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PubMed1977

Serum alkaline phosphatase isoenzymes in epileptic children receving anticonvulsant drugs.

In 40 epileptic children on long-term anticonvulsant treatment, serum alkaline phosphatase (AP) isoenzymes were separated semiquantitatively using a combination of L-phenylalanine inhibition and heat inactivation. Though mean total serum AP activity was significantly increased compared to age matched controls, only 4 individual values exceeded the upper limit (mean + 2SD) of the reference sample. In epileptics the mean activity of the heat-sensitive non L-phenylalanine sensitive AP fraction (non-LPSAP) was significantly increased (P less than 0.01) and the mean Q-value (i.e. percentage ratio of heat-stable non-LPSAP/non-LPSAP) was significantly decreased (P less than 0.05), thus indicating an enhancement of the bone isoenzymes during anticonvulsant treatment. In 4 patients the isoenzyme pattern was abnormal although total serum AP activity was normal and in 3 of them the deviation indicated enhanced bone isoenzyme. The data provide evidence that in anticonvulsant treated children the bone isoenzyme, rather than hepatobiliary isoenzyme fraction, may be increased even when total serum AP activity is normal. Thus, semiquantitative separation of serum AP isoenzymes may be a helpful guide as to whether or not an eplieptic child should be given vitamin D.

Adolescent↗

PubMed1983

Analysis of isoenzyme patterns of acid phosphatase in acute leukemias.

The status of acid phosphatase isoenzymes was evaluated in cells of patients with acute lymphocytic leukemias or lymphomas by analytical isoelectric focusing in polyacrylamide gels (IEF) on horizontal thin-layer slabs. The isoenzyme patterns were correlated with routine immunological cell surface markers and the relationship of enzyme activity to specific immunological subclasses of ALL is discussed. By isoelectric focusing up to five isoenzyme groups (I-V) containing several isoenzyme were observed. No leukemia specific or additional isoenzyme could be demonstrated. This biochemical characterization showed a marked heterogeneity within two major immunologic subgroups indicating that various differentiation stages of cell maturation could be involved in cALL and T-ALL. According to their degree of maturation along T-cell differentiation axis the leukemic cells displayed no enzyme activity, weak isoenzyme bands or the incomplete or complete isoenzyme pattern seen with normal lymphocytes from human tonsils which were used as controls. The investigation of specific enzymatic patterns can lead to a further definition of subsets of acute leukemias and give insight into lymphopoietic differentiation.

Acid Phosphatase↗

PubMed1976

The further heterogeneity of creatine kinase. Presence of isoenzymes of cathodic mobility in rat tissues.

Supernatant fractions from rat tissue homogenates were examined for creatine kinase (EC 2.7.3.2) isoenzymes by agarose gel electrophoresis at pH 8.6. The distribution of the various isoenzymes, and their relative contributions to the total tissue creatine kinase, is stated. A previously unreported isoenzyme associated with brain and kidney preparations is described. It is a minor contributor to brain creatine-kinase activity but is the major isoenzyme for kidney. The presence of an additional isoenzyme for cardiac tissue was confirmed; this isoenzyme exhibits similar levels of activity to that of the MB isoenzyme.

Animals↗

PubMed1987

Studies on the ontogenesis of the different isoenzymes of Na+, K+-ATPase in rat brain in vivo and in vitro in relation to their regulation and cellular localisation.

Na+,K+ ATPase isoenzyme activities (alpha(+)-high ouabain affinity; alpha low ouabain affinity) were investigated in developing rat brain in vivo and in whole rat brain reaggregating cultures in vitro. The perinatal profile of the two isoenzyme forms in vivo revealed that, although alpha activity predominates in immature (P14.5-P16) brain, the activity alpha(+) form increases more increases more rapidly such that it is predominant at birth in both cerebellum and forebrain. No regional variation in the proportional activities of the two isoenzyme forms was seen perinatally to explain the previously reported, differential sensitivity of the cerebellar alpha isoenzyme to neonatally induced hypothyroidism. Whole rat brain reaggregating cultures seeded at P16 show a normal development of Na+,K+ ATPase isoenzyme activity if grown for 14 days in a serum supplemented medium (S+). Cultivation of whole rat brain reaggregates in serum deprived medium (S-) leads to a retarded development of alpha isoenzyme activity possibly due to the absence of T3 from the medium. Hormonally-induced changes in the development of the brain Na+, K+-ATPase isoenzymes are discussed in relation to their possible function and cellular localization.

Animals↗

PubMed1988

Antibodies to a synthetic peptide that react specifically with a common surface region on two hydrocarbon-inducible isoenzymes of cytochrome P-450 in the rat.

An antibody that reacts with two hydrocarbon-inducible isoenzymes of rat cytochrome P-450 (c and d) in the rat was produced by immunising with a synthetic peptide, Leu-Ile-Ser-Lys-Phe-Gln-Lys-Leu-Met, which has the same primary structure as that of a region of both of these isoenzymes. There was no crossreactivity with hydrocarbon-inducible isoenzymes in liver microsomes from rabbit, mouse or in man. Nor was there any crossreactivity detected with liver microsomes from uninduced rats, or rats induced with phenobarbitone or isonicotinic acid hydrazide. This is consistent with the primary structure of these isoenzymes in the regions aligned with amino acids 174-182 (the immunising peptide) in rat isoenzyme c and demonstrates the ability to produce antibodies of defined specificity against isoenzymes of cytochrome P-450 by using synthetic peptide. As the antibody preparation is able to bind to isoenzymes c and d in their native conformations, either as partially purified enzymes, or in microsomes, it is suggested that this region is present on the surface of these cytochromes P-450.

Animals↗

PubMed1979

Relationship of thyroid status and serum N-acetyl-beta-glucosaminidase isoenzyme activities in humans.

Serum from 28 hyperthyroid, hypothyroid, and euthyroid pre- or early puberty females was examined for N-acetyl-beta-glucosaminidase (HEX) activity. Total, isoenzyme A (labile), and isoenzyme B (stable) levels were determined for this enzyme. A high degree of correlation (r = 0.76; p less than 0.001) exists between total hexosaminidase activity and thyroid hormone levels (as reflected by the Free Thyroxine Index). Examining each isoenzyme individually, A is selectively enhanced (r = 0.84; p less than 0.0005) whereas B displays no significant change regardless of thyroid activity. In hyperthyroid individuals, levels of total hexosaminidase (730 +/- 67 units) (mean +/- S.D.) and isoenzyme A (516 +/- 46) were significantly higher than levels found in either the hypothyroid (total: 547 +/- 30; isoenzyme A: 352 +/- 31) or euthyroid (total: 620 +/- 81; isoenzyme A: 423 +/- 45) groups. However, no change was observed in levels of isoenzyme B among hypothyroid (195 +/- 19), euthyroid (197 +/- 43) and hyperthyroid (215 +/- 32) groups. These data substantiate our earlier findings in the rat, wherein thyroxine administration evoked a similar response in the liver. They are of particular interest in light of the deficiency of HEX A in variants of GM2 gangliosidosis.

Acetylglucosaminidase↗

PubMed1986

Affinity electrophoresis of human serum alkaline phosphatase isoenzymes in agarose gel containing lectin.

Separation of alkaline phosphatase isoenzymes using affinity electrophoresis in agarose gel containing lectin is described. The bone and biliary isoenzymes precipitate during electrophoresis and are clearly separated from the liver isoenzyme. The liver, intestinal and placental alkaline phosphatases are essentially not affected by the lectin. The migration distances of the precipitating bone and biliary fractions vary with their alkaline phosphatase activity. The bone isoenzyme is more heterogeneous than the biliary isoenzyme with respect to interaction with lectin forming both insoluble and soluble complexes. Affinity electrophoresis in agarose gel containing lectin can be used for quantitation by densitometry of liver and bone isoenzymes in sera containing only these two fractions but must be combined with conventional electrophoresis, preferably in agar gel, if biliary, intestinal, or placental isoenzymes are also present.

Alkaline Phosphatase↗

PubMed1986

Sandwich electroimmunofixation (SEIF) for the assessment of isoenzyme specificities of immunoglobulins isolated from enzyme-immunoglobulin complexes.

Sandwich electroimmunofixation (SEIF) was used to determine immunologic specificity for isoenzymes of immunoglobulins isolated from enzyme-immunoglobulin complexes. After electrophoretic separation of isoenzymes, isoenzyme-specific human immunoglobulins obtained from complexes and antihuman immunoglobulin antibodies were applied to the supporting medium and allowed to react. Complexes of isoenzyme-immunoglobulins-anti-immunoglobulin antibodies formed immunoprecipitates and were fixed in the supporting medium. After washout of the unreacted enzymes and proteins with buffer, the immunoprecipitates were stained. A comparison with control enzymograms allowed for a determination as to whether the immunoglobulins reacted with specific isoenzymes. When an immunologic reaction with more than 2 isoenzymes occurred, the specificity was quantitated by densitometry. SEIF, used to examine immunoglobulins isolated from aspartate aminotransferase-, lactate dehydrogenase-, alkaline phosphatase- and amylase-immunoglobulin complexes, was found to be a rapid and reliable technique. This approach showed that immunoglobulins differ in their specificities for various isoenzymes.

