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L H Jin, J H Bahn,
W S Eum,
H Y Kwon,
S H Jang,
K H Han,
T C Kang,
M H Won,
J H Kang,
S W Cho,
J Park,
S Y Choi
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ABSTRACT: Antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) have been considered to have a beneficial effect against various diseases mediated by reactive oxygen species (ROS). Although a variety of modified recombinant antioxidant enzymes have been generated to protect against the oxidative stresses, the lack of their transduction ability into cells resulted in limited ability to detoxify intracellular ROS. To render the catalase enzyme capable of detoxifying intracellular ROS when added extracellularly, cell-permeable recombinant catalase proteins were generated. A human liver catalase gene was cloned and fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) and arginine-rich peptides (RRRRRRRRR) in a bacterial expression vector to produce genetic in-frame Tat-CAT and 9Arg-CAT fusion proteins, respectively. The expressed and purified fusion proteins can be transduced into mammalian cells (HeLa and PC12 cells) in a time- and dose-dependent manner when added exogenously in culture medium, and transduced fusion proteins were enzymatically active and stable for 60 h. When exposed to H(2)O(2), the viability of HeLa cells transduced with Tat-CAT or 9Arg-CAT fusion proteins was significantly increased. In combination with transduced SOD, transduced catalase also resulted in a cooperative increase in cell viability when the cells were treated with paraquat, an intracellular antioxide anion generator. We then evaluated the ability of the catalase fusion proteins to transduce into animal skin. This analysis showed that Tat-CAT and 9Arg-CAT fusion proteins efficiently penetrated the epidermis as well as the dermis of the subcutaneous layer when sprayed on animal skin, as judged by immunohistochemistry and specific enzyme activities. These results suggest that Tat-CAT and 9Arg-CAT fusion proteins can be used in protein therapy for various disorders related to this antioxidant enzyme.
Free Radical Biology and Medicine 01/2002; 31(11):1509-19. · 5.42 Impact Factor
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ABSTRACT: Several oligopeptides, derived from certain proteins, translocate as a form fused to small molecules or exogenous proteins across the plasma membrane into cells. Some of these oligopeptides, the so-called protein-transduction domains (PTDs), contain a high proportion of basic residues. The translocation of some of these basic PTDs, such as oligoarginines, has been studied as chemically fused forms to other organic compounds. In this study, we also tested to determine whether or not oligoarginines, when fused genetically to an exogenous protein such as GFP, are also able to translocate efficiently across the plasma membrane. The oligoarginine Rn (n = 5,6,7,8,9)-GFP fusion proteins were translocated quite efficiently, and the transduction efficiency increased in proportion to the number of arginine residues. However, the cellular uptake of the oligolysine-GFP fusion proteins was less efficient than that of the corresponding oligoarginine-GFP fusion proteins. When fused to GFP, the translocation efficiency of R5 was similar to that of Tat(49-57)(RKKRRQRRR). This finding suggests that the arginine homo-oligopeptide is more efficient than other PTDs which contain a mixture of basic residues. On the other hand, both the K9- and Tat(49-57)-GFP fusion proteins were transduced with similar efficiencies. It appears that basic oligopeptides may be useful for the efficient translocation of diverse exogenous proteins as genetically fused forms.
Molecules and Cells 11/2001; 12(2):267-71. · 2.18 Impact Factor
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S G Jeon, J H Bahn,
J S Jang,
S H Jang,
B R Lee,
K S Lee,
J Park,
T C Kang,
M H Won,
H B Kim,
O S Kwo,
S W Cho,
S Y Choi
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ABSTRACT: We isolated a cDNA that encodes the bovine brain gamma-aminobutyrate transaminase (GABA-T; EC 2.6.1.19) from the lambda gt 11 cDNA library, which showed a high degree of sequence similarity to the corresponding enzymes from various sources. Northern blot analysis revealed two differentially expressed GABA-T transcripts of approximately 2.0 and 6.0 kb in the bovine tissues. Southern blot analysis indicates that the two GABA-T transcripts are encoded in a greater-than 10-kb, single-copy gene. Bovine GABA-T cDNA was expressed in E. coli using the pGEX bacterial- expression vector system. The overexpressed GABA-T was enzymatically active after purification, and it had very similar kinetic parameters when compared with those of other mammalian GABA-Ts.
Molecules and Cells 09/2001; 12(1):91-6. · 2.18 Impact Factor
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ABSTRACT: Gamma-aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the central nervous system (CNS). Degradation of GABA in the CNS is catalyzed by the action of GABA transaminase (GABA-T). However, the neuroanatomical characteristics of GABA-T in the gerbil, which is a useful experimental animal in neuroscience, are still unknown. Therefore, we performed a comparative analysis of the distribution of GABA-T in rat and gerbil brains using immunohistochemistry. GABA-T immunoreactive neurons were observed in the regions which contained GABAergic neurons of both animals: corpus striatum; substantia nigra, pars reticulata; septal nucleus; and accumbens nucleus. GABA-T + neurons were restricted to layers III and V in the rat. Unlike the rat GABA-T + neurons were observed in layers II, III, and V of the gerbil cerebral cortex. These results suggest that the expression of GABA-T in the gerbil brain may be similar to that in the rat brain, except in the cerebral cortex.
