Biochimica et Biophysica Acta (BBA) - Enzymology最新文献

Soluble extracts from encysted dormant embryos of Artemia contain a poly(A) polymerase activity which has been partially purified and characterized. The enzyme requires manganese, an RNA primer and ATP for maximal activity. The K_m for ATP is 0.04 mM and the enzyme is inhibited by concentrations higher than 0.5 mM ATP. dATP replaces ATP with a 15% efficiency. The K_m for dATP is 0.06 mM. Natural RNAs, poly(A) and poly(AG) are the best RNA primers among several homopolymers and copolymers tested. The poly(A) polymerase does not show any specificity for the nucleotide in the 3′ end of the RNA primer. The product of the reaction is a polyadenylic acid chain covalently bound to the RNA primer molecule. The length of the poly(A) chain is about ten nucleotides, but this length is dependent on the incubation time and the RNA primer concentration. The molecular weight of the enzyme is 70 000 and its isoelectric point is 6.0. The existence of an active poly(A) polymerase in dormant embryos of Artemia, likely with a cytoplasmic localization, suggests a role for this enzyme in the processing and activation of the stored mRNAs after resumption of development.

An NAD-dependent alcohol dehydrogenase (alcohol : NAD⁺ oxidoreductase, EC 1.1.1.1) active towards l-octan-2-ol but not towards the corresponding d-isomer was purified to homogeneity from the soil bacterium Comamonas terrigena. The enzyme is a tetramer (molecular weight 125 000–141 000) and is most active at pH 8.5–9.9. Preferred alcohol substrates are l-alkan-2-ols, activity towards which was inhibited by EDTA, 1,10-phenanthroline and 2,2′-bipyridine. The enzyme exhibits much weaker activity towards primary alcohols, symmetrical secondary alcohols and asymmetric secondary alcohols in which the hydroxyl moiety is located at positions other than C-2, and little or no activity towards d-alkan-2-ols. For l-alkan-2-ols, symmetrical secondary alcohols and primary alcohols, log K_m values decrease linearly with increase in the number of carbon atoms in the alkyl chain. A plot of standard free-energy of binding (ΔG⁰′) of substrates against the number of carbon atoms in the alkyl chain (primary alcohols) or the longer of the two portions of the alkyl chain (secondary alcohols) gives a single straight-line relationship, suggesting that hydrophobic interactions make an important contribution to substrate binding. The observed specificity was interpreted in terms of a model in which secondary alcohols interact with the enzyme through the hydrogen and hydroxyl group that participate in NAD⁺ reduction, and one of the two alkyl segments. The size of the unbound alkyl segment markedly affects V, the optimum being a single methyl unit. This specificity was correlated with that of the CS2 secondary alkylsulphohydrolase that catalyses the production of l-alkan-2-ols from d-alkan-2-yl sulphate surfactants.

Iduronate sulfatase of human placenta separates on DEAE Bio-Gel A chromatography into two components, a less acidic form A and a more acidic form B. The two forms have different mobilities on gel electrophoresis and different isoelectric points, pH 5.0 for form A and pH 4.5 for form B. They show the same pH optima in sodium acetate buffer and similar K_m values for [³H]disulfated disaccharide substrate. Iduronate sulfatase A is more heat labile than iduronate sulfatase B. Different molecular weights were found by gel filtration while similar values were estimated by sucrose gradient centrifugation. Neuraminidase treatment of the two forms gives evidence that these enzymes contain sialic acid residues.

Phosphofructokinase (ATP : d-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from Streptococcus thermophilus has been purified. It is a tetramer composed of identical subunits of molecular weight 36 000 and exhibits Michaelis-Menten kinetics. Compared to the phosphofructokinases from taxonomically related bacteria, the enzyme from S. thermophilus is more stable at high temperatures. In addition, it has been demonstrated that the phosphofructokinases from lactobacteria and also from Bacillus stearothermophilus show immunologic cross-reaction. In spite of the significantly different kinetic properties and the different thermostability of these enzymes, this finding indicates great structural resemblance.

β-Glucuronidase (β-d-glucuronide glucuronosohydrolase, EC 3.2.1.31) was covalently attached to alkylamine-controlled pore glass via a glutaraldehyde immobilization scheme. The activity of this immobilized β-glucuronidase was studied with respect to several kinetic parameters in comparison with the behavior of the soluble enzyme. K_m values for p-nitrophenyl glucuronide, estriol-3-glucuronide, and estriol-16β-glucuronide were determined. For each substrate the K_m was essentially the same, 0.2 mM, and this value did not change when the enzyme was immobilized. The soluble and immobilized enzyme both displayed a relatively broad pH maximum centered at pH 6.8 for all substrates. Several organic-aqueous mixtures including methanol, ethanol, acetonitrile and ethylene glycol were tested, and their effects on the activity of immobilized β-glucuronidase were similar to those found for the soluble enzyme. Long-term (1 year) storage stability tests of the immobilized enzyme were carried out. The immobilized enzyme retained 40% of its initial activity after 1 year and was very robust towards most of the organic solvents tested.

tRNA, 18 S and 28 S ribosomal RNAs were found to activate muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) but inhibit liver and heart AMP deaminases. The macromolecular structures are essential for modulation of enzyme activity, since the effects of RNA disappeared after RNAase treatment. Sucrose density centrifugation experiments clearly demonstrated the binding of purified muscle AMP deaminase to tRNA, 18 S and 28 S RNAs. The binding is reversible and responsive to alterations of pH and KCl concentration. The binding was stable at pH 5.1–7.0 in 0.1 M KCl, but most of the enzyme dissociated at pH 7.5. KCl below 0.1 M concentration had no effect on dissociation of enzyme-RNA complex, but in 0.15 M KCl the complex was partially dissociated and in 0.2 M KCl most of the enzyme was released. Various nucleotides were also effective in dissociation of the enzyme from complex. The binding is saturable and the maximum number of muscle AMP deaminase molecules bound per mol 28 S RNA was calculated to be approx. 30. Liver and heart AMP deaminases were also found to interact with RNA.