Alkaline Phosphatase↗

PubMed1985

Isoenzyme studies in human leukemia-lymphoma cell lines--III. beta-Hexosaminidase (E.C. 3.2.1.30).

The hexosaminidase (beta-N-acetylglucosaminidase) isoenzyme profiles of 86 human hematopoietic cell lines grown actively in suspension culture were analysed by isoelectric focusing and by conventional disc electrophoresis on horizontal thin-layer polyacrylamide gels. A maximum of three hexosaminidase (Hex) isoenzymes (A = anodic, I = intermediate, B = basic) could be demonstrated. The immunological phenotyping of 74 leukemia-lymphoma derived cell lines had led to a categorization into four groups with a subclassification of the T- and B-cell lines into several stages of differentiation: 26 T-cell, 34 B-cell, 6 myelomonocytic and 8 Non-T, Non-B cell leukemia-lymphoma cell lines. Twelve so-called 'normal' B-lymphoblastoid cell lines were also available. Distinct isoenzyme profiles were seen in the different stages of differentiation in the T- and B-leukemia-lymphoma cell lines. Among the 12 normal B-lymphoblastoid cell lines heterogeneity in the isoenzymatic phenotypes was detected. Hex isoenzyme expression in normal and neoplastic lymphoid cell lines represents hypothetically sequential stages of T- and B-cell differentiation. Myelomonocytic cell lines displayed strongly stained bands of all three isoenzymes. Heterogeneity was seen in the group of Non-T, Non-B cell lines. Four out of 5 pre B-cell lines and 4 out of 4 Non-T, Non-B cell lines which are comparable to cases of pre B- and common ALL revealed a high Hex I/Hex A ratio in terms of intensity of the isoenzyme bands. The analysis of Hex isoenzymes is useful for characterizing lymphoid and myeloid populations (both normal and malignant, cultured or fresh), particularly with regard to their stage of differentiation. But this enzyme should be part of a multiple enzyme study where the information obtained is complementary. In turn, enzyme marker analysis should be included in the multiple marker analysis for an optimized characterization of leukemic cells.

Antigens, Surface↗

PubMed1987

Purification procedure and N-terminal amino acid sequence of yeast malate dehydrogenase isoenzymes.

A method has been devised for the rapid isolation of malate dehydrogenase isoenzymes. First, anionic proteins were precipitated with polyethyleneimine, whilst hydrophobic malate dehydrogenase remained in the supernatant fluid. Secondly, the supernatant was 30% saturated with ammonium sulfate and the two isoenzymes were separated by hydrophobic phenyl-Sepharose CL-4B chromatography. For further purification the enzymes were chromatofocused, and polybuffer was removed by hydrophobic chromatography. Affinity chromatography with blue Sepharose CL-6B [1] was used as final purification step. The purified isoenzymes were homogeneous as shown by isoelectric focusing and could be used for N-terminal sequencing. 34 amino acid residues could be identified for the cytoplasmic isoenzyme and 56 amino acid residues for the mitochondrial isoenzyme. Although there are regions of strong homology between both isoenzymes, the sequence differences clearly showed support that both isoenzymes are coded by different genes. Sequence comparison clearly indicated that the N-terminus of the cytoplasmic enzyme extended that of the mitochondrial enzyme by 12 amino acid residues. The amino acid sequence of the extending sequence resembled that of leading sequences known for enzymes which are transported into the mitochondria. The assumed leading sequence is discussed with respect to its possible role in glucose inactivation.

Amino Acid Sequence↗

PubMed1984

Serum galactosyltransferase isoenzymes as markers for solid tumours in humans.

