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PubMed1980

Immunology and biochemistry of the Regan isoenzyme.

The Regan isoenzyme is a placental-type alkaline phosphatase that is expressed in a number of human tumors, particularly in gonadal and urologic cancers. Attention is given to the unique gene that codes for placental alkaline phosphatase and the similarities and differences in the tumor and placental gene products. The separation and identification of individual organ-specific isoenzymes is accomplished by a variety of biochemical, immunologic, and electrophoretic techniques and the correlation of the Regan isoenzyme, non-Regan isoenzyme, and Nagao isoenzyme, and the Kasahara isoenzyme is made with their developmental counterparts. The L-leucine-sensitivity phenotypes of placental and tumor alkaline phosphatases and the non-Regan early placental type alkaline phosphatases appear to be developmental phase-specific. Oncotrophoblast gene expression has been investigated with monophenotypic cell culture lines as a consequence of modulation by prednisolone and hyperosmolarity. Finally, general discussion of oncodevelopmental proteins as tumor markers precedes a current opinion of Regan isoenzyme as a tumor marker. Evidence now points to seminoma as a consistent producer of Regan isoenzyme although much more work will be required to establish its clinical utility.

Alkaline Phosphatase↗
PubMed1991

Profiles of creatine kinase isoenzyme compositions in single muscle fibres of different types.

Creatine kinase (CK) isoenzyme compositions of different types of single muscle fibres dissected from soleus (SOL) and extensor digitorum longus (EDL) muscles from rats were examined. CK isoenzymes were separated into cytoplasmic (CK-MM, CK-MB, CK-BB) (muscle, brain and hybrid types, respectively) and mitochondrial (m-CK) isoenzymes. Total CK and CK-MM activities showed the highest activities in fast-twitch glycolytic fibres (FG), lower in fast-twitch oxidative glycolytic (FOG) and the lowest in slow-twitch oxidative (SO) fibres. Conversely, the activity of m-CK was highest in SO, lowest in FG and intermediate in FOG fibres. The activity of CK-MB was highest in SO and lower in FOG and FG fibres. However, the activities of total CK and CK isoenzymes in a single muscle fibre type were not distinguishable from those of another type, and the profiles of CK isoenzyme compositions from the same type of single muscle fibres overlapped over a considerable range. The relationships between the four CK isoenzymes activities in single muscle fibres of different types were not similar. These results suggest that CK isoenzymes of single muscle fibres of different types play different roles in intracellular energy metabolism. Therefore, it is supposed that the CK isoenzyme compositions of single muscle fibres are suitable for their contractive and metabolic properties.

Animals↗
PubMed2001

Isoenzyme profile of glutathione S-transferases in human kidney.

Novel glutathione S-transferase (GST) isoenzymes, which do not bind to the glutathione (GSH) affinity column, were recently identified in dog kidney and dog renal cell lines. In humans, similar affinity flow-through GST has been previously found only in the urinary bladder. To ascertain whether these affinity flow-through GST isoenzymes also exist in the human kidney, we separated GST isoenzymes from five kidney samples on the basis of their affinity to GSH affinity resin. GSTs were further purified by anion exchange chromatography and chromatofocusing and characterized with specific substrates. Our results show that the human kidney has both affinity flow-through GST isoenzymes and those which bind tightly to the GSH affinity column. Purification of affinity-bound GST resulted in a rich profile of different isoenzymes with balanced expression of both anionic and cationic forms. Affinity flow-through GST was represented by one isoenzyme (pI-7.9) in all kidney samples tested, but one kidney specimen also contained another GST isoenzyme (pI-7.0). Our results for the first time show the presence of GST isoenzymes that do not bind to GSH-affinity resin in the human kidney. Although the assessment of similarity between the human kidney and urinary bladder affinity flow-through GST requires further elucidation, it can be speculated that these particular GSTs may play an important role in providing protection against the common carcinogens.

Chromatography, Affinity↗
PubMed1989

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.

Catalysis↗
PubMed1991

Isolation and characterization of a homogeneous isoenzyme of wheat germ acid phosphatase.

