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Molecular and Enzymatic Characterization of Three
Phosphoinositide-Specific Phospholipase C Isoforms from
Potato 总被引:19,自引:0,他引:19 下载免费PDF全文
Joachim Kopka Christophe Pical Julie E. Gray Bernd Müller-R?ber 《Plant physiology》1998,116(1):239-250
Many cellular responses to stimulation of cell-surface receptors by extracellular signals are transmitted across the plasma membrane by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), which is cleaved into diacylglycerol and inositol-1,4,5-tris-phosphate by phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X and Y domains, as well as C2-like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP2 hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP2-hydrolyzing activity at 10 μm Ca2+ and were inhibited by Al3+ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg2+. All three stplc genes are expressed in various tissues of potato, including leaves, flowers, tubers, and roots, and are affected by drought stress in a gene-specific manner. 相似文献
4.
Identification of a Functional Homolog of the Yeast Copper
Homeostasis Gene ATX1 from
Arabidopsis 总被引:11,自引:0,他引:11 下载免费PDF全文
Edward Himelblau Helena Mira Su-Ju Lin Valeria Cizewski Culotta Lola Pe?arrubia Richard M. Amasino 《Plant physiology》1998,117(4):1227-1234
A cDNA clone encoding a homolog of the yeast (Saccharomyces cerevisiae) gene Anti-oxidant 1 (ATX1) has been identified from Arabidopsis. This gene, referred to as Copper CHaperone (CCH), encodes a protein that is 36% identical to the amino acid sequence of ATX1 and has a 48-amino acid extension at the C-terminal end, which is absent from ATX1 homologs identified in animals. ATX1-deficient yeast (atx1) displayed a loss of high-affinity iron uptake. Expression of CCH in the atx1 strain restored high-affinity iron uptake, demonstrating that CCH is a functional homolog of ATX1. When overexpressed in yeast lacking the superoxide dismutase gene SOD1, both ATX1 and CCH protected the cell from the reactive oxygen toxicity that results from superoxide dismutase deficiency. CCH was unable to rescue the sod1 phenotype in the absence of copper, indicating that CCH function is copper dependent. In Arabidopsis CCH mRNA is present in the root, leaf, and inflorescence and is up-regulated 7-fold in leaves undergoing senescence. In plants treated with 800 nL/L ozone for 30 min, CCH mRNA levels increased by 30%. In excised leaves and whole plants treated with high levels of exogenous CuSO4, CCH mRNA levels decreased, indicating that CCH is regulated differently than characterized metallothionein proteins in Arabidopsis. 相似文献
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《Bioscience, biotechnology, and biochemistry》2013,77(7):1168-1171
Glutamate decarboxylase (GAD) [EC 4.1.1.15] was purified from a cell-free extract of Lactobadllus brevis TFO 12005 by chromatographies on Sephadex G-100, DEAE-Sepharose CL-6B, and Mono Q. About 9 mg of purified GAD was obtained from 90.2 g of wet cells. The purified preparation showed a single protein band on SDS-PAGE. The molecular weights of purified GAD by SDS-PAGE and gel filtration on Superdex 200 were 60,000 and 120,000, respectively, indicating that GAD from L. brevis exists as a dimer. The N-terminal amino acid sequence of the purified GAD was NH2-Met-Asn-Lys-Asn-Asp-Gln-Glu-Gln-Thr-. The optimum pH and temperature of GAD were at pH 4.2 and at 30°C. The GAD activity was increased by the addition of sulfate ions in a dose-dependent manner. The order of effect was as follows: ammonium sulfate?>?sodium sulfate?>?magnesium sulfate, indicating that the increase of hydrophobic interaction between subunits causes the increase of GAD activity. The purified GAD reacted only with l-glutamic acid as a substrate and the Km, kcat, and kcat/Km values were 9.3 mm, 6.5 s?1, and 7 × 102 m?1 s?1, respectively. 相似文献
7.