High resolution agarose isoelectric focusing was used to compare the galactosyltransferase isoenzyme patterns of serum from 9 healthy controls with those from 38 patients with either breast, lung, ovarian, stomach or colonic cancer. At least 12 peaks of enzyme activity were found in every sample, the healthy controls having major forms with isoelectric points of 4.74, 4.87, 4.96, 5.16 and 5.23. Thirty patients (79%) had elevated levels of at least one isoenzyme and 23 (61%) had at least 3 isoenzymes elevated compared to only 10 (26%) patients who had elevated total serum galactosyltransferase activity. The isoenzymes which were most often elevated in the cancer patient group had isoelectric points of 4.93, 5.16 and 4.61. One isoenzyme with an isoelectric point of 4.43 was preferentially elevated in patients with ovarian cancer. Those isoenzymes containing little or no sialic acid were rarely elevated in cancer patients. Although no cancer-associated isoenzyme was detected the quantitative differences observed in the cancer patient group were striking.

Female↗

PubMed1996

Activity of creatine kinase MB-isoenzyme in rat serum after heart irradiation and/or farmorubicin (4'-epidoxorubicin) treatment.

Activity of creatine kinase (CK, EC 2.7.3.2) and its CK-MB isoenzyme were evaluated in rat serum after external irradiation of the heart with 20 Gy given as A single dose or 4 x 5 Gy and also after treating the animals with farmorubicin 10 mg/kg in a single dose of 4 x 2.5 mg/kg. Fractionated irradiation or repeated injection of farmorubicin induced high five-fold transient increment of CK-MB isoenzyme activity in rat serum. Combined treatment (4 x 5 Gy heart irradiation, week 1, after 2 days pause 4 x 2.5 mg/kg farmorubicin, week 2) showed increase of MB isoenzyme activity in serum roughly comparable with that measured in animals treated with farmorubicin alone. The behaviour of MB isoenzyme activity after single doses of radiation 1 x 20 Gy or farmorubicin 1 x 10 mg/kg was dissimilar to that after repeated doses and was generally lower. Specially, a high single dose of farmorubicin led at first to suppression of CK-MB isoenzyme activity and then to an increase, however, slower than after fractionated treatment. Generally, elevation of CK-MB isoenzyme activity in rat serum after heart irradiation and farmorubicin injection (scheme independent) was many times higher than the total CK activity increment. Results suggest that estimation of CK-MB isoenzyme activity in serum is more sensitive and definitive evidence of early cardiac damage exerted by gamma-rays and/or farmorubicin than total CK activity.

Animals↗

PubMed1992

Interactions of human alkaline phosphatase isoenzymes with triazine dyes using affinity partitioning, affinity chromatography and difference spectroscopy.

Aqueous two-phase systems consisting of dextran, polyethylene glycol and dye-liganded polyethylene glycol were employed to investigate the affinity partitioning behaviour of isoenzymes of human alkaline phosphatase. Whereas in the system without a dye ligand the partition coefficients of the isoenzymes from human intestine and placenta were identical, the isoenzyme from human liver showed a significantly lower partition coefficient under the same conditions. After addition of dye-liganded polyethylene glycol two groups of dyes possessing substantial affinities to the isoenzymes were found. One, represented by Procion Yellow HE-3G, interacts specifically with the active centre of the isoenzymes. Differences in the affinity of the isoenzymes towards the individual dye ligands are caused only by the carbohydrate content, especially by the terminal sialic acid residues. The other group of dye ligands, represented by Procion Navy MX-RB, binds obviously in a more complex fashion involving other binding sites, which are only present in alkaline phosphatase of human liver. Procion Navy MX-RB was found to function as a suitable affinity ligand for the separation of human liver alkaline phosphatase from the other isoenzymes. Differences in the primary structure of two allelic forms of human placental alkaline phosphatase [(SS) and (F)] are not recognized in aqueous two-phase systems with or without dye-liganded polyethylene glycol.

Alkaline Phosphatase↗

PubMed1986

Differential inhibition of rat and human glutathione S-transferase isoenzymes by plant phenols.

Glutathione S-transferase (GST) isoenzymes isolated from human tissues and rat liver are differentially inhibited by quercetin, alizarin, purpurogallin and ellagic acid. Rat liver GST isoenzymes are far more sensitive to these compounds as compared to the human GST isoenzymes. Among human GST, the anionic isoenzymes containing C type and A' type subunits are inhibited to a greater extent as compared to the cationic isoenzymes containing A and B type subunits. The anionic GST isoenzymes of human erythrocytes and placenta are differentially inhibited by these plant phenols indicating that the placental and erythrocyte isoenzymes may be distinct proteins.

Animals↗

PubMed1982

The development and evaluation of a homogeneous immunoassay for the isoenzymes of aspartate aminotransferase.

Purified isoenzymes of aspartate aminotransferase (AST) from human liver (mitochondrial) and erythrocytes (cytoplasmic) were used to elicit antisera in rabbits. Each antiserum was characterized for titer and specificity. Complexes formed upon addition of each isoenzyme to its specific antiserum were demonstrated to be catalytically inactive. Results obtained when either filtration or centrifugation was used for separating the complexes (heterogeneous assay) were comparable to those obtained when the complexes were not separated (homogeneous assay) from the mixture before assay. A quality control system was designed to monitor specificity in addition to the usual parameters. The precision for the inhibition of cytoplasmic (CV 4.8%) and mitochondrial (CV 3.5%) isoenzymes was within that of the enzymatic assay. Several parametric conditions of the enzyme-antibody reactions were examined, and the assay was adapted for semi-automation. The homogeneous assay was evaluated with a series of pseudo specimens containing known mixtures of pure isoenzymes to determine the extent of recovery (99.8%) for a particular isoenzyme in the presence of varying concentrations of the other isoenzyme. In addition, sera from patients having elevated AST concentrations were examined for isoenzyme contributions to total AST activity. A mean recovery of 96% was obtained for these specimens.

Antibody Specificity↗

PubMed1983

Abnormal electrophoretic mobility of creatine kinase isoenzyme in serum of an otherwise healthy person.

We describe the abnormal electrophoretic mobility of a creatine kinase isoenzyme in the serum of an apparently healthy individual. In agarose gel electrophoresis this isoenzyme migrates more toward the cathode than does the MM isoenzyme. It is more resistant to heat and more strongly inhibited by urea than the normal MM isoenzyme. We performed gel filtration, on a Sephadex G-200 column, of the patient's serum and observed two distinct isoenzymes with different relative molecular masses; a normal isoenzyme of 80,000 daltons and another abnormal one of 240,000 daltons. We performed serum immunoelectrophoresis but did not observe any immune complex formation involving the abnormal isoenzyme.

Adult↗

PubMed1989

Alkaline phosphatase isoenzymes in rheumatic diseases.

Serum alkaline phosphatase isoenzymes were determined quantitatively by electrophoresis on cellulose acetate in 168 patients with rheumatic diseases subgrouped for disease activity. Median values of total alkaline phosphatase and bone isoenzyme activity, as well as frequency of patients showing pathological values, increased gradually and significantly corresponding to disease activity in rheumatoid arthritis and ankylosing spondylitis, from 0% in inactive to 90% in very active forms. Bone isoenzyme was much more sensitive than total alkaline phosphatase in moderate disease activity and was also correlated to the number of involved extravertebral joints and pain in ankylosing spondylitis. No correlation was found with stage or duration of disease, age, sex, and erythrocyte sedimentation rate. Additional to bone isoenzyme, liver isoenzymes were elevated in some patients, but with only a weak correlation with disease activity. The intestinal isoenzymes were always normal. We conclude that quantitative determination of serum alkaline phosphatase bone isoenzyme activity is a major indicator for the assessment of disease activity and therapeutic monitoring in rheumatoid arthritis and ankylosing spondylitis.

Alkaline Phosphatase↗

PubMed2002

Isoenzyme-directed selection and characterization of anti-creatine kinase single chain Fv antibodies from a human phage display library.

Epitopes differing among isoenzymes of creatine kinase (CK) are apparently limited in number and poorly immunogenic in vivo. Especially for the BB-CK isoenzyme, very few monoclonal antibodies (mAb) are available. Here, we use in vitro selection with a synthetic human phage display antibody library and develop isoenzyme competition and peptide panning strategies to obtain human single chain Fv (scFv) antibodies against specific CK isoenzymes. We isolated and characterized seven scFv clones that recognize native as well as denatured cytosolic BB-CK in ELISA, immunoblot, immunofluorescence histochemistry and surface plasmon resonance (SPR) spectroscopy. To a variable but minor degree, they also react with cytosolic MM-CK, but not with mitochondrial CK isoenzymes. Epitope mapping revealed that the scFv antibodies recognize different BB-CK epitopes, including the N-terminus and the isoenzyme-specific box, a highly conserved sequence of unknown function for which no mAb were available so far. With a K(D) of 3.5-9.6 x 10(-7) M, the isolated scFv compare favorably with mouse mAb and may overcome certain of their limitations. Our results demonstrate the advantages of in vitro antibody selection for the generation of isoenzyme-specific antibodies.

Amino Acid Sequence↗