Isozyme-specific Antibodies Research Articles

Aldehyde dehydrogenase isozymes: markers of cancer stem cells in human melanoma.

Aldehyde dehydrogenase isozymes: markers of cancer stem cells in human melanoma.

Soybean vegetative lipoxygenases are not vacuolar storage proteins.

The paraveinal mesophyll (PVM) of soybean is a distinctive uniseriate layer of branched cells situated between the spongy and palisade chlorenchyma of leaves that contains an abundance of putative vegetative storage proteins, Vspα and Vspβ, in its vacuoles. Soybean vegetative lipoxygenases (five isozymes designated as Vlx(A-E)) have been reported to co-localise with Vsp in PVM vacuoles; however, conflicting results regarding the tissue-level and subcellular localisations of specific Vlx isozymes have been reported. We employed immuno-cytochemistry with affinity-purified, isozyme-specific antibodies to reinvestigate the subcellular locations of soybean Vlx isozymes during a sink limitation experiment. VlxB and VlxC were localised to the cytoplasm and nucleoplasm of PVM cells, whereas VlxD was present in the cytoplasm and nucleoplasm of mesophyll chlorenchyma (MC) cells. Label was not associated with storage vacuoles or any evident protein bodies, so our results cast doubt on the hypothesis that Vlx isozymes function as vegetative storage proteins.

Immunohistochemical Localization of Glycogen Phosphorylase Isozymes in the Rat Gastrointestinal Muscle Layers and Enteric Nervous System

Glycogen represents the major brain energy reserve though its precise functions are still under debate. Glycogen has also been found in different cell types of the enteric nervous system (ENS), the largest and most complex component of the peripheral nervous system. In the present work we have demonstrated, by application of isozyme-specific antibodies, the presence of isozymes of glycogen phosphorylase (GP), one of the major control sites in glycogen metabolism, in the rat ENS. Immunohistochemistry revealed that isoform BB (brain) is the predominant isozyme expressed in enteric glial cells (EGC) and rare neurons of the myenteric and submucosal plexuses. Isoform MM (muscle) appears in cells which are, according to their location and morphology, probably interstitial cells of Cajal (ICC). In addition, both GP isoforms are expressed in longitudinal and circular intestinal smooth muscle layers. As GP BB is mainly regulated by the cellular AMP level, a special function of glycogen in the energy supply of neural gut functions is suggested.

Immunocytochemical Analysis of Glycogen Phosphorylase Isozymes in the Developing and Adult Retina of the Domestic Chicken (Gallus domesticus)

Glycogen is the major energy reserve in neural tissues including the retina. A key-enzyme in glycogen metabolism is glycogen phosphorylase (GP) which exists in three differentially regulated isoforms. By applying isozyme-specific antibodies it could be demonstrated that the GP BB (brain), but not the GP MM (muscle) isoform is expressed in the chicken retina in neuronal and glial (Müller) cells. In the embryonic chicken retina, GP showed a development-dependent expression pattern. Double-labeling experiments with cell type-specific antibodies revealed that GP is expressed in various layers of the retina some of which, e.g., the photoreceptor inner segments, are known to be sites of high energy consumption. This suggests important roles of GP BB, and therefore glycogen, in early differentiation, spontaneous wave generation and in formation and stabilization of synapses.

Glycogen phosphorylase isozymes and energy metabolism in the rat peripheral nervous system—An immunocytochemical study

Glycogen represents the major brain energy reserve which is located mainly in astrocytes. Though the role of brain glycogen has drawn increasing attention, little is known about glycogen metabolism in the peripheral nervous system. In the present work, we have demonstrated immunocytochemically the ubiquitous presence of glycogen phosphorylase (GP), one of the major control sites in glycogen metabolism, in the axons of rat spinal and sciatic nerves, but not in Schwann cells. Application of isozyme-specific antibodies revealed the presence of the GP BB (brain) isoform, but not the MM (muscle) isoform. This is in accord with previous results demonstrating the presence of isoform BB, but not MM, in the few GP-containing brain and spinal cord neurons and in vagus nerve axons. In contrast, brain astrocytes express both isoforms. As GP BB is mainly regulated by the cellular AMP level, a special role of glycogen in the energization of the nerve axons is suggested. The cellular locations of hexokinase, pyruvate dehydrogenase and glucose transporters are discussed in respect to possible metabolic roles of glycogen in peripheral nerves.