Aluminum-Resistant Arabidopsis Mutants That Exhibit Altered
Patterns of Aluminum Accumulation and Organic Acid Release from
Roots 总被引:15,自引:0,他引:15 下载免费PDF全文
Al-resistant (alr) mutants of Arabidopsis thaliana were isolated and characterized to gain a better understanding of the genetic and physiological mechanisms of Al resistance. alr mutants were identified on the basis of enhanced root growth in the presence of levels of Al that strongly inhibited root growth in wild-type seedlings. Genetic analysis of the alr mutants showed that Al resistance was semidominant, and chromosome mapping of the mutants with microsatellite and random amplified polymorphic DNA markers indicated that the mutants mapped to two sites in the Arabidopsis genome: one locus on chromosome 1 (alr-108, alr-128, alr-131, and alr-139) and another on chromosome 4 (alr-104). Al accumulation in roots of mutant seedlings was studied by staining with the fluorescent Al-indicator dye morin and quantified via inductively coupled argon plasma mass spectrometry. It was found that the alr mutants accumulated lower levels of Al in the root tips compared with wild type. The possibility that the mutants released Al-chelating organic acids was examined. The mutants that mapped together on chromosome 1 released greater amounts of citrate or malate (as well as pyruvate) compared with wild type, suggesting that Al exclusion from roots of these alr mutants results from enhanced organic acid exudation. Roots of alr-104, on the other hand, did not exhibit increased release of malate or citrate, but did alkalinize the rhizosphere to a greater extent than wild-type roots. A detailed examination of Al resistance in this mutant is described in an accompanying paper (J. Degenhardt, P.B. Larsen, S.H. Howell, L.V. Kochian [1998] Plant Physiol 117: 19–27). 相似文献
8.
Purification and cDNA Cloning of Isochorismate Synthase from
Elicited Cell Cultures of Catharanthus roseus 下载免费PDF全文
Léon J.P. van Tegelen Paolo R.H. Moreno Anton F. Croes Robert Verpoorte George J. Wullems 《Plant physiology》1999,119(2):705-712
Isochorismate is an important
metabolite formed at the end of the shikimate pathway, which is
involved in the synthesis of both primary and secondary metabolites. It
is synthesized from chorismate in a reaction catalyzed by the enzyme
isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS to
homogeneity from elicited Catharanthus roseus cell
cultures. Two isoforms with an apparent molecular mass of 64 kD were
purified and characterized. The Km values
for chorismate were 558 and 319 μm for isoforms I and II,
respectively. The isoforms were not inhibited by aromatic amino acids
and required Mg2+ for enzyme activity. Polymerase chain
reaction on a cDNA library from elicited C. roseus cells
with a degenerated primer based on the sequence of an internal peptide
from isoform II resulted in an amplification product that was used to
screen the cDNA library. This led to the first isolation, to our
knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD
with an N-terminal chloroplast-targeting signal. The deduced amino acid
sequence shares homology with bacterial ICS and also with anthranilate
synthases from plants. Southern analysis indicates the existence of
only one ICS gene in C. roseus.The shikimate pathway is a major pathway in primary and secondary
plant metabolism (Herrmann, 1995). It provides chorismate for the
synthesis of the aromatic amino acids Phe, Tyr, and Trp, which are used
in protein biosynthesis, but also serves as a precursor for a wide
variety of aromatic substances (Herrmann, 1995; Weaver and Hermann,
1997; Fig. Fig.1a).1a). Chorismate is also the starting point of a biosynthetic
pathway leading to phylloquinones (vitamin K1)
and anthraquinones (Poulsen and Verpoorte, 1991). The first committed
step in this pathway is the conversion of chorismate into
isochorismate, which is catalyzed by ICS (Poulsen and Verpoorte, 1991;
Fig. Fig.1b).1b). Its substrate, chorismate, plays a pivotal role in the
synthesis of shikimate-pathway-derived compounds, and its distribution
over the various pathways is expected to be tightly regulated. Elicited
cell cultures of Catharanthus roseus provide an example of
the partitioning of chorismate. Concurrently, these cultures produce
both Trp-derived indole alkaloids and DHBA (Moreno et al., 1994). In
bacteria DHBA is synthesized from isochorismate (Young et al.,
1969). Elicitation of C. roseus cell cultures with a fungal
extract induces not only several enzymes of the indole alkaloid
biosynthetic pathway (Pasquali et al., 1992) but also ICS
(Moreno et al., 1994). Information concerning the expression and
biochemical characteristics of the enzymes that compete for available
chorismate (ICS, CM, and AS) may help us to understand the regulation
of the distribution of this precursor over the various pathways. Such
information is already available for CM (Eberhard et al., 1996) and AS
(Poulsen et al., 1993; Bohlmann et al., 1995) but not for ICS.