The participation of Mg complex of nucleoside diphosphates and nucleoside triphosphates in the reverse and forward reactions catalyzed by purified carbamyl phosphokinase (ATP : carbamate phosphotransferase, EC 2.7.2.2) of Streptococcus faecalis R, ATCC-8043 were studied. The results of initial velocity studies at approx. 1 mM free Mg²⁺ concentration have indicated that in the reverse reaction MgdADP was as effective a substrate as MgADP. The phosphoryl group transfer from carbamyl phosphate to MgGDP, MgCDP and MgUDP was also observed at relatively higher concentrations of the enzyme and respective magnesium nucleoside diphosphate. In the forward direction MgdATP was found to be as efficient a phosphate donor as MgATP. On the other hand, Mg complexes of GTP, CTP and UTP were ineffective even at higher concentrations of the enzyme and respective magnesium nucleoside triphosphate. Product inhibition studies carried out at non-inhibitory level of approx. 1 mM free Mg²⁺ concentration have revealed that the enzyme has two distinct sites, one for nucleoside diphosphate or nucleoside triphosphate and the other for carbamyl phosphate or carbamate, and its reaction with the substrates is of the random type. Further tests of numerical values for kinetic constants have indicated that they are partially consistent with the Haldane relationship which is characteristic of rapid equilibrium and random mechanism.

‘Malic’ enzyme (l-malate:NADP⁺ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) was purified from Clostridium thermocellum by DEAE-cellulose, agarose-NADP and Sephadex G-200 column chromatography. The 117-fold purified ‘malic’ enzyme displayed a maximum activity of 135 units/mg at 40°C and represented 0.8% of the total cell protein. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis of the protein suggested 90% purity and an approximate tetrameric subunit molecular weight of 40 000. The enzyme absolutely required both bivalent and monovalent cations for catalysis. Mn²⁺ and NH₄⁺ were the most effective cationic activators examined. Increasing NH₄⁺ concentration increased both enzyme activity and affinity toward l-malate. The apparent K_m for l-malate was 3 · 10⁻⁴ M at 0.4 mM NH₄Cl. Enzyme activity increased linearly when temperature was raised between 22–60°C and a Q₁₀ of 2.1 was calculated from an Arrhenius plot. The enzyme was stable to heating at 60°C but was denatured at higher temperatures. The enzyme half-life was 10 min at 72°C. The enzyme displayed a broad pH optimum (7.2–8.2 for Tris-HCl buffer) but was inactivated by p-chloromercuribenzoate. The high thermal stability, low apparent molecular weight and NH₄⁺ activation are properties not common to all previously described ‘malic’ enzymes.

In order to obtain an efficacious and safe immunoglobulin G (IgG) preparation for intravenous use, the digestion of IgG with an immobilized pepsin (EC 3.4.23.1) preparation was studied. Thus, pepsin was immobilized onto glutaraldehyde-activated AH-Sepharose 4B under acidic conditions. The enzymatic properties, such as proteolytic activity, pH-activity profile and heat stability, of the immobilized pepsin preparation were examined. The immobilized pepsin retained more than 40% of its proteolytic activity toward $\text{N-}\text{acetyl-}\text{l}\text{-phenylalanyl-}\text{l}\text{-3,5-diiodotyrosine}$ and more than 30% toward IgG, and also remarkable stability as compared with free pepsin. The immobilized pepsin thus prepared was efficiently used for the limited cleavage of IgG and the gel-filtration effect of the column made it easily possible to yield the F(ab′)₂-rich fraction for intravenous use.

The possibility of a functional complex formation between glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) and 3-phosphoglycerate kinase (EC 2.7.2.3), enzymes catalysing two consecutive reactions in glycolysis has been investigated. Kinetic analysis of the coupled enzymatic reaction did not reveal any kinetic sign of the assumed interaction up to 4 · 10⁻⁶ M kinase and 10⁻⁴ M dehydrogenase. Fluorescence anisotropy of 10⁻⁷ M or 2 · 10⁻⁵ M glyceraldehyde-3-phosphate dehydrogenase labeled with fluorescein isothiocynate did not change in the presence of non-labeled 3-phosphoglycerate kinase (up to 4 · 10⁻⁵ M). The frontal gel chromatographic analysis of a mixture of the two enzymes (10⁻⁴ M dehydrogenase and 10⁻⁵ M kinase) could not reveal any molecular species with the kinase activity having a molecular weight higher than that of 3-phosphoglycerate kinase. Both types of physicochemical measurements were also performed in the presence of substrates of the kinase and gave the same results. The data seem to invalidate the hypothesis that there is a complex between purified pig muscle glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase.