Lasse Uotila

University of Helsinki, Helsinki, Province of Southern Finland, Finland

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Publications (33)80.41 Total impact

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    Lasse UOTILA · Martti KOIVUSALO
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    ABSTRACT: 1Glyoxalase I has been obtained in electrophoretically pure form from sheep liver by a procedure which includes ammonium sulfate and poly(ethylene glycol) fractionations and column chromatographies on hydroxyapatite, Cibacron Blue–Sephadex G-100 and DEAE-cellulose. The specific activity of the homogeneous preparations is about 4000 units/mg of protein (25° C). Three separate peaks of activity were obtained in the last column on DEAE-cellulose (DE-32). No signs of heterogeneity were seen in the previous steps. Purified but not crude preparations gave two activity peaks on disc gel electrophoresis.2The isoelectric point of metal-free apoglyoxalase I is 5.0 by isoelectric focusing. The apparent molecular weight of glyoxalase I is 45 900 from gel chromatography. From dodecylsulfate gel electrophoresis the enzyme has a subunit molecular weight of 21000.3In addition to methylglyoxal, phenylglyoxal and kethoxal are good substrates of sheep liver glyoxalase I. Hydroxypyruvaldehyde and glyoxal react more slowly.4Catalytically inactive apoenzyme of sheep liver glyoxalase I has been prepared by dialysis against EDTA and Chelex-100. All of the activity is restored by Mg2+ ions; Zn2+, Mn2+, Co2+, Ni2+ and Ca2+ ions, in decreasing order of maximum velocity, give partial reactivation. Of these metals, Mg2+ has the highest (2.8 mM) and Zn2+ the lowest (0.007 mM) apparent half-saturation concentration when assayed in 80 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonate (Hepes) buffer pH 6.8.5Several chelating agents are inhibitors of sheep liver glyoxalase I. In all cases Mg2+ reverses the inhibition after a short incubation time.
    Preview · Article · Mar 2005
  • Lasse Uotila · Martti Koivusalo

    No preview · Article · Feb 1997 · Advances in Experimental Medicine and Biology
  • M Koivusalo · R Lapatto · L Uotila

    No preview · Article · Feb 1995 · Advances in Experimental Medicine and Biology
  • M Koivusalo · L Uotila

    No preview · Article · Feb 1993 · Advances in Experimental Medicine and Biology
  • M Koivusalo · L Uotila

    No preview · Article · Feb 1991 · Advances in Experimental Medicine and Biology
  • Lasse Uotila
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    ABSTRACT: Vitamin K functions in animal cells as the cofactor of the enzyme vitamin K-dependent carboxylase which catalyzes the post-translational formation of gamma-carboxyglutamyl (Gla) residues in specific vitamin K-dependent proteins. These proteins include four blood coagulation factors (prothrombin and Factors VII, IX and X), other plasma proteins (protein C, protein S and protein Z), two proteins from bone (osteocalcin or bone Gla-protein and matrix Gla-protein), and other proteins from lung, kidney, spleen, testis, placenta and other tissues. In the proteins involved in blood coagulation the Gla residues are mandatory for the activation of the inactive proenzymes; this process occurs on phospholipid surfaces to which the proenzymes are bound via Gla residues and calcium ions. The energy needed in the carboxylation reaction is obtained from the oxidation of vitamin K hydroquinone to 2,3-epoxide of the vitamin. Specific enzymes, vitamin K epoxide reductase and vitamin K quinone reductases, catalyze consecutive reactions in which the vitamin K hydroquinone is regenerated, thus allowing continued use of the vitamin K molecule for the carboxylations. The oral anticoagulants, derivatives of 4-hydroxycoumarin and indan-1,3-dione, used as therapeutic agents in thromboembolic disease, are antagonists to vitamin K preventing the catalytic use of vitamin K in the carboxylations by irreversibly inhibiting vitamin K epoxide reductase.
    No preview · Article · Feb 1990 · Scandinavian journal of clinical and laboratory investigation. Supplementum
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    ABSTRACT: Two separate pools of glyoxalase II were demonstrated in rat liver mitochondria, one in the intermembrane space and the other in the matrix. The enzyme was purified from both sources by affinity chromatography on S-(carbobenzoxy)glutathione-Affi-Gel 40. From both crude and purified preparations polyacrylamide gel-electrophoresis resolved multiple forms of glyoxalase II, two from the intermembrane space and five from the matrix. Among the thioesters of glutathione tested as substrates, S-D-lactoylglutathione was hydrolyzed most efficiently by the enzymes from both sources. Significant differences were observed in the specificities between the intermembrane space and matrix enzymes with S-acetoacetylglutathione, S-acetylglutathione, S-propionylglutathione and S-succinylglutathione as substrates. Pure glyoxalase II from rat liver cytosol was chemically polymerized and used as antigen. Antibodies were raised in rabbits and the antiserum was used for comparison of the two purified mitochondrial enzymes with cytosolic glyoxalase II by immunoblotting. The enzyme purified from the intermembrane space cross-reacted with the antiserum, but the matrix glyoxalase II did not. The results give evidence for the presence in rat liver mitochondria of two species of glyoxalase II with differing characteristics. Only the enzyme from the intermembrane space appears to resemble the cytosolic glyoxalase II forms.
    No preview · Article · Nov 1989 · Biochimica et Biophysica Acta
  • Source
    Martti Koivusalo · Marc Baumann · Lasse Uotila
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    ABSTRACT: Formaldehyde dehydrogenase (EC 1.2.1.1) is a widely occurring enzyme which catalyzes the oxidation of S-hydroxymethylglutathione, formed from formaldehyde and glutathione, into S-formyglutathione in the presence of NAD. We determined the amino acid sequences for 5 tryptic peptides (containing altogether 57 amino acids) from electrophoretically homogeneous rat liver formaldehyde dehydrogenase and found that they all were exactly homologous to the sequence of rat liver class III alcohol dehydrogenase (ADH-2). Formaldehyde dehydrogenase was found to be able at high pH values to catalyze the NAD-dependent oxidation of long-chain aliphatic alcohols like n-octanol and 12-hydroxydodecanoate but ethanol was used only at very high substrate concentrations and pyrazole was not inhibitory. The amino acid sequence homology and identical structural and kinetic properties indicate that formaldehyde dehydrogenase and the mammalian class III alcohol dehydrogenases are identical enzymes.
    Preview · Article · Nov 1989 · FEBS Letters
  • Lasse Uotila · Martti Koivusalo