Figure 1a, Position of ICS in the plant metabolism. SA,
Salicylic acid, OSB, o-succinylbenzoic acid. b, Reaction
catalyzed by ICS.Isochorismate plays an important role in bacterial and plant metabolism
as a precursor of o-succinylbenzoic acid, an intermediate in
the biosynthesis of menaquinones (vitamin K2)
(Weische and Leistner, 1985) and phylloquinones (vitamin
K1; Poulsen and Verpoorte, 1991). In bacteria
isochorismate is also a precursor of siderophores such as
DHBA (Young et al., 1969), enterobactin (Walsh et
al., 1990), amonabactin (Barghouthi et al., 1991), and salicylic acid
(Serino et al., 1995). Although evidence from tobacco would indicate
that salicylic acid in plants is derived from Phe via benzoic acid
(Yalpani et al., 1993; Lee et al., 1995; Coquoz et al., 1998), it
cannot be excluded that it is also synthesized from isochorismate. In
the secondary metabolism of higher plants, isochorismate is a precursor
for the biosynthesis of anthraquinones (Inoue et al., 1984; Sieweke and
Leistner, 1992), naphthoquinones (Müller and Leistner, 1978),
catalpalactone (Inouye et al., 1975), and certain alkaloids in orchids
(Leete and Bodem, 1976).ICS was first extracted and partially purified from crude extracts of
Aerobacter aerogenes (Young and Gibson, 1969). Later, ICS
activity was detected in protein extracts of cell cultures from plants
of the Rubiaceae, Celastraceae, and Apocynaceae families (Ledüc
et al., 1991; Poulsen et al., 1991; Poulsen and Verpoorte, 1992). Genes
encoding ICS have been cloned from bacteria such as Escherichia
coli (Ozenberger et al., 1989), Pseudomonas aeruginosa
(Serino et al., 1995), Aeromonas hydrophila (Barghouthi et
al., 1991), Flavobacterium K3–15
(Schaaf et al., 1993), Hemophilus influenzae
(Fleischmann et al., 1995), and Bacillus subtilis
(Rowland and Taber, 1996). Both E. coli and B.
subtilis have two distinct ICS genes; one is involved in
siderophore biosynthesis and the other is involved in menaquinone
production (Daruwala et al., 1996, 1997; Müller et al., 1996;
Rowland and Taber, 1996). The biochemical properties of the two ICS
enzymes from E. coli are different (Daruwala et al., 1997;
Liu et al., 1990). Sequence analysis has revealed that the bacterial
ICS enzymes share homology with the chorismate-utilizing
enzymes AS and p-aminobenzoate synthase, suggesting that
they share a common evolutionary origin (Ozenberger et al.,
1989).Much biochemical and molecular data concerning the shikimate pathway in
plants have accumulated in recent years (Schmid and Amrhein, 1995;
Weaver and Hermann, 1997), but relatively little work has been done on
ICS from higher plants. The enzyme has been partially purified from
Galium mollugo cell cultures (Ledüc et al., 1991,
1997), but purification of the ICS protein to homogeneity has remained
elusive, probably because of instability of the enzyme.Our interests focus on the role of ICS in the regulation of chorismate
partitioning over the various pathways. Furthermore, we studied ICS in
C. roseus to gain insight into the biosynthesis of DHBA in
higher plants (Moreno et al., 1994). In this paper we report the first
purification, to our knowledge, of ICS to homogeneity from a plant
source and the cloning of the corresponding cDNA. 相似文献
9.
Biosynthesis of Lipoic Acid in Arabidopsis:
Cloning and Characterization of the cDNA for Lipoic Acid
Synthase 总被引:3,自引:0,他引:3 下载免费PDF全文
Lipoic acid is a coenzyme that is essential for the activity of enzyme complexes such as those of pyruvate dehydrogenase and glycine decarboxylase. We report here the isolation and characterization of LIP1 cDNA for lipoic acid synthase of Arabidopsis. The Arabidopsis LIP1 cDNA was isolated using an expressed sequence tag homologous to the lipoic acid synthase of Escherichia coli. This cDNA was shown to code for Arabidopsis lipoic acid synthase by its ability to complement a lipA mutant of E. coli defective in lipoic acid synthase. DNA-sequence analysis of the LIP1 cDNA revealed an open reading frame predicting a protein of 374 amino acids. Comparisons of the deduced amino acid sequence with those of E. coli and yeast lipoic acid synthase homologs showed a high degree of sequence similarity and the presence of a leader sequence presumably required for import into the mitochondria. Southern-hybridization analysis suggested that LIP1 is a single-copy gene in Arabidopsis. Western analysis with an antibody against lipoic acid synthase demonstrated that this enzyme is located in the mitochondrial compartment in Arabidopsis cells as a 43-kD polypeptide. 相似文献
10.
Characterization of the Polyoxin Biosynthetic Gene Cluster from
Streptomyces cacaoi and Engineered Production of Polyoxin
H 总被引:1,自引:0,他引:1
Wenqing Chen Tingting Huang Xinyi He Qingqing Meng Delin You Linquan Bai Jialiang Li Mingxuan Wu Rui Li Zhoujie Xie Huchen Zhou Xiufen Zhou Huarong Tan Zixin Deng 《The Journal of biological chemistry》2009,284(16):10627-10638
11.