Molecules and Cells 07/2001; 11(3):321-5. · 2.18 Impact Factor
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ABSTRACT: It is well established that GABA degradation may play a key role in epileptogenesis. However, whether or not the expression of GABA-transaminase (GABA-T), which catalyzes GABA degradation and participates in the neuronal metabolism via GABA shunt, changes chronologically after on-set of seizure remains to be clarified. To identify the change of GABA-T expression in seizure, GABA-T expression in the gerbil hippocampus, associated with different sequelae of spontaneous seizures, was investigated. The distribution pattern of GABA-T immunoreactive neurons in the hippocampus between the seizure-resistant and pre-seizure group of seizure sensitive gerbils was similar. Interestingly, at 30 min postictal, the enhancement of GABA-T immunoreactivity in the perikarya was apparently observed. This contrasted with the decline in GABA-T immunoreactivity in the granular and pyramidal layer. At 12-24 h postictal, GABA-T immunoreactivity in the hilar neurons had declined significantly. However, the GABA-T immunoreactivity in the granular layer increased. These findings suggest that in the gerbil, the alteration in GABA-T expressions may play an important role in the self-recovery mechanism from seizure attack via both GABA degradation and regulation of neuronal metabolism.
Neurochemistry International 07/2001; 38(7):609-14. · 2.86 Impact Factor
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S Y Choi, J H Bahn,
B R Lee,
S G Jeon,
J S Jang,
C K Kim,
L H Jin,
K H Kim,
J S Park,
J Park,
S W Cho
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ABSTRACT: An NAD+ dependent succinic semialdehyde dehydrogenase from bovine brain was inactivated by pyridoxal-5'- phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After NaBH(4) reduction of the pyridoxal-5'-phosphate inactivated enzyme, it was observed that 3.8 mol phosphopyridoxyl residues were incorporated/enzyme tetramer. The coenzyme, NAD+, protected the enzyme against inactivation by pyridoxal-5'-phosphate. The absorption spectrum of the reduced and dialyzed pyridoxal-5'-phosphate-inactivated enzyme showed a characteristic peak at 325 nm, which was absent in the spectrum of the native enzyme. The fluorescence spectrum of the pyridoxyl enzyme differs completely from that of the native enzyme. After tryptic digestion of the enzyme modified with pyridoxal-5'-phosphate followed by [3H]NaBH4 reduction, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. The sequences of the peptide containing the phosphopyridoxyllysine were clearly identical to sequences of other mammalian succinic semialdehyde dehydrogenase brain species including human. It is suggested that the catalytic function of succinic semialdehyde dehydrogenase is modulated by binding of pyridoxal-5'-phosphate to specific Lys(347) residue at or near the coenzyme-binding site of the protein.
Journal of Neurochemistry 03/2001; 76(3):919-25. · 4.06 Impact Factor
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ABSTRACT: gamma-Aminobutyric acid-transaminase (GABA-T) plays an important role in the metabolism of GABA, particularly in the neurons or glial cells. The present study was undertaken to determine the alteration of GABA-T expression in the gerbil hippocampus after ischemia-reperfusion. In the sham, GABA-T(+) neurons were scattered in the hippocampus proper and dentate gyrus. The intensity of the GABA-T immunoreactivity had nearly disappeared in the interneurons at 12 h after ischemia. In contrast, 24 h post-ischemia the dramatic augmentation of GABA-T immunoreactivity in the pyramidal cells was observed in the CA1 area but not in the CA2 or CA3 areas. Forty-eight hours after ischemia-reperfusion, its immunoreactivity was preserved in the CA1 neurons. These results suggest that the over-expression of GABA-T in the CA1 area may be related to delayed neuronal death after ischemia-reperfusion insult.
Neuroscience Letters 12/2000; 294(1):33-6. · 2.11 Impact Factor
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ABSTRACT: Human brain gamma-aminobutyrate transaminase is differentially expressed in a tissue-specific manner. mRNA master dot-blot analysis for 50 different human tissues, including different brain regions and fetal tissues, provided a complete map of the tissue distribution. Genomic Southern analysis revealed that the gamma-aminobutyrate transaminase gene is a single copy, at least 15 kb in size. In addition, human brain gamma-aminobutyrate transaminase cDNA was expressed in Escherichia coli using a pGEX expression vector system. Catalytically active gamma-aminobutyrate transaminase was expressed in large quantities and the purified recombinant enzyme had kinetic parameters that were indistinguishable from those isolated from other mammalian brains. The human enzyme was inactivated by a well-known antiepileptic drug vigabatrin. Values of Ki and kinact were 1 mM and 0.35 min-1, respectively. Results from inactivation kinetics suggested that human gamma-aminobutyrate transaminase is more sensitive to the vigabatrin drug than the enzyme isolated from bovine brain.
European Journal of Biochemistry 10/2000; 267(17):5601-7. · 3.58 Impact Factor
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ABSTRACT: 80% Aqueous MeOH extracts from the wood of Caesalpinia sappan, which showed remarkable anticonvulsant activity, were fractionated using EtOAc, n-BuOH, and H2O. Among them, the EtOAc fraction significantly inhibited the activities of two GABA degradative enzymes, succinic semialdehyde dehydrogenase (SSADH) and succinic semialdehyde reductase (SSAR). Repeated column chromatographies for the fraction guided by activity test led to the isolation of the two active principal components. Their chemical structures were determined to be sappanchalcone and brazilin based on spectral data. The pure compounds, sappanchalcone (1) and brazilin (2), inactivated the SSAR activities in a dose dependent manner, whereas SSADH was inhibited partially by sappanchalcone and not by brazilin.
Archives of Pharmacal Research 09/2000; 23(4):344-8. · 1.59 Impact Factor
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ABSTRACT: The structural differences between two types of glutamate dehydrogenase (GDH) isoproteins (GDH I and GDH II), homogeneously isolated from bovine brain, were investigated using a biosensor technology and monoclonal antibodies. A total of seven monoclonal antibodies raised against GDH II were produced, and the antibodies recognized a single protein band that comigrates with purified GDH II on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot. Of seven anti-GDH II monoclonal antibodies tested in the immunoblot analysis, all seven antibodies interacted with GDH II, whereas only four antibodies recognized the protein band of the other GDH isoprotein, GDH I. When inhibition tests of the GDH isoproteins were performed with the seven anti-GDH II monoclonal antibodies, three antibodies inhibited GDH II activity, whereas only one antibody inhibited GDH I activity. The binding affinity of anti-GDH II monoclonal antibodies for GDH II (K(D) = 1.0 nM) determined using a biosensor technology (Pharmacia BIAcore) was fivefold higher than for GDH I (K(D) = 5.3 nM). These results, together with epitope mapping analysis, suggest that there may be structural differences between the two GDH isoproteins, in addition to their different biochemical properties. Using the anti-GDH II antibodies as probes, we also investigated the cross-reactivities of brain GDHs from some mammalian and an avian species, showing that the mammalian brain GDH enzymes are related immunologically to each other.
Journal of Neurochemistry 06/1999; 72(5):2162-9. · 4.06 Impact Factor
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ABSTRACT: In our search for the anticonvulsant constituent of Gastrodia elata repeated column chromatographies guided by activity assay led to isolation of an active compound, which was identified as gastrodin on the basis of spectral data. Brain succinic semialdehyde dehydrogenase (SSADH) was inactivated by preincubation with gastrodin in a time-dependent manner and the reaction was monitored by absorption and fluorescence spectroscopic methods. The inactivation followed pseudo-first-order kinetics with the second-rate order constant of 1.2 x 10(3) M-1min-1. The time course of the reaction was significantly affected by the coenzyme NAD+, which affected complete protection against the loss of the catalytic activity, whereas substrate succinic semialdehyde failed to prevent the inactivation of the enzyme. It is postulated that the gastrodin is able to elevate the neurotransmitter GABA levels in central nervous system by inhibitory action on one of the GABA degradative enzymes, SSADH.
Archives of Pharmacal Research 05/1999; 22(2):219-24. · 1.59 Impact Factor
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ABSTRACT: Six monoclonal antibodies that recognize porcine brain pyridoxal kinase have been selected and designated as PK67, PK86, PK91, PK144, PK252 and PK275. A total of six monoclonal antibodies recognizing different epitopes of the enzyme were obtained, of which four inhibited the enzyme activity. When total proteins of porcine brain homogenate separated by SDS-PAGE were subjected to monoclonal antibodies, a single reactive protein band of molecular weight 39 kDa which comigrated with purified porcine pyridoxal kinase was detected. Using the anti-pyridoxal kinase antibodies as probes, the cross reactivities of brain pyridoxal kinase from human and other mammalian tissues and from avian sources were also investigated. Among human and all animal tissues tested, immunoreactive bands on Western blots appeared to have the same molecular mass of 39 kDa. These results indicate that mammalian brains contain only one major type of immunologically similar pyridoxal kinase, although some properties of the enzymes reported previously differed from one another.
BioFactors 02/1999; 10(1):35-42. · 4.93 Impact Factor