Immunocytochemical analysis of rat vagus nerve by antibodies against glycogen phosphorylase isozymes

Glycogen is an endogenous store of glucose equivalents for energy metabolism in many tissues. The brain contains a significant amount of glycogen the role of which as an energy reserve is currently under debate. Apparently little is known concerning a possible role of glycogen in peripheral nerves. We have demonstrated immunocytochemically the presence of glycogen phosphorylase (GP), a key enzyme in glycogen metabolism, in large and small axons of the rat vagus nerve, but not in Schwann cells. Furthermore, the isozyme-specific antibodies applied detected only the presence of the brain isoform BB of GP, but not the muscle isoform MM. This is in agreement with the occurrence of solely the BB isoform in the few brain and spinal cord neurons that contain GP. In contrast, astroglial cells in brain and spinal cord have previously been shown to contain both isoforms. Since GP isozymes are regulated differentially, the expression of isoform BB may provide hints to possible functions of glycogen in the vagus nerve.

Subcellular localization of Strboh proteins and NADPH-dependent O2−-generating activity in potato tuber tissues

Rapid generation of reactive oxygen species (ROS) at the cell surface has been implicated in plant defence responses. Genetic evidence indicates that a plant NADPH oxidase (Rboh; respiratory burst oxidase homologue) is associated with oxidative burst. However, there is not enough physiological evidence of Rboh localization available yet. Isozyme-specific antibodies against potato StrbohA and StrbohB (St; Solanum tuberosum) were prepared to investigate the localization of these proteins. Immunoblot analyses using potato microsomal proteins revealed that StrbohA was expressed constitutively at a low level, whereas the accumulation of StrbohB protein was induced by the cell wall elicitor of the potato pathogen Phytophthora infestans. It is demonstrated here that StrbohA and StrbohB are distributed in plasma membrane fractions which have been separated by sucrose density-gradient centrifugation using their specific antibodies. Green fluorescent protein-tagged Strboh proteins were also located on the plasma membrane by transient expression assay in onion epidermal cells. Additionally, NADPH-dependent O2(-)-generating activities in plasma membrane fractions were diphenylene iodonium-sensitive and NaN3-insensitive. These data suggest that StrbohA and StrbohB are predominantly localized on the plasma membrane and regulate ROS production in defence signalling.

Generation of specific antibodies and their use to characterize sex differences in four rat P450 3A enzymes following vehicle and pregnenolone 16α-carbonitrile treatment

The purpose of this study was to identify isozyme-specific antibodies and use them to determine the expression levels of four P450 3A enzymes in the livers of vehicle- and pregnenolone 16α-carbonitrile (PCN)-treated rats of both sexes, since previous work on mRNA levels has shown considerable sexual dimorphism. Using Western blot analysis with four isozyme-specific antibodies, we show that P450 3A1, 3A2, and 3A9 were expressed in vehicle-treated adult female rats at very low levels whereas P450 3A18 was not detected. PCN treatment of females strongly induced the expression of P450 3A1 in the livers with protein product increases of 214-, 3-, and 5-fold for P450 3A1, 3A2, and 3A9, respectively, and P450 3A18 was induced to 3.7 pmol/mg protein. In contrast, all four P450 3As were detected in livers of vehicle-treated males, in the order of 3A2 ≫ 3A18 > 3A9 ≅ 3A1. The protein product increases induced by PCN treatment of male rats were 92-, 3-, 6-, and 16-fold for P450 3A1, 3A2, 3A9, and 3A18, respectively.

Protein kinase C isozymes in hypertension and hypertrophy: Insight from SHHF rat hearts

Chronic hypertension results in cardiac hypertrophy and may lead to congestive heart failure. The protein kinase C (PKC) family has been identified as a signaling component promoting cardiac hypertrophy. We hypothesized that PKC activation may play a role mediating hypertrophy in the spontaneously hypertensive heart failure (SHHF) rat heart. Six-month-old SHHF and normotensive control Wistar Furth (WF) rats were used. Hypertension and cardiac hypertrophy were confirmed in SHHF rats. PKC expression and activation were analyzed by Western blots using isozyme-specific antibodies. Compared to WF, untreated SHHF rats had increased phospho-active alpha (10-fold), delta (4-fold), and epsilon (3-fold) isozyme expression. Furthermore, we analyzed the effect of an angiotensin II type 1 receptor blocker (ARB) and hydralazine (Hy) on PKC regulation in SHHF rat left ventricle (LV). Both the ARB and Hy normalized LV blood pressure, but only the ARB reduced heart mass. Neither treatment affected PKC expression or activity. Our data show differential activation of PKC in the hypertensive, hypertrophic SHHF rat heart. Regression of hypertrophy elicited by an ARB in this model occurred independently of changes in the expression and activity of the PKC isoforms examined.