An acid phosphatase (orthophosphoric monoester phosphohydrolase, acid optimum; EC 3.1.3.2) isoenzyme from wheat germ was purified 7000-fold to homogeneity. The effect of wheat germ sources and their relationship to the isoenzyme content and purification behavior of acid phosphatases was investigated. Extensive information about the purification and stabilization of the enzyme is provided. The instability of isoenzymes in the latter stages of purification appeared to be the result of surface inactivation together with a sensitivity to dilution that could be partially offset by addition of Triton X-100 during chromatographic procedures. Added sulfhydryl protecting reagents had no effect on activity or stability, which was greatest in the pH range 4-7. The purified isoenzyme was homogeneous by polyacrylamide gel electrophoresis and exhibited the highest specific activity and turnover number reported for any acid phosphatase. The molecular weights of the pure isoenzyme and of related isoenzymes from wheat germ were found to be identical (58,000). The pure isoenzyme contained a single polypeptide chain and had a negligible carbohydrate content. The amino acid composition was determined. Of the various reasons that were considered to explain isoenzyme occurrence, a genetic basis was considered most likely. The enzyme was found to exhibit substrate inhibition with some substrates below pH 6, while above pH 8 it exhibited downwardly curving Lineweaver-Burk plots of the type that are generally described as "substrate activation". The observation of a phosphotransferase activity was consistent with the formation of a covalent phosphoenzyme intermediate, while inactivation by diethyl pyrocarbonate was consistent with the presence of an active site histidine.

Acid Phosphatase↗
PubMed1990

Differences in the kinetic properties and sensitivity to inhibitors of human placental, erythrocyte, and major hepatic aldehyde dehydrogenase isoenzymes.

(i) The characteristics of the major human hepatic isoenzymes of aldehyde dehydrogenase (ALDH), ALDH I and ALDH II, were compared with the ALDH activities found in human placenta and erythrocytes, (ii) In human liver biopsies, the Km of ALDH I was approximately 7 mumol/L whereas it was 32 mumol/L for ALDH II. The Vmax for ALDH I was 2-3 times greater than the ALDH II Vmax. Human liver ALDH I and II also differed in their sensitivity in inhibitors. Namely, ALDH I was less sensitive to disulfiram than the ALDH II isoenzyme. (iii) ALDH activity in human placenta and erythrocytes was much lower than in liver tissue. Kinetic data showed that placental ALDH isoenzyme had a high Km (in the millimolar range) and increased its activity raising the pH from 7.4 to 8.8, more than the hepatic ALDH I and ALDH II isoenzymes did. Erythrocyte ALDH activity presented a dual component; the smaller one was characterized by a low Km (micromolar range), whereas most of the ALDH activity showed a high Km (millimolar range). (iv) Placental ALDH was resistant to nitrefazole inhibition and was inhibited by disulfiram in a manner similar to the hepatic ALDH I isoenzyme; erythrocyte ALDH was more sensitive to the inhibitory action of disulfiram and nitrefazole. (v) It is concluded that erythrocyte and placental ALDH isoenzymes are different from the hepatic ALDH I and ALDH II forms. It is also suggested that placental and erythrocyte ALDH isoenzymes are different high-Km isoenzymes.

Acetaldehyde↗
PubMed1994

Examination of glutathione S-transferase isoenzyme profiles in human liver using high-performance affinity chromatography.

A method for the examination of the glutathione S-transferase isoenzyme profiles in human liver using a new HPLC affinity support is described. Liver cytosol was injected directly onto an HPLC column (5 x 0.46 cm) containing a support with a covalently bound affinity ligand (S-octylglutathione) specific for the isoenzymes. Contaminating cytosolic proteins were removed in a washing step. The isoenzymes were eluted with a linear gradient of a different affinity ligand in the mobile phase. Coinciding with the affinity ligand gradient, a salt gradient (0-200 mM sodium chloride) was applied. In this manner the isoenzymes were fractionated into the enzymatically active homodimers and heterodimers. The classes of the affinity fractionated isoenzymes were determined by SDS-PAGE and ELISA while the subunit content was determined by reversed-phase chromatography. For one liver three Alpha class isoenzyme subunits, forming three heterodimers and two homodimers, were detected. Five livers were examined, and the homodimer A1-1 was found to be the predominant glutathione S-transferase isoenzyme. Minor amounts of Pi and Mu class isoenzymes were also detected. This non-denaturing high-performance affinity chromatography method reduced analysis time by a factor of ten when compared to other affinity analysis methods for the glutathione S-transferases.

Chromatography, Affinity↗
PubMed1987

Isolation and partial characterization of the hexokinase isoenzymes from pathogenic and non-pathogenic strains of Entamoeba histolytica.

Isoenzyme electrophoretic patterns (zymodemes) are increasingly used to distinguish between pathogenic and non-pathogenic strains of Entamoeba histolytica. Isolates of E. histolytica from asymptomatic and symptomatic cases have been shown to differ in the electrophoretic mobility of their hexokinase and phosphoglucomutase isoenzymes. The hexokinase isoenzymes from a non-pathogenic strain and from a pathogenic strain of E. histolytica were purified by fast protein liquid chromatography in several steps, which included a separation by size, chromatofocusing, and anion exchange chromatography. The isoenzymes differed in their isoelectric points, which ranged from pH 4.8-5.4, but had very similar kinetic properties and almost identical apparent molecular weights (48,000) in sodium dodecyl sulfate polyacrylamide gels, as well as on gel filtration columns. Comparison of tryptic peptide analysis of each of the isoenzymes indicated considerable homology between the non-pathogenic and pathogenic forms. Antibodies produced against each of the two pathogenic hexokinase isoenzymes inhibited their enzymatic activity. The antibodies also inhibited the activity of the isoenzymes of the non-pathogenic strain. Our findings suggest that the isoenzymes have structural similarities, and that the pathogenic ones differ from the non-pathogenic ones in their electromobility due to post-translational modifications.

Animals↗
PubMed1985

Expression of a monocyte-specific esterase isoenzyme in cases of acute myeloid leukemias.

The carboxylic esterase (E.C. 3.1.1.1) isoenzymes from cases of acute myeloid leukemias were separated by analytical isoelectric focusing on horizontal thin-layer gels. One isoenzyme consisting of one or two components (bands) could be completely and selectively inhibited by addition of 40 mM sodium fluoride (NaF) to the staining bath. The 105 cases were classified into the groups M1-M6 according to the FAB proposals. The NaF-sensitive isoenzyme was not detected in cases of FAB groups M1/2 (acute myeloblastic leukemia without or with maturation), group M3 (acute promyelocytic leukemia) or group M6 (erythroleukemia). Thirty-one out of 33 cases in the FAB group M4 (acute myelomonocytic leukemia) and 9/9 cases in FAB group M5 (acute monocytic leukemia) expressed the NaF-sensitive isoenzyme. The NaF-sensitive isoenzyme was found at different staining intensities; all M5 cases showed the isoenzyme at strong or very strong intensity, whereas most of the M4 cases displayed the isoenzyme at weak, medium or strong staining intensity. The data presented are further evidence that the presence of the NaF-sensitive esterase isoenzyme indicates monocytic involvement or differentiation in cases of myeloid leukemias. The easy and fast to perform method of isoelectric focusing can be used to distinguish the monocytic variants among the acute myeloid leukemias and can supplement the morphological analysis of these cases.

Carboxylic Ester Hydrolases↗
PubMed1996

Selective translocation of non-conventional protein kinase C isoenzymes by gonadotropin-releasing hormone (GnRH) in the gonadotrope-derived alpha T3-1 cell line.

