Native Isoenzymes Research Articles

Properties of purified cytosolic isoenzyme I of Cu,Zn-superoxide dismutase from Nicotiana plumbaginifolia leaves.

The isoenzyme I of cytosolic Cu,Zn-superoxide dismutase (SOD) from Nicotiana plumbaginifolia (tobacco) leaves has been purified to apparent homogeneity. The relative molecular mass of the native isoenzyme, determined by gel filtration chromatography, is about 33.2 kDa. SDS-polyacrylamide gel electrophoresis shows that the enzyme is composed of two equal subunits of 16.6 kDa The isolectric point, assayed by isoelectric focusing, in the pH range of 3.5-6.5, is 4.3. The enzyme stability was tested at different temperatures, pH, and concentration of inhibitors (KCN and H(2)O(2)). The catalytic constant (k(cat)) was 1.17 +/- 0.14 x 10(9) M(-1) s(-1) at pH 9.9 and 0.1 M ionic strength. The activation energy of the thermal denaturation process is 263 kJ mol(-1). The electrostatic surface potential of the modeled tobacco Cu,Zn-SOD I was calculated showing that the functional spatial network of charges on the protein surface has been maintained, independently of the amino acid substitution around the active sites.

Production of Recombinant Human Creatine Kinase (r-hCK) Isozymes by Tandem Repeat Expression of M and B Genes and Characterization of r-hCK-MB

Serum creatine kinase-MB isoenzyme (CK-MB) is widely used as a marker of myocardial injury. We prepared recombinant human CK (r-hCK) MB isoenzyme and examined its potential for use as a control material for assay of CK-MB in serum. cDNAs encoding CK-M and CK-B subunits were inserted into the same plasmid vector, followed by transformation of Escherichia coli. The resulting three types of CK isoenzymes were purified by conventional chromatography. The ratio of MB to MM to BB was 50:40:10 on the basis of CK activity. Highly purified CK-MB with a specific activity of 533 U/mg was produced in a yield of 5.7 mg/g of packed cells. Purified r-hCK-MB had the isoelectric point (pI 5.3) and molecular size (46 kDa for the subunit) of native CK-MB. Its immunoreactivity in an ELISA using antibody against native heart enzyme was similar to that of cardiac CK-MB. The r-hCK-MB retained >90% activity for at least 4 months at 11 degrees C in a delipidated serum matrix in a liquid form at a concentration of 118 U/L. r-hCK-MB shows key properties of the native cardiac isoenzyme and may be useful as a control and calibrator for serum assays of CK-MB.

Differences in catalytic properties between native isoenzymes of xyloglucan endotransglycosylase (XET)

Four isoenzymes of xyloglucan endotransglycosylase (XET; EC 2.4.1.207) were isolated from sprouting mung bean seedlings (M35, M45, M55a, M55b) and two from cauliflower florets (C30, C45). Purification in each case was by ammonium sulphate precipitation, reversible formation of a covalent xyloglucan–enzyme complex, and cation-exchange chromatography. The isoenzymes differed in pH optimum (range 5.0–6.5), K m for the nonasaccharide XLLGol (Gal 2.Xyl 3.Glc 3.glucitol) as acceptor substrate, ability to utilise diverse oligosaccharides as acceptor substrate, and ability to bind to carboxymethyl-cellulose (and thus possibly to other polyanions such as pectin in the cell wall). None of the isoenzymes was particularly cold-tolerant, unlike one XET (TCH4) of Arabidopsis. The two cauliflower isoenzymes had higher K m values for XLLGol (70–130 μM) than the four mung bean isoenzymes (16–35 μM). We suggest that this difference is related to the major roles of the XETs in these two tissues: integration of new xyloglucan into the walls of the densely cytoplasmic cauliflower florets, and re-structuring of existing wall material in the rapidly vacuolating bean shoots.

Glutathione S-transferase isoenzymes expressed in the three developmental stages of the insect Tenebrio molitor

The isoenzymes of glutathione S-transferase expressed in the three developmental stages [larvae( L), pupae( P), adults( A)] of Tenebrio molitor, an insect of economic significance, were studied. Four major isoenzymes [A (L,P,A), B (L,P,A), C (L,P,A) and D (L,P,A)] were present throughout development as judged by chromatofocusing at pH ranges 7–4 and 9–6. GSTs were purified by means of S-hexyl-GSH-Epoxy activated Sepharose 6B affinity chromatography and subsequent chromatofocusing at pH range 7–4. The molecular weight of the native isoenzymes was estimated to be 50.5 kDa, after gel filtration chromatography, while that of the subunits was 27 kDa, indicating that the enzyme was dimeric. With the exception of the molecular weights which remained constant, the isoelectric points, substrate specificities, sensitivities to inhibitors, pH optima of CDNB-conjugating activity and kinetic parameters of the isoenzymes altered in the three developmental stages.

Regenerated and denatured peroxidase as potential lipid oxidation catalysts

The aim of this study was to determine the relative lipid oxidation activities of regenerated and denatured peroxidase using horseradish peroxidase (HRP) as a model. Poor correlation between peroxidase activity regeneration and enhancement of lipid oxidation activity suggested that the regenerated enzyme did not possess increased potential to catalyse lipid oxidation. Heat denatured peroxidase, not containing any regenerated enzyme, was shown to have a greater lipid oxidation activity than the native enzyme. The denatured peroxidase comprised three haemoproteins with similar isoelectric points that were significantly lower than that of the major native isoenzyme. Two of the proteins, present in relatively minor amounts, possessed most of the lipid oxidation activity. Evidence for the release of haem on heating HRP under conditions that led to regeneration of activity of the cooled enzyme was provided by circular dichroism (CD) spectroscopy. The CD study also suggested that irreversible polymerisation of haem occurred on heating, which may have restricted subsequent activity regeneration. The freed haem appeared to have only a low lipid oxidation activity compared with the denatured holoenzyme. Nuclear magnetic resonance (NMR) spectroscopy detected ‘structural regeneration’ of the haem pocket under conditions that did not lead to regeneration of functional enzyme. ‘Structurally regenerated’ forms could potentially enhance lipid oxidation on storage of heat processed foods.

Concentration of enzyme-dependent activation of PLC-β1 and PLC-β2 by Gα 11 and βγ-subunits

Differential regulation of PLC-β1 and −β2 by the G-protein α-subunit, Gα 11, and by G-protein βγ-subunits was studied utilizing recombinant PLC-β1 and −β2. Rat PLC-β1 and human PLC-β2 were purified after recombinant baculovirus-mediated expression in Sf9 cells. The catalytic properties of the purified recombinant isoenzymes were directly compared to PLC-β1 purified from bovine brain and PLC-β2 partially purified from HL60 polymorphonuclear neutrophils. The recombinant isoenzymes were indistinguishable from the native isoenzymes with respect to dependence of reaction velocity on bulk PtdIns(4,5)P 2 substrate concentration, pH, and free Ca 2+ concentration. Marked AlF 4 t--dependent activation was observed upon reconstitution of rPLC-β1 with the G-protein α-subunit, Gα 11. Activation occurred with a concentration dependence on Gα 11 for activation and elevation in reaction velocity that was similar to that of native PLC-β1. In contrast, Gα 11 promoted only a small elevation in the catalytic rate of recombinant PLC-β2, which was also typical of the native isoenzyme. Maximal reaction rates with respect to PLC-β isoenzyme concentration were achieved and indicated that rPLC-β2 required 10-fold greater concentrations of both Gα 11 and of rPLC-β2 for activation than did rPLC-β1. rPLC-β1 and rPLC-β2 were also differentially regulated by βγ-subunits. This differential activation was not the result of different concentration dependencies on βγ-subunit for activation, but rather, the result of the greater degree to which the catalytic rate of PLC-β2 was elevated by βγ-subunits when compared to PLC-β1.

Re-evaluation of the structure and physiological function of guanidino kinases in fruitfly (Drosophila), sea urchin (Psammechinus miliaris) and man.

Purification and biophysical characterization of mitochondrial creatine kinase (Mi-CK) from sperm of the sea urchin Psammechinus miliaris, as well as gel-permeation chromatography of human heart Mi-CK demonstrate that these two Mi-CK isoenzymes form highly symmetrical octameric molecules with an M(r) of approx. 350,000, a value similar to that found for all other Mi-CK isoenzymes investigated so far. The absolute evolutionary conservation of this oligomeric form from sea urchins to mammals points both to its essentiality for Mi-CK function and to an important role of octameric Mi-CK in the energy metabolism of tissues and cells with high and fluctuating energy demands. To investigate whether a similar physiological principle also operates in an even more distantly related animal phylum, the arginine kinase (ArgK) isoenzyme system of Drosophila flight muscle was investigated with two independent subcellular fractionation procedures and subsequent analysis of the fractions by SDS/PAGE, immunoblotting and native isoenzyme electrophoresis. In contrast with a previous report [Munneke and Collier (1988) Biochem. Genet. 26, 131-141], strong evidence against the occurrence of a Mi-ArgK isoenzyme in Drosophila was obtained. The findings of the present study are discussed in the context of CK and ArgK function in general and of structural and bioenergetic differences between vertebrate striated muscles and arthropod flight muscles.

Rat Plasma Clearance Rate and Organ Distribution of β-Hexosaminidase Isoenzymes from Human Serum

beta-Hexosaminidase (EC 3.2.1.30) is markedly increased in human serum in liver disease, chronic alcoholism, and pregnancy. Knowledge of the clearance rate of plasma/serum beta-hexosaminidase is necessary to evaluate this increase. We studied the plasma clearance of beta-hexosaminidase isoenzymes (purified from human serum and placenta) after their infusion into rat circulation. A recently developed enzyme immunoassay method was used to measure the human beta-hexosaminidase isoenzymes; this method relies on both immunoreactivity and enzyme activity, so intact human isoenzymes were measured. In comparison with the placental isoenzymes (t1/2 less than 2 min), the serum forms showed a considerably slower clearance (t1/2 = 2-4 h). However, desialylation of the serum forms resulted in their rapid clearance (t1/2 less than 2 min). The organ localization of the enzyme eliminated from the circulation was investigated 24 h after infusion. Placental and native serum isoenzymes accumulated mainly in the liver and the spleen. Desialylated serum forms were almost exclusively localized to the liver.

The cDNA sequence of a neutral horseradish peroxidase

A cDNA clone encoding a horseradish ( Armoracia rusticana) peroxidase has been isolated and characterized. The cDNA contains 1378 nucleotides excluding the poly(A) tail and the deduced protein contains 327 amino acids which includes a 28 amino acid leader sequence. The predicted amino acid sequence is nine amino acids shorter than the major isoenzyme belonging to the horseradish peroxidase C group (HRP-C) and the sequence shows 53.7% identity with this isoenzyme. The described clone encodes nine cysteines of which eight correspond well with the cysteines found in HRP-C. Five potential N-glycosylation sites with the general sequence Asn-X-Thr/Ser are present in the deduced sequence. Compared to the earlier described HRP-C this is three glycosylation sites less. The shorter sequence and fewer N-glycosylation sites give the native isoenzyme a molecular weight of several thousands less than the horseradish peroxidase C isoenzymes. Comparison with the net charge value of HRP-C indicates that the described cDNA clone encodes a peroxidase which has either the same or a slightly less basic p I value, depending on whether the encoded protein is N-terminally blocked or not. This excludes the possibility that HRP-n could belong to either the HRP-A, -D or -E groups. The low sequence identity (53.7%) with HRP-C indicates that the described clone does not belong to the HRP-C isoenzyme group and comparison of the total amino acid composition withe HRP-B group does not place the described clone within this isoenzyme group. Our conclusion is that the described cDNA clone encodes a neutral horseradish peroxidase which belongs to a new, not earlier described, horseradish peroxidase group.

Lipoxygenase isoenzymes: A spectroscopic and structural characterization of soybean seed enzymes

Applying recent developments in protein purification techniques, a number of lipoxygenase isoenzymes have been isolated in satisfactory quantities for a detailed physical and structural characterization. Four seed isoenzymes from two soybean cultivars have been studied by peptide mapping, free thiol and iron content determinations, and C-terminal analysis as well as by uv-visible absorption and EPR spectroscopy. While differences between the type 1 enzyme and the other isoenzymes were readily detected using proteolytic peptide mapping, digestion with dilute hydrochloric acid and C-terminal analysis both revealed structural features which were similar in all of the isoenzymes. One clear difference between the lipoxygenases was in their free sulfhydryl group content. The uv-visible absorption spectrum of each native isoenzyme was consistent with expectations for the experimental aromatic amino acid content. All of the isoenzymes contained one non-heme iron atom per molecule of protein. The oxidation of each isoenzyme with product hydroperoxide resulted in the conversion of the iron from an EPR silent state into several forms with EPR signals characteristic of high spin iron(III). The EPR spectra of all isoenzymes were remarkably similar. A time course EPR and catalytic activity study revealed that the various EPR active states represent a complex equilibrium between iron atoms in different environments. The pH dependence for the EPR and absorption spectroscopy lends support to the hypothesis that acid/base chemistry represents an important aspect of lipoxygenase catalysis.

Analysis of the phosphofructokinase subunits and isoenzymes in human tissues.

The 6-phosphofructo-1-kinase (PFK) subunits and isoenzymes were studied in human muscle, heart, brain, liver, platelets, fibroblasts, erythrocytes, placenta and umbilical cord. In each tissue, the subunit types in the native isoenzymes were characterized by immunological titration with subunit-specific antibodies and by column chromatography on QAE (quaternary aminoethyl)-Sephadex. Further, the subunits of the partially purified native isoenzymes were resolved by SDS/polyacrylamide-gel electrophoresis, identified by immunoblotting, and quantified by scanning gel densitometry of silver-stained gels and immunoblots. Depending on the type of tissue, one to three subunits were detected. The Mr values of the L, M and C subunits regardless of tissue were 76,700 +/- 1400, 82,500 +/- 1640 and 86,500 +/- 1620. Of the tissues studied, only the muscle PFK isoenzymes exhibited one subunit, which was the M-type subunit. Of the other tissues studied, the PFK isoenzymes contained various amounts of all three subunits. Considering the properties of the native PFK isoenzymes, it is clear that, in human tissues, they are not simply various combinations of two or three homotetrameric isoenzymes, but complex mixtures of homotetramers and heterotetramers. The kinetic/regulatory properties of the various isoenzyme pools were found to be dependent on subunit composition.

A comparison of the kinetic properties of native bovine muscle carbonic anhydrase and an activated derivative with modified thiol groups

Steady-state and equilibrium kinetic properties of native bovine carbonic anhydrase III (carbonate hydrolyase, EC 4.2.1.1) and a derivative modified with methyl methanethiosulfonate were investigated. The modified enzyme has a markedly increased CO 2 hydration activity compared to the native form with a 3-times higher value of k cat and a 6–10-times higher value of k cat K m . Qualitatively, the activated enzyme shows the same kinetic behavior as native isoenzyme III. This is reflected in similar pH dependences of the kinetic parameters for CO 2 hydration, similar solvent hydrogen isotope effects on these parameters, similar deviations from Michaelis-Menten kinetics for the HCO 3 − dehydration reaction, and similar behavior of the kinetics of CO 2 HCO 3 − exchange at chemical equilibrium as measured by a 13C-NMR magnetization transfer technique. It is concluded that the conversion of -SH groups to -S-S-CH 3 moieties does not change the catalytic mechanism, but leads to an increased rate of CO 2 HCO 3 − interconversion as well as to an increased rate of proton transfer between the active site and the reaction medium.

Calcium-activated, phospholipid-dependent protein kinases from rat liver: subcellular distribution, purification, and characterization of multiple forms.

Three forms of Ca2+- and phospholipid-dependent protein kinase (protein kinase C) were extensively purified from rat liver homogenate. Subcellular fractionation analysis indicated that the majority (approximately 85%) of the activity was associated with particulate fractions of the liver. Among these, the microsomal and nuclear fractions accounted for approximately 63% and approximately 10% of total activity. The remaining 15% of protein kinase C was recovered in the soluble fraction following differential centrifugation. It was also found that most of the membrane-associated protein kinase C was latent, with 4-6-fold stimulation with detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, octyl beta-glucoside, or Triton X-100. The activity of both the bound form and the soluble enzyme was enhanced by the addition of Ca2+ and phosphatidylserine, when histone H1 was used as substrate. The bound protein kinase C activity was dissociated by homogenization of liver in buffer containing ethylene glycol bis(beta-aminoethyl ether)-N,-N,N',N'-tetraacetic acid, ethylenediaminetetraacetic acid, and various proteolytic inhibitors, and the solubilized extract was used to purify multiple forms of the enzyme. The purification procedure sequentially utilized (NH4)2SO4 fractionation, ion-exchange chromatography on DEAE-cellulose, gel permeation chromatography on Fractogel TSK HW-55 (F), ion-exchange chromatography on hydroxylapatite, gel permeation chromatography on Ultrogel AcA34, and affinity chromatography on polyacrylamide-immobilized phosphatidylserine. On hydroxylapatite columns, protein kinase C activity was resolved into three isoenzymic forms designated C-I, C-II, and C-III. The molecular weights of the three isoenzymic forms were in the range of 208,000-225,000 as shown by chromatography on calibrated Ultrogel AcA34 columns and sucrose density gradient centrifugation. Furthermore, all three isoenzymes demonstrated a single peak with a sedimentation coefficient (s20.w) in the range of 9.0-9.2. However, with polyacrylamide gel electrophoresis, all the forms showed a single protein component with average molecular weight of 64K, suggesting that the native isoenzymes may be composed by subunits. Finally, all three isoenzymes exhibited nearly identical enzymatic properties.

Rat heart glycogen phosphorylase II is genetically distinct from phosphorylase I

Previous studies in our laboratory have shown that rat heart glycogen phosphorylase (1,4-α- d-glucan:orthophosphate α- d-glucosyltransferase, EC 2.4.1.1) separates into two forms upon ion-exchange chromatography. Both forms could be shown to have the same subunit M r and to incorporate one molecule of phosphate per subunit. The studies reported here were done to check whether both forms are native isoenzymes and, further, which form might represent the heart-specific phosphorylase. Firstly, the iso-electric points of the purified enzymes are compared with those associated with phosphorylase activity in crude extracts from rat heart. Two out of four major bands coincided with the bands of purified phosphorylase I b and II b (isoelectric points: 5.5 and 6.25), indicating apparent identity. Secondly, antibodies to rat skeletal muscle phosphorylase reacted with rat heart phosphorylase I, whereas phosphorylase II was neither inhibited nor precipitated by the antibody. Thirdly, peptide maps obtained after proteolytic digestion of SDS-denatured phosphorylase I and II showed different patterns. In addition to the kinetic differences between these two forms reported earlier, phosphorylase II a was inhibited by glucose 6-phosphate, whereas phosphorylase I a was not. These results suggest that phosphorylase II is a heart-specific isoenzyme which is presumably encoded by a different gene.

Purification and spectral characterization of a paraperoxidase from Cucurbita pepo ripe fruits

The isolation and purification of the two major anionic (A 1) and cationic (C 1) peroxidase isoenzymes extracted from the ripe fruit of Curcubita pepo have been described. The anionic isoenzyme A 1 has spectral properties very similar to those of normal horseradish peroxidase isoenzymes. The cationic isoenzyme C 1 has a paraperoxidase nature and has been characterized by electronic and circular dichroism spectroscopy, together with several of its derivatives. The spectra suggest that native isoenzyme C 1 may exist as equilibrium mixture of high-spin and low-spin forms, but such equilibria are apparently absent in the various derivatives of the enzyme. Careful comparison of the spectra of the native isoenzyme C 1 and its reduced form with those of the corresponding cyanide derivatives seems to exclude the possibility that the paraperoxidase character could arise from a cyanide-bound form of the enzyme. The spectra of isoenzyme C 1 recorded in the presence of various substrates and H 2O 2 indicate an accumulation of compound III.

The purification of human creatine kinase BB with high specific activity

This paper describes the purification of human creatine kinase BB with high specific activity (1,122 U/mg). The procedure used resulted in a protein yield of 5.4 mg (21% recovery) from 150 g of brain tissue. Two-dimensional electrophoresis and PAGE studies indicated that purified CK-BB might exist as native isoenzyme along with structural aggregates since the multi-banded appearance was reduced to a single band with sodium dodecyl sulfate treatment but not with 2-mercaptoethanol. Investigators are cautioned not to store brain tissue for prolonged periods of time before isolation of the isoenzyme as this may lead to protein redistribution with additional bands becoming evident on PAGE.

Structural studies of two apyrases from Solanum tuberosum

The proportion of acid and basic amino acid residues obtained for two homogeneous isoenzymes of apyrase isolated from different clonal varieties of Solanum tuberosum (Pimpernel and Desirée) was essentially the same. This does not agree with the difference in pI values observed. Treatment with asparaginase and glutaminase caused partial inactivation of both enzyme activities in both isoenzymes, and pI values were changed, but not equalized. The differences in pI values of the native isoenzymes may still be attributed to different proportions of glutamine and asparagine in the primary structure. Leucine is the amino-terminal residue in both isoenzymes. Both have two disulphide bridges and one buried sulphydryl group which is not essential for enzyme activity. Differences in pI values should thus be attributed to factors other than amino acid composition.

Synthesis of rat muscle carbonic anhydrase III in a cell-free translation system

Carbonic anhydrase III (CA III) was identified in the products of rat soleus muscle RNA translation in vitro by both a radioimmunoassay and a specific immunoprecipitation technique followed by SDS—polyacrylamide gel electrophoresis analysis of the precipitated antigen. The primary translation product has the same M r-value as the native isoenzyme. CA III mRNA was found to represent ∼0.55% of the total mRNA present in rat soleus muscle.

Organelle-Bound Malate Dehydrogenase Isoenzymes Are Synthesized as Higher Molecular Weight Precursors

Biosynthesis of malate dehydrogenase isoenzymes was studied in cotyledons of watermelons (Citrullus vulgaris Schrad., var. Stone Mountain). The glyoxysomal and mitochondrial isoenzymes are synthesized as higher molecular weight precursors which can be immunoprecipitated by mono-specific antibodies from the products of in vitro translation in reticulocyte lysates programed with cotyledonary mRNA and with the same size from enzyme extracts of pulse-labeled cotyledons. During translocation from the cytosol into the organelles, processing takes place. An 8 kilodalton extra sequence is cleaved from the glyoxysomal precursor and a 3.3 kilodalton extra sequence from the mitochondrial precursor producing the native subunits of 33 and 38 kilodaltons, respectively. The data support a post-translational translocation of the organelle-destined malate dehydrogenase isoenzymes. The in vitro translation of the cytosolic malate dehydrogenase I yields a product which has the same molecular weight as the subunit of the native isoenzyme (39.5 kilodaltons).

Three atypical serum creatine kinases in a patient with carcinoma of the colon

Three electrophoretically atypical creatine kinase (CK) bands were found simultaneously in the serum of a patient with terminal carcinoma of the colon. The atypical CKs were found at the cathodal side of CK-MB (CK-X1), at a sample application point (CK-X2), and at the anodal side of CK-MM (CK-X3) on agar plates. Our data seemed to suggest that CK-X1 was predominantly a CK-IgA/β-lipoprotein complex, CK-X3 was a CK-IgG complex, and CK-X2 was macromolecular, different from both CK-X1 and CK-X3. The light chains of IgA and IgG were both of the λ type. All native isoenzymes comprising these atypical CKs were identified as MM type. There was no change of the electrophoretic pattern of serum CK isoenzymes throughout the course of the study.