Marcel Tappaz Marc Bitoun Isabelle Reymond & Alain Sergeant 《Journal of neurochemistry》1999,73(3):903-912
Cysteine sulfinate decarboxylase (CSD) is considered as the rate-limiting enzyme in the biosynthesis of taurine, a possible osmoregulator in brain. Through cloning and sequencing of RT-PCR and RACE-PCR products of rat brain mRNAs, a 2,396-bp cDNA sequence was obtained encoding a protein of 493 amino acids (calculated molecular mass, 55.2 kDa). The corresponding fusion protein showed a substrate specificity similar to that of the endogenous enzyme. The sequence of the encoded protein is identical to that encoded by liver CSD cDNA. Among other characterized amino acid decarboxylases, CSD shows the highest homology (54%) with either isoform of glutamic acid decarboxylase (GAD65 and GAD67). A single mRNA band, approximately 2.5 kb, was detected by northern blot in RNA extracts of brain, liver, and kidney. However, brain and liver CSD cDNA sequences differed in the 5' untranslated region. This indicates two forms of CSD mRNA. Analysis of PCR-amplified products of genomic DNA suggests that the brain form results from the use of a 3' alternative internal splicing site within an exon specifically found in liver CSD mRNA. Through selective RT-PCR the brain form was detected in brain only, whereas the liver form was found in liver and kidney. These results indicate a tissue-specific regulation of CSD genomic expression. 相似文献
12.
我们从重组的人α干扰素处理的单层HeLa细胞常规提取Poly(A)~+RNA作为逆转录合成cDNA第一链之模板,用引物-适配接头法在噬菌质粒pTz19R中构建cDNA文库。以~(32)p-标记的480bpIL-6cDNA片段作探针进行菌落原位及狭缝印迹杂交,筛选出6个阳性克隆。其中两个克隆并用限制酶切分析及DNA序列测定做进一步鉴定。结果证明,一个克隆的cDNA片段长1.3kb,含有人白介素6全长编码区;另一个的cDNA插入片段为0.9kb,缺乏信号肽及成熟IL-6N端30个残基的编码序列。 相似文献
13.
Wenli Li Jianhua Ju Scott R. Rajski Hiroyuki Osada Ben Shen 《The Journal of biological chemistry》2008,283(42):28607-28617
Tautomycin (TTM) is a highly potent and specific protein phosphatase
inhibitor isolated from Streptomyces spiroverticillatus. The
biological activity of TTM makes it an important lead for drug discovery,
whereas its spiroketal-containing polyketide chain and rare dialkylmaleic
anhydride moiety draw attention to novel biosynthetic chemistries responsible
for its production. To elucidate the biosynthetic machinery associated with
these novel molecular features, the ttm biosynthetic gene cluster
from S. spiroverticillatus was isolated and characterized, and its
involvement in TTM biosynthesis was confirmed by gene inactivation and
complementation experiments. The ttm cluster was localized to a 86-kb
DNA region, consisting of 20 open reading frames that encode three modular
type I polyketide synthases (TtmHIJ), one type II thioesterase (TtmT), five
proteins for methoxymalonyl-S-acyl carrier protein biosynthesis
(Ttm-ABCDE), eight proteins for dialkylmaleic anhydride biosynthesis and
regulation (TtmKLMNOPRS), as well as two additional regulatory proteins (TtmF
and TtmQ) and one tailoring enzyme (TtmG). A model for TTM biosynthesis is
proposed based on functional assignments from sequence analysis, which agrees
well with previous feeding experiments, and has been further supported by
in vivo gene inactivation experiments. These findings set the stage
to fully investigate TTM biosynthesis and to biosynthetically engineer new TTM
analogs.Tautomycin (TTM)2
is a polyketide natural product first isolated in 1987 from Streptomyces
spiroverticillatus (1).
The structure and stereochemistry of TTM were established on the basis of
chemical degradation and spectroscopic evidence
(2-4).
TTM contains several features not common to polyketide natural products,
including a spiroketal group, a methoxymalonate-derived unit, and an acyl
chain bearing a dialkylmaleic anhydride moiety. Structurally related to TTM is
tautomycetin (TTN), which was first isolated in 1989 from Streptomyces
griseochromogenes following the discovery of TTM
(5,
6). The structure of TTN was
deduced by chemical degradation and spectroscopic analysis
(6), and its stereochemistry
was established by comparison of spectral data with those of TTN degradation
products and synthetic fragments
(7). Both TTM and TTN exist as
tautomeric mixtures composed of two interconverting anhydride and diacid forms
in approximately a 5:4 ratio under neutral conditions
(Fig. 1A)
(1,
2).Open in a separate windowFIGURE 1.A, structures of TTM and TTN in anhydride or diacid forms, and
biosynthetic origin of the dialkylmaleic anhydride by feeding experiments
using 13C-labeled acetate and propionate. The
methoxymalonate-derived unit in TTM is highlighted by the dotted oval.