Gonadotropin-releasing hormone acts via G-protein coupled receptors to stimulate polyphosphoinositide-specific phospholipase C (PIC) with consequent elevation of cytosolic Ca2+ and activation of protein kinase C (PKC). Whereas Ca2+ is known to mediate stimulation of exocytotic gonadotropin release by GnRH, the identity of the PKC isoenzymes activated by GnRH and their physiological role in gonadotropes are poorly understood. In many systems translocation of PKC (from cytosolic to particulate fractions of cellular homogenates) has been taken as evidence of hormonal activation of PKC and down regulation of PKC (by prolonged treatment with PKC-activating phorbol esters) has been used extensively to investigate the role of PKC in hormone action. Here we have assessed the influence of GnRH and phorbol esters on translocation and down regulation of PKC isoenzymes identified by Western blotting with isoenzyme-specific antibodies in alpha T3-1 cells (a gonadotrope-derived cell line). These cells were found to posses PKCs alpha, epsilon and zeta but not beta, delta (present in rat pituitaries) or gamma (present in rat brains). In short-term stimulations (10 min), the PKC-activating phorbol esters, PMA and PDBu, caused concentration-dependent increases in the proportion of PKC alpha and PKC epsilon recovered from the particulate fraction of alpha T3-1 cells, but did not induce measurable translocation of PKC zeta. The inactive phorbol ester 4 alpha PDBu did not cause translocation of any of these isoenzymes. GnRH treatment induced a concentration-dependent increase in the proportion of particulate PKC epsilon and PKC zeta but had no measurable effect on PKC alpha translocation. In longer incubations (6-48 h) GnRH failed to cause measurable down-regulation of these isoenzymes whereas PMA treatment led to a clear down regulation of PKCs alpha and epsilon (albeit with different kinetics). The data demonstrate the differential activation and down regulation of PKC isoenzymes by GnRH versus PMA, which are clearly pertinent to the design of experiments intended to address the role of such isoenzymes in GnRH action. Moreover, they provide the first demonstration of hormonal regulation of an atypical PKC isoenzyme (PKC zeta) in pituitary cells.

Biological Transport↗
PubMed1978

Distribution and homologies of subunits in the LDH isoenzyme pattern of urodeles.

1. The subunit distribution and homologies of LDH isoenzymes are investigated in the species Triturus vulgaris, cristatus, alpestris and helveticus, and Ambystoma mexicanum by means of immunoprecipitation and starch gel electrophoresis. 2. Fresh tissue extracts contain the following patterns: (a) Trit. vulgaris--Two isoenzymes: the M4 and H4 tetramers. No hybrid formation is observed between H and M subunits. (b) Trit. cristatus--Two isoenzymes: the M4 and H4 tetramers. Occasional hybrid formation between H and M subunits takes place. (c) Trit. alpestris--Six isoenzymes: the M4, H4, H3H', H2H'2, HH'3 and H'4 tetramers. No hybrid formation between the M and the H and H' subunits is detected. (d) Trit. helveticus--Six isoenzymes: the M4, H4, H3H', H2H'2, HH'3, and H'4 tetramers; the H' subunit is more positively charged than the M subunit, which leads to pattern reversal in heart and skeletal muscle tissues. No hybrid formation between the M and the H and H' subunits is observed. (e) Ambyst. mex.--Eleven isoenzymes in heart, eye and brain, six isoenzymes in all other tissues tested. The presence of two M subunits, which form hybrids with the H subunit in a restricted way, is suggested. 3. In tissue extracts of the tested species the tendency of all present LDH subunits to form hybrids with each other without restriction is increased after prolonged storage at 2 degrees C. 4. The most acidic of the major isoenzymes (LDH1) in Trit. vulgaris, cristatus and helveticus tends to split into a series of sub-bands which migrate faster to the anode than the original main band.

Ambystoma↗
PubMed1984

Immunohistochemical localization of creatine kinase BB-isoenzyme and tubulin in gerbil brain.

Cellular distribution of creatine kinase BB-isoenzyme and tubulin was investigated immunohistochemically with tissue sections from various areas of the gerbil brain. Both the reaction for creatine kinase BB-isoenzyme and tubulin visualized nerve cell bodies, dendrites and axons. While the reaction for tubulin was similar between nerve cell bodies and their dendrites, the reaction for creatine kinase BB-isoenzyme tended to be more intense in dendrites. While the reaction for tubulin could be seen in nerve cell bodies of different sizes, the reaction for creatine kinase BB-isoenzyme was more intense in larger neurons. Both the reaction for creatine kinase BB-isoenzyme and tubulin visualized astrocytic cytoplasm but only in the corpus callosum and in the white matter of limited areas. Visualization of dendrites to their peripheries with the reaction for creatine kinase BB-isoenzyme may indicate participation of this isoenzyme in maintainance of high energy-phosphates locally within dendrites. A combination of the immunohistochemical procedure for creatine kinase BB-isoenzyme and tubulin may be very useful for demonstration of various pathophysiological states of the central nervous system which affect dendrites.

Animals↗
PubMed2002

Kinetics of bromodichloromethane metabolism by cytochrome P450 isoenzymes in human liver microsomes.