    No preview · Chapter · Jan 1989
  • Lasse Uotila
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    ABSTRACT: The steady-state kinetic mechanism of vitamin K-dependent carboxylase from calf liver has been investigated by initial-velocity measurements with varying concentrations of two carboxylase substrates and constant, nonsaturating concentrations of the other two substrates. With all combinations of the varied substrates tested linear kinetics were obtained with lines intersecting on the left side of the 1/v axis in double-reciprocal plots. Thus the carboxylase has a sequential reaction mechanism which includes the quinternary complex of the enzyme with its four substrates. A mechanism with the ordered steady-state addition of all substrates to the enzyme accords well with the results. A totally random mechanism was excluded but the alternative possibility remained that part of the substrates are added in a rapid-equilibrium random reaction. Experiments with saturating constant concentrations of sodium bicarbonate and varying concentrations of the other substrates suggest that bicarbonate (CO2) is either the first or, more probably, the last substrate bound to the enzyme.
    No preview · Article · Aug 1988 · Archives of Biochemistry and Biophysics
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    ABSTRACT: Glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6), which has been regarded as a cytosolic enzyme, was also found in rat liver mitochondria. The mitochondrial fraction contained about 10-15% of the total glyoxalase II activity in liver. The actual existence of the specific mitochondrial glyoxalase II was verified by showing that all of the activity of the crude mitochondrial pellet was still present in purified mitochondria prepared in a Ficoll gradient. Subfractionation of the mitochondria by digitonin treatment showed that 56% of the activity resided in the mitochondrial matrix and 19% in the intermembrane space. Partial purification of the enzyme (420-fold) was also achieved. Statistically significant differences were found in the substrate specificities of the mitochondrial and the cytosolic glyoxalase II. Electrophoresis and isoelectric focusing of either the crude mitochondrial extract or of the purified mitochondrial glyoxalase II resolved the enzyme activity into five forms with the respective pI values of 8.1, 7.5, 7.0, 6.85 and 6.6. Three of these forms (pI values 7.0-6.6) were exclusively mitochondrial, with no counterpart in the cytosol. The relative molecular mass of the partially purified enzyme, as estimated by Superose 12 gel chromatography, was 21,000. These results give evidence for the presence of mitochondrial glyoxalase II which is different from the cytosolic enzymes in several characteristics.
    No preview · Article · Jul 1988 · Biochimica et Biophysica Acta
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    ABSTRACT: Glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6) was purified to homogeneity and separated into two forms (alpha, pI = 8.0; beta, pI = 7.4) from both liver and brain of wistar rats by column isoelectric focusing. These forms were also found to have different electrophoretic mobilities. No significant differences were found between the alpha and beta forms from either source in the relative molecular mass (about 24,000) or in Km values using three substrates. The temperature-inactivation profiles were also similar, the two forms being stable up to 50 degrees C. Chemical modification studies with phenylglyoxal suggest that these enzyme forms probably contain arginine residues near the active site. Inactivation of alpha and beta forms by diethylpyrocarbonate and by photooxidation with methylene blue, and protection by S-D-mandeloylglutathione, a slowly reacting substrate, suggest the presence of histidine at the active site. The alpha and beta forms show different half-life values in inactivation by histidine reagents, which may be due to a difference in the active-site structures of these enzymes. The results probably indicate distinct structures (sequences) for alpha and beta forms.
    No preview · Article · Mar 1987 · Biochimica et Biophysica Acta
  • Lasse Uotila · Martti Koivusalo
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    ABSTRACT: Red cell hemolysates from nonrelated Finns were analyzed by electrofocusing on polyacrylamide gel, and formaldehyde dehydrogenase (EC 1.2.1.1) was located by an activity-staining method. Three forms of the enzyme were constantly found for all the individuals studied but no variants were observed in this population (n = 217). Human liver also had three formaldehyde dehydrogenase forms with locations identical to those of the red cell formaldehyde dehydrogenase. Population genetic studies of formaldehyde dehydrogenase can easily be performed with red cell hemolysates with the techniques described here, and there is no need to use liver biopsy samples.
    No preview · Article · Feb 1987 · Human Heredity
  • L Uotila · M Koivusalo