Both chloroplastic and cytosolic phosphoglycerate kinase isozymes are present in the pea leaf nucleus.

Phosphoglycerate kinase (EC 2.7.2.3) occurs in chloroplasts, cytosol, and nuclei in higher plants. Immunocytolocalization experiments with isozyme-specific antibodies indicate that both the chloroplastic and the cytosolic forms of the enzyme are present in the pea (Pisum sativum L.) leaf nucleus.

Two-layer antibody capture of enzymes on the surface of microtiter plates: application to the study of the regulation of phospholipase C-γ1 catalytic activity

In vitro quantification of the catalytic activity of an enzyme isoform requires the availability of selective agents that allow for either measurements in the presence of the other enzyme isoforms or purification of the isoform and subsequent performance of these measurements on the purified enzyme. Isozyme-specific antibodies are useful tools for these types of analyses. In the present report, we detail a method for the measurement of phospholipase C-γ1 enzyme activity employing native enzyme that is immobilized on microtiter plates. The method uses biotinylated antiglobulin bound to streptavidin-coated microtiter plates to immobilize antiphospholipase C-γ1 antibody and subsequently capture phospholipase C-γ1 from brain tissue lysates. This method avoids biotinylation of the primary (antiphospholipase C-γ1) antibody, making it less labor intensive than previously described methods for using streptavidin-coated plates; in addition, it is highly reproducible and sensitive and allows for quantification of enzyme activity. We employ the technique to show that one or more tyrosine kinases copurify with rat brain phospholipase C-γ1. The method is applicable to the study of any enzyme isoform for which antibodies that capture the native form of the enzyme are available and could easily be employed in high-throughput procedures.

Immunocytochemical localization of glycogen phosphorylase isozymes in rat nervous tissues by using isozyme-specific antibodies.

Isozyme-specific antibodies were raised against peptides from the low-homology regions of the sequences of rat glycogen phosphorylase BB and MM isozymes by immunization of rabbits and guinea pigs. Immunocytochemical double-labelling experiments on frozen sections of rat nervous tissues were performed to investigate the isozyme localization pattern. Astrocytes throughout the brain and spinal cord expressed both isozymes in perfect co-localization. Ependymal cells only expressed the BB isozyme. Most neurones were not immunoreactive. The rare neurones that contained glycogen phosphorylase only expressed the BB isozyme. Nearly all of these neurones formed part of the afferent somatosensory system. These findings stress the general importance of glycogen in neural energy metabolism and indicate a special role for the glycogen phosphorylase BB isozyme in neurones in the somatosensory system.

Carbonic anhydrase isozymes IX and XII in gastric tumors.

To systematically study the expression of carbonic anhydrase (CA) isozymes IX and XII in gastric tumors. We analyzed a representative series of specimens from non-neoplastic gastric mucosa and from various dysplastic and neoplastic gastric lesions for the expression of CA IX and XII. Immunohistochemical staining was performed using isozyme-specific antibodies and biotin-streptavidin complex method. CA IX was highly expressed in the normal gastric mucosa and remained positive in many gastric tumors. In adenomas, CA IX expression significantly decreased towards the high grade dysplasia. However, the expression resumed back to the normal level in well differentiated adenocarcinomas, while it again declined in carcinomas with less differentiation. In comparison, CA XII showed no or weak immunoreaction in the normal gastric mucosa and was slightly increased in tumors. These results demonstrate that CA IX expression is sustained in several types of gastric tumors. The variations observed in the CA IX levels support the concept that gastric adenomas and carcinomas are distinct entities and do not represent progressive steps of a single pathway.

Expression of the transmembrane carbonic anhydrases, CA IX and CA XII, in the human male excurrent ducts.

Testicular fluid is concentrated and acidified during its passage through the excurrent ducts. These processes involve bicarbonate absorption, in which carbonic anhydrases are implicated. In this study, the distribution of two transmembrane carbonic anhydrase isozymes (CA IX and CA XII) in the human excurrent ducts was investigated using isozyme-specific antibodies in conjunction with immunohistochemical and immunoblotting techniques. Specific staining for CA XII was present in the basolateral plasma membrane of the epithelial cells in the efferent ducts, predominantly in the non-ciliated cells. In the epididymal duct, CA XII was detected only in sporadic cells, which also contained CA II, thus suggesting that they are apical mitochondria-rich cells. CA IX was also localized to the basolateral plasma membrane of the epithelium in the efferent ducts, but its staining was weaker and less uniform compared to CA XII. No signal for CA IX was detected in the epididymal duct. Western blot analysis from efferent duct samples revealed specific bands for CA IX and CA XII, confirming that the immunohistochemical stainings represent these isozymes. The expression of CA XII and CA IX in the excurrent duct system and co-expression of CA XII with Aquaporin-1 in the same efferent duct epithelial cells suggest their functional involvement in ion transport and concentration processes of testicular fluid.

Expression and characterization of protein kinase C in isolated rabbit parietal cells.

We investigated the expression, characterization and distribution of protein kinase C (PKC) isozymes in isolated rabbit parietal cells (PC). Cellular extracts of PC were analyzed by Western blot using isozyme-specific antibodies. The Ca2+-independent PKC-epsilon was detected in cytosolic, membrane and cytoskeletal fractions of basal and histamine-stimulated PC, whereas the Ca2+-dependent PKC-alpha was confined to the cytosolic and membrane fractions. Cytosolic and membrane fractions were partially purified by DEAE cellulose column chromatography with elution of increasing NaCl concentration. Eluates of 0.15 M and 0.3 M NaCl PC fractions were identified as PKC-alpha and -epsilon isoforms, respectively. Phorbol 12-myristate 13-acetate (TPA) treatment of PC for 15, 30 and 60 sec decreased significantly cytosolic PKC-alpha and increased membrane-associated PKC-alpha. In contrast to the distribution of PKC-alpha, TPA did not alter membrane or cytosolic level of PKC-epsilon. Comparison of the dose-response curves between TPA-induced hydrogen (H+) secretion, as measured by aminopyrine (AP) uptake, and the membrane-associated PKC-alpha suggests that translocation of PKC-alpha is not involved in the H+ secretory process in PC. Furthermore, a PKC inhibitor, staurosporine, produced a concentration-dependent enhancement of histamine-stimulated H+ secretion. These findings suggest that PKC-alpha plays a negative modulatory role, rather than an obligatory role, in H+ secretion. The localization and distribution of PKC-epsilon into the cytoskeletal fraction of PC also suggests that this isozyme may be involved in the cellular regulation of reversible morphological transformation during stimulation.

Detection of Heme Oxygenase 1 and 2 Proteins and Bilirubin Formation

It is possible to detect changes in the level of expression of the two isozymes of heme oxygenase (HO). The sequence differences allow the production of isozyme-specific antibodies that can be used in immunoblot analysis. Total HO activity in tissue microsomal fractions or extracts of bacteria transformed with an HO gene can be measured as described in this unit using a coupled assay employing biliverdin reductase, which converts the biliverdin product to bilirubin. Bilirubin is then quantified with a scanning spectrophotometer.

Cell-specific expression of mitochondrial carbonic anhydrase in the human and rat gastrointestinal tract.

Mitochondrial carbonic anhydrase V (CA V) in liver provides HCO3- to pyruvate carboxylase for the first step in gluconeogenesis and HCO3- to carbamyl phosphate synthetase I for the first step in ureagenesis. Because carbamyl phosphate synthetase I and ornithine transcarbamylase are also expressed in enterocytes, we tested the hypothesis that CA V is expressed in the gastrointestinal tract in addition to liver. Polyclonal rabbit antisera were raised against a polypeptide of 17 C-terminal amino acids of human CA V and against purified recombinant mouse isozyme and were used in Western blotting and immunoperoxidase staining of human and rat tissues. Immunohistochemistry showed that CA V is expressed cell-specifically in the alimentary canal mucosa from stomach to rectum. Immunoreactions for CA V were detected in the parietal cells and gastrin-producing G-cells of the stomach and in intestinal enterocytes. Western blotting of human and rat gastrointestinal tissues with isozyme-specific antibodies showed positive signals for CA V with the expected molecular mass. The findings in human tissues paralleled those in rat. The cell-specific pattern of CA V expression suggests a role for CA V in alimentary canal physiology. We propose that mitochondrial CA V participates in the detoxification of ammonia produced in the gastrointestinal tract by providing bicarbonate to carbamyl phosphate synthetase I. (J Histochem Cytochem 47:517-524, 1999)

Differential subcellular redistribution of protein kinase C isozymes in the rat hippocampus induced by kainic acid.