The kinetic constants for the metabolism of bromodichloromethane (BDCM) by three cytochrome P450 (CYP) isoenzymes have been measured in human liver microsomes. The three CYP isoenzymes, CYP2E1, CYP1A2 and CYP3A4, have been identified previously as important in the metabolism of this compound. To measure the constants for each isoenzyme, enzyme-specific inhibitory antibodies were used to block the activities for two of the three isoenzymes. CYP2E1 was found to have the lowest K(m), 2.9 microM, and the highest catalytic activity, k(cat). The K(m) for the other isoenzymes, CYP1A2 and CYP3A4, were about 60 microM with lower values of k(cat). Apparent kinetic constants obtained from two microsomal samples that were not inhibited were consistent with these results. In addition, 11 human microsome samples characterized for 10 CYP activities were correlated with the metabolism of 9.7 microM BDCM by each sample; statistical analysis showed a correlation with CYP2E1 activity only. This result is consistent with the finding that CYP2E1 is the only isoenzyme with a K(m) lower than the BDCM concentration used. The kinetic constants obtained from the inhibited microsomes were compared to similar results from recombinant human isoenzyme preparations containing only one CYP isoenzyme. The results for CYP2E1 were very similar, while the results for CYP1A2 were somewhat less similar and there was a substantial divergence for CYP3A4 in the two systems. Possible reasons for these differences are differing levels of CYP reductase and/or differing makeup of the membrane lipid environment for the CYPs. Because of the low levels of BDCM exposure from drinking water, it appears likely that CYP2E1 will dominate hepatic CYP-mediated BDCM metabolism in humans.

Carcinogens↗
PubMed1997

Method for the simultaneous assay of initial velocities of lactate dehydrogenase isoenzymes following gel electrophoresis.

A method was developed for determining initial isoenzyme velocity in a model based on lactate dehydrogenase (LD, EC. 1.1.1.27). First, purified LD was fractionated by polyacrylamide disc gel electrophoresis. Following electrophoresis, the gel was cut into slices, each containing an isoenzyme, and the isoenzymes were subsequently extracted by slice homogenization into a buffer and their activity was measured by the reverse (pyruvate to lactate) LD reaction. The volume of LD solution positively correlated with the activity of all five isoenzymes. Isoenzyme recovery in all cases exceeded 80%, compared to isoenzyme activity before electrophoresis. The lower limit of detection was 6.5 mU/microliter for LD 1, as measured using this method. The kinetic parameters of isoenzymes following recovery were similar to literature values. The results indicate that the method described here represents an improvement in the measurement of initial LD isoenzyme velocity.

Acrylamide↗
PubMed1985

Serum amylase isoenzyme alterations in acute abdominal conditions.

To determine the accuracy of the serum amylase in identifying a pancreatic source, amylase isoenzymes were determined prospectively in 65 patients initially evaluated with a complaint of abdominal pain and associated hyperamylasemia. Isoenzyme patterns were demonstrated by an electrophoretic technique, and the results were correlated with clinical diagnoses. Patients were divided into two diagnostic groups. Group I consisted of 42 patients with clinical findings suggesting pancreatitis. P-type isoenzymes were normal or elevated in 31 of these patients (74%), and s-type isoenzymes were normal or elevated in 11 (26%). Group 2 consisted of 23 patients with abdominal pain attributed to causes other than pancreatitis. Four patients (17%) had elevation of p-type isoenzymes, and 19 patients (83%) had predominantly s-type patterns. We conclude that amylase isoenzymes cannot determine unequivocally the cause of hyperamylasemia, but they can enhance the diagnostic specificity of the serum amylase. Elevated serum amylase with a predominant p-type pattern suggests pancreatic disease; elevation of s-type isoenzymes suggests but is not conclusive for, diagnoses other than pancreatitis. Hyperamylasemia with a normal isoenzyme pattern occurred in a few patients in both groups, and it was nondiagnostic.

Abdomen↗
PubMed1999

Different changes in the cytosole creatine kinase isoenzymes from heart of adult and old rats during stress.