    No preview · Article · Feb 1987 · Progress in clinical and biological research
  • Lasse Uotila
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    ABSTRACT: Red cell hemolysates from a Finnish population sample (n = 242) were fractionated by isoelectric focusing on polyacrylamide gel, and S-formylglutathione hydrolase (EC 3.1.2.12) was located by activity staining. Polymorphism, which is probably genetically determined, was found. The samples from most persons studied gave one major enzyme band, whereas for 6 persons three enzyme bands were found. The enzyme is a dimer, and the polymorphism observed appears to result from two alleles, FGH1 and FGH2 at an autosomal locus. The frequency found for FGH1 was 0.988.
    No preview · Article · Feb 1984 · Human Heredity
  • M Koivusalo · T Koivula · L Uotila

    No preview · Article · Feb 1982 · Progress in clinical and biological research
  • Lasse Uotila
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    ABSTRACT: This chapter discusses enzymatic and chemical methods for the synthesis of purified glutathione thiol esters. S-2-Hydroxyacylglutathione is prepared most conveniently by the reaction catalyzed by glyoxalase I. The reaction mixture contains potassium phosphate, glutathione (GSH), α-ketoaldehyde, and yeast glyoxalase I. S-Acetylglutathione synthesis involve addition of glutathione in water and the pH of the solution adjusted. Ethanol is added to 35%. Thiolacetic acid is added in a hood with constant stirring. The pH of the solution is adjusted to 4.5. The reaction mixture is stirred and progress of the reaction is followed by measuring the increase of A240 of small aliquots after extraction with ether. The product, in about 80% yield, is cleared by filtration and concentrated in a rotary evaporator at 30°. S-propionylglutathione is analogous to that for S-acetylglutathione is used in which thiolpropionic acid is the acylating agent. It is found that S-acylglutathione content can be determined by completely hydrolyzing the thiol ester by glyoxalase II or neutral hydroxylamine and assaying the amount of GSH formed with 5,5′-dithiobis(2-nitrobenzoate), 2,2′-dithiodipyridine, or 4,4′-dithiodipyridine. The hydrolysis time must be short to prevent significant oxidation of thiols. The disulfide chosen should be included directly in the hydrolysis mixture.
    No preview · Article · Dec 1981 · Methods in enzymology
  • Lasse Uotila · Martti Koivusalo

    No preview · Article · Feb 1981 · Methods in Enzymology
  • Lasse Uotila · Martti Koivusalo

    No preview · Article · Feb 1981 · Methods in Enzymology
  • Lasse Uotila · Bengt Mannervik
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    ABSTRACT: The steady-state kinetic mechanism of human liver formaldehyde dehydrogenase (formaldehyde:NAD+ oxidoreductase (glutathione-formylating), EC 1.2.1.1) was investigated by product inhibition of the forward and the reverse reactions catalyzed by the enzyme. The results are compatible with a mechanism which contains the random addition to the enzyme of NAD+ and S-hydroxymethylglutathione (the adduct of glutathione and formaldehyde), or NADH and S-formylglutathione, and free glutathione as the allosteric activator of the enzyme (Uotila, L. and Mannervik, B. (1979) Biochem. J. 177, 869-878).
    No preview · Article · Jan 1981 · Biochimica et Biophysica Acta