Protein kinase C (PKC) consists of a family of Ca2+/phospholipid-dependent isozymes that has been implicated in the delayed neurotoxic effects of glutamate in vitro. In the present study, we assessed the effect of the glutamate analogue kainic acid (KA) on the subcellular expression of PKC isozymes in the hippocampus (HPC) in the period preceding (0.5, 1.5, 12, and 24 h) and during (120 h) hippocampal necrosis using western blot analysis and PKC isozyme-specific antibodies. Before subcellular fractionation (cytosol + membrane), hippocampi were microdissected into "HPC" (fields CA1-CA3) and "dentate gyrus" (DG; granule cells + hilus) regions. Four general patterns of alterations in PKC isozyme expression/distribution were observed following KA treatment. The first pattern was a relative stability in expression following KA treatment and was most apparent for cytosol PKCalpha (HPC + DG) and membrane (HPC) and cytosol (DG) PKCbetaII. The second pattern, observed with PKCgamma and PKCepsilon, was characterized by an initial increase in expression in both membrane and cytosolic fractions before seizure activity (0.5 h) followed by a gradual decrease until significant reductions are observed by 120 h. The third pattern, exhibited by PKCdelta, involved an apparent translocation, increasing in the membrane and decreasing in the cytosol, followed by down-regulation in both fractions and subsequent recovery. The fourth pattern was observed with PKCzeta only and entailed a significant reduction in expression before and during limbic motor seizures followed by a dramatic fivefold increase in the membrane fraction during the period of hippocampal necrosis (120 h). Although these patterns did not segregate according to conventional PKC isozyme classifications, they do indicate dynamic isozyme-specific regulation by KA. The subcellular redistribution of PKC isozymes may contribute to the histopathological sequelae produced by KA in the hippocampus and may model the pathogenesis associated with diseases involving glutamate-induced neurotoxicity.

Tissue Distribution of Human Aldehyde Dehydrogenase E3 (ALDH9): Comparison of Enzyme Activity with E3 Protein and mRNA Distribution

The tissue distribution of the E3 isozyme of human aldehyde dehydrogenase has been investigated by three methods: enzyme activity assay employing betaine aldehyde as substrate, Western blotting employing E3 isozyme-specific antibodies, and Northern blotting using a human liver E3 cDNA as probe. All three methods showed that E3 isozyme was universally distributed among all tissues tested. The highest levels of the E3 isozyme activity were found in liver, adrenal gland, and kidney. These same tissues also showed highest levels of the E3 protein via the Western blot. This distribution is consistent with the possible physiological role of E3 isozyme in the synthesis of the osmolyte, betaine, and the neurotransmitter, GABA. Northern blot analysis, however, differed from that of enzyme assay and the Western blot in that it showed highest mRNA levels in skeletal and heart muscles, which had low enzyme activities and E3 protein levels.

Organization of DNA topoisomerase II isotypes during the cell cycle of human lymphocytes and HeLa cells.

We have monitored the organization of DNA topoisomerase II (Topo II) in relation to chromatin disaggregation during mitogen stimulation of lymphocytes and to the mitotic chromosome condensation cycle by immunofluorescence microscopy with isozyme-specific antibodies. Labelling for both Topo II alpha and Topo II beta was diffusely nucleoplasmic and non-nucleolar in resting lymphocytes and the pattern changed little during stimulation. Topo II alpha labelling intensity increased in parallel with the extent of cell stimulation, but a fraction of fully stimulated cells was labelled very brightly. Topo II beta labelling intensity was also greater in stimulated cells, but all partially and fully stimulated cells were labelled at the same, higher, intensity. In addition, anti-Topo II beta detected a few small spots within nucleoli of stimulated cells that coincided with regions containing fibrillarin. In lymphocytes and HeLa, chromosome association of Topo II alpha began in prophase and lasted throughout mitosis. In contrast, Topo II beta stayed nucleoplasmic in prophase, was diffusely cytoplasmic during mitosis, and was first detected post-mitotically in nuclei with decondensing chromosomes and a reformed nuclear envelope. The results are consistent with a role for Topo II alpha, but not for Topo II beta, in mitotic chromosome condensation, and indicate that the isotypes may play independent roles in the reorganization of chromatin structure during lymphocyte mitogenic activation.