We have shown that immobilization stress causes changes in the spectrum of the heart's cytosole creatine kinase isoenzymes. In myocardium adult immobilized rats, MM isoenzyme is decreased and MB and BB isoenzymes are increased compared to intact animals. The pattern of isoenzyme response in old and adult rats was different. Immobilization of old rat caused an increase of MM and a decrease of MB isoenzyme in the heart's creatine kinase isoenzyme spectrum. We suggest that this change in the creatine kinase isoenzyme spectrum in the myocardium of adult rats plays an adaptive role, increasing the effectiveness of its performance during stress. In old rats, apparent changes in the creatine kinase isoenzyme spectrum promote a disturbance of myocardial energy metabolism.

Aging↗
PubMed2000

Isoenzymes of glutathione S-transferase from the mosquito Anopheles dirus species B: the purification, partial characterization and interaction with various insecticides.

Previously we have purified and characterized a major glutathione S-transferase (GST) activity, GST-4a, from the Thai mosquito Anopheles dirus B, a model mosquito for study of anopheline malaria vectors [Prapanthadara, L. Koottathep, S., Promtet, N., Hemingway, J. and Ketterman, A.J. (1996) Insect Biochem. Mol. Biol. 26:3, 277-285]. In this report we have purified an isoenzyme, GST-4c, which has the greatest DDT-dehydrochlorinase activity. Three additional isoenzymes, GST-4b, GST-5 and GST-6, were also partially purified and characterized for comparison. All of the Anopheles GST isoenzymes preferred 1-chloro-2,4-dinitrobenzene (CDNB) as an electrophilic substrate. In kinetic studies with CDNB as an electrophilic substrate, the V(max) of GST-4c was 24.38 micromole/min/mg which was seven-fold less than GST-4a. The two isoenzymes also possessed different K(m)s for CDNB and glutathione. Despite being only partially pure GST-4b had nearly a four-fold greater V(max) for CDNB than GST-4c. In contrast, GST-4c possessed the greatest DDT-dehydrochlorinase specific activity among the purified insect GST isoenzymes and no activity was detected for GST-5. Seven putative GST substrates used in this study were not utilized by An. dirus GSTs, although they were capable of inhibiting CDNB conjugating activity to different extents for the different isoenzymes. Bromosulfophthalein and ethacrynic acid were the most potent inhibitors. The inhibition studies demonstrate different degrees of interaction of the An. dirus isoenzymes with various insecticides. The GSTs were inhibited more readily by organochlorines and pyrethroids than by the phosphorothioates and carbamate. In a comparison between An. dirus and previous data from An. gambiae the two anopheline species possess a similar pattern of GST isoenzymes although the individual enzymes differ significantly at the functional level. The available data suggests there may be a minimum of three GST classes in anopheline insects.

Animals↗
PubMed2000

Evidence for the existence of two soluble NAD(+) kinase isoenzymes in Euglena gracilis Z.

Two soluble NAD(+) kinase isoenzymes (isoenzymes 1 and 2) from Euglena gracilis were separated by preparative electrophoresis and characterized. They display several similar properties: both have an identical apparent molecular weight of 68 kDa and their activities are independent on calmodulin, insensitive to 2-mercaptoethanol but inhibited by p-chloromercurybenzoate, 5, 5'-dithiobis(2-nitrobenzoate) and, surprisingly, by low dithiothreitol concentrations, the inhibition by dithiothreitol being irreversible for isoenzyme 1 but reversible for isoenzyme 2. Nevertheless, the two isoenzymes mainly differ by their specificities towards triphosphate nucleotides and their catalytic mechanisms. Isoenzyme 1 is as active in the presence of ATP as of GTP and acts by a ping-pong mechanism with a k(M) for NAD(+) of 0.26 mM and a k(M) for low MgATP(2-)concentrations of 0.03 mM. Isoenzyme 2 is three-fold more active in the presence of GTP than of ATP and operates by a sequential mechanism with k(M)s for NAD(+) and MgGTP(2-) of 1.03 and 0.20 mM, respectively. This study shows the evidence for the existence of two structurally similar but catalytically different NAD(+) kinase isoenzymes in E. gracilis. One resembles the enzyme previously described in bacteria. The other displays a catalytic mechanism identical to that of NAD(+) kinase from other organisms but remains unique among all the NAD(+) kinases studied to-date regarding its specificity towards GTP.

Adenosine Triphosphate↗