic acid or glutamine, 15 proline, 16 glycine, 14 alanine, 15 valine, 4 methionine, 12 isoleucine, 28 leucine, 7 tyrosine, 7 phenylalanine, 5 histidine, 14 lysine, 16 arginine, 2 tryptophan, no --SS-- bonds or free --SH. Guanosine(5')pentaphospho(5')adenosine is a very strong inhibitor similar to adenosine(5')pentaphospho(5')adenosine as an inhibitor of cytosolic adenylate kinase.     

Nucleoside phosphotransferase in animal tissues. Tissue distribution and kinetic properties

Summary Amphibian, avian and mammal tissues contain a nucleoside phosphotransferase clearly different from those previously described in vegetables and bacteria.Whatever the animal source, the enzyme showed many similar characteristics as far as substrate specificity, dependence upon Mg²⁺ instability at 37 °C, and the protecting effect of nucleotides were concerned. Moreover, when submitted to gel filtration, the enzyme behaved in all cases as a dissociable high molecular weight protein, whose degree of association was controlled by nucleotides.In amphibian and avian tissues multiple forms of the enzyme seem to be present which differ for the substrate concentration at half-maximal velocity (S_0.5); the concentration of nucleotide effector which affords half-maximal protection at 37 °C (P_0.5); and the Hill coefficient for monophosphate donor. Within each single species, the higher the interaction coefficient was, the lower S_0.5 and P_0.5 values were.In mammalian tissues one form of nucleoside phosphotransferase seems to prevail where cooperative interactions are almost absent and whose S_0.5 as well as P_0.5 values do not vary significantly from one tissue to another.     

The purification and properties of nucleoside phosphotransferase from mucosa of chicken intestine

Nucleoside phosphotransferase (nucleotide: 3'-deoxynucleoside 5'-phosphotransferase, EC 2.7.1.77) has been purified from chicken intestine mucosa to apparent homogeneity. The enzyme is represented by a multisubunit protein at different degrees of association. It can dissociate into a component with a marked fall in catalytic activity. The associated forms are similar to the enzyme previously purified from chick embryo as regards: substrate specificity both with respect to nucleoside monophosphate donors and to deoxyribonucleoside acceptors; sigmoidicity in the rate curve with a variable phosphate donor; instability to heat, dilution and lowering of pH; the activating and protecting effect of nucleotides, particularly the diphosphate forms. The dissociated form displays lower Vmax and higher S0.5 than the associated ones; and the Hill constants are always about 1. With this form, nucleotides show only a modest activating effect and do not protect. Mg2+, Mn2+ or Co2+ are required for catalytic activity, whereas the protective effect of nucleotides is independent of divalent metals. Inorganic phosphate stabilizes associated forms of the enzyme, but inhibits its activity by competing with nucleotide effectors. The enzyme behaves also as a phosphohydrolase, particularly with respect to deoxyribonucleoside monophosphates; deoxyuridine and deoxythymidine inhibit hydrolytic activity.     

Purification and properties of suppressor seryl-tRNA: ATP phosphotransferase from bovine liver

Seryl-tRNASerCmCA: ATP phosphotransferase was purified 1200-fold from bovine liver by ultracentrifugation at 150 000 X g, chromatography on DEAE-cellulose, fractional precipitation with ammonium sulfate, chromatography on hydroxyapatite, gel filtration on Sephacryl S-300 and affinity chromatography on Blue Sepharose. Molecular mass was estimated as 135-145 kDa. The Km values for ATP and ser-tRNASerCmCA were 2 mM and 21 nM, respectively. This enzyme did not react with ser-tRNASerIGA, tyr-tRNA or thr-tRNA.     

ATP:AMP phosphotransferase from baker''s yeast. Purification and properties

Synchronous cells of the green alga, Scenedesmus obliquus, cultured in a 14-h/10-h light/dark regime, contain a peak of ribonucleoside-diphosphate reductase activity and maximum deoxyribonucleoside 5'-triphosphate concentrations at the 12th hour of the cell cycle, coinciding with DNA synthesis and preceding the formation of eight daughter cells. The intracellular dTTP pool reaches 4.5 pmol and the other pools 2-3 pmol/10(6) cells. Algal reductase activity is sensitive to cycloheximide, but not to lincomycin. These correlations demonstrate the functioning of the NDP leads to dNDP leads to dNTP pathway of DNA precursor biosynthesis in plant cells. In the presence of 20 micrograms 5-fluorodeoxyuridine/ml, an inhibitor of thymidylate synthesis, the dTTP pool is rapidly depleted and DNA synthesis ceases. 5-Fluorouracil and methotrexate produce similar effects. At the same time the ribonucleotide reductase activity and also the dATP pool are greatly increased, especially when fluorodeoxyuridine treatment is combined with continued illumination of the algae. In contrast, arabinosylcytosine, an inhibitor of DNA replication, has no effect on ribonucleotide reduction. The control of de novo enzyme synthesis in the eucaryotic algae therefore appears to depend on the presence of dTTP (or a related nucleotide), but not directly coupled to DNA synthesis. This interdependence resembles the situation observed in HeLa cells, while it may differ in detail from control mechanisms of ribonucleotide reductase studied in bacteria.     

Evolved neomycin phosphotransferase from an isolate of Klebsiella pneumoniae

A new aminoglycoside resistance gene (aphA1-IAB) confers high-level resistance to neomycin. The sequence of aphA1-IAB is closely related to aphA1 found in the transposons Tn4352, Tn903 and Tn602. For example, aphA1-IAB differs from aphA1-903 at five nucleotides that result in four amino acid replacements. The enzyme encoded by aphA1-IAB has a significantly higher turnover number with neomycin, kanamycin and G418 as substrates than does the aphA1-903 enzyme. A parsimonious phylogenetic tree suggests that aphA1-IAB evolved from an ancestral form that is closely related or identical to the aphA1 found in Tn903. The excess of replacement substitutions over silent substitutions in aphA1-IAB, as well as its convergence toward aphA3 from Staphylococcus aureus, is indicative of selective evolution. Our hypothesis to explain these results is that aphA1-IAB evolved under the selective pressure of neomycin use in relatively recent times.     

Preparation and properties of an immobilized derivative of penicillinase.

Penicillinase (beta-lactamase I, EC 3.5.2.6) secreted by Bacillus cereus, strain 569/H, was covalently attached to aminoethyl cellulose via glutaraldehyde. The immobilized derivative shows increased thermostability and decreased susceptibility to conformational changes induced by certain substrates of penicillinase. The decline in the rate of hydrolysis of such substrates was consequently suppressed by immobilization. A marked increase in Km was observed with all substrates except for the unsubstituted 6-aminopenicillanic acid. The altered properties of the new derivative are attributed to the constraint imposed by immobilization on the conformational flexibility of the enzyme molecule. Thus, apart from obvious technological interest, immobilized penicillinase provides a useful model for the study of the role of flexibility in the function of an enzyme.     

Structural analyses of nucleotide binding to an aminoglycoside phosphotransferase

3',5"-Aminoglycoside phosphotransferase type IIIa [APH(3')-IIIa] is a bacterial enzyme that confers resistance to a range of aminoglycoside antibiotics while exhibiting striking homology to eukaryotic protein kinases (ePK). The structures of APH(3')-IIIa in its apoenzyme form and in complex with the nonhydrolyzable ATP analogue AMPPNP were determined to 3.2 and 2.4 A resolution, respectively. Furthermore, refinement of the previously determined ADP complex was completed. The structure of the apoenzyme revealed alternate positioning of a flexible loop (analogous to the P-loop of ePK's), occupying part of the nucleotide-binding pocket of the enzyme. Despite structural similarity to protein kinases, there was no evidence of domain movement associated with nucleotide binding. This rigidity is due to the presence of more extensive interlobe interactions in the APH(3')-IIIa structure than in the ePK's. Differences between the ADP and AMPPNP complexes are confined to the area of the nucleotide-binding pocket. The position of conserved active site residues and magnesium ions remains unchanged, but there are differences in metal coordination between the two nucleotide complexes. Comparison of the di/triphosphate binding site of APH(3')-IIIa with that of ePK's suggests that the reaction mechanism of APH(3")-IIIa and related aminoglycoside kinases will closely resemble that of eukaryotic protein kinases. However, the orientation of the adenine ring in the binding pocket differs between APH(3')-IIIa and the ePK's by a rotation of approximately 40 degrees. This alternate binding mode is likely a conserved feature among aminoglycoside kinases and could be exploited for the structure-based drug design of compounds to combat antibiotic resistance.