R, polyketide moiety of TTM or TTN. B, selected natural product
inhibitors of PP-1 and PP-2A featuring a spiroketal or dialkylmaleric
anhydride moiety. C, selected natural products containing a
dialkylmaleic anhydride moiety.Early studies of TTM revealed its ability to induce morphological changes
in leukemia cells (8). However,
it was later realized that TTM is a potent and specific inhibitor of protein
phosphatases (PPs) PP-1 and PP-2A
(9). PP-1 and PP-2A are two of
the four major serine/threonine protein phosphatases that regulate diverse
cellular events such as cell division, gene expression, muscle contraction,
glycogen metabolism, and neuronal signaling in eukaryotic cells
(10-12).
Many natural product PP-1 and PP-2A inhibitors are known, including okadaic
acid (13), calyculin-A
(14), phoslactomycin,
spirastrellolide, and cantharidin
(15)
(Fig. 1B), as well as
TTM (16,
17), and TTN
(18). They have served as
useful tools to study PP-involved intracellular events in vivo and as
novel leads for drug discovery
(10-12).
Among these PP inhibitors, TTM and TTN are unique because of their PP-1
selectivity. Despite their structural similarities, TTM exhibits potent
specific inhibition of PP-1 and PP-2A with IC50 values of 22-32
nm and only a slight preference for PP-1
(18). Conversely, TTN shows
nearly a 40-fold higher binding affinity to PP-1 (IC50 = 1.6
nm) than to PP-2A (IC50 = 62 nm)
(18). Because the major
structural differences between TTM and TTN reside in the region distal to the
dialkylmaleic anhydride moiety (Fig.
1A), it has been proposed that differences in these
moieties might be responsible for the PP-1 selectivity
(17-19).
Finally, TTN also has an impressive immuno-suppressive activity
(20,
21), which is apparently
devoid for TTM. Clearly, the structural differences between these two
polyketides translate into large, exploitable differences in bio-activities,
yet an understanding of the biosynthetic origins of these differences remains
elusive.The spiroketal and dialkylmaleic anhydride features of TTM are uncommon for
polyketide natural products, as is the methoxymalonate-derived unit
(Fig. 1A). Few studies
have been carried out for spiroketal biosynthesis, yet it is reasonably common
among the phosphatase inhibitors such as calyculin A, okadaic acid, and a few
others (Fig. 1B). Less
common, but still found in the phosphatase inhibitor cantharidin, as well as
TTM and TTN, is the dialkylmaleic anhydride moiety
(Fig. 1B); this unit
appears in a number of other natural products
(Fig. 1C), although
the biosynthetic steps leading to this reactive moiety (a protected version of
a dicarboxylate) have not been rigorously investigated. Feeding experiments
with 13C-labeled precursors indicated that the anhydride of TTM and
TTN is assembled from a propionate and an as yet undefined C-5 unit
(Fig. 1A), which would
require novel chemistry for polyketide biosynthesis
(22). TTM differentiates
itself from all known PP-1 and PP-2A inhibitors by virtue of its unique
combination of both the dialkymaleic anhydride and spiroketal
functionalities.Multiple total syntheses of TTM and a small number of analogs have been
reported, confirming the predicted structure and absolute stereochemistry and
facilitating structure-activity relationship studies on PP inhibition and
apoptosis induction (19,
23-25).
These studies revealed that: (i) the C22-C26 carbon chain and the
dialkylmaleic anhydride are the minimum requirements for TTM bioactivity; (ii)
the C18-C21 carbon chain and 22-hydroxy group are important for PP inhibition;
(iii) the spiroketal moiety determines the affinity to specific protein
phosphatases; (iv) the active form is most likely the dicarboxylate; and (v)
3′-epi-TTM exhibits 1,000-fold less activity than TTM. However, taken as
a whole, none of the analogs had an improved potency or selectivity for PP-1
inhibition than the natural TTM
(19,
22-25).
As a result, a more specific inhibitor of PP-1 is urgently awaited to
differentiate the physiological roles of PP-1 and PP-2A in vivo and
to explore PPs as therapeutic targets for drug discovery.We have undertaken the cloning and characterization of the TTM biosynthetic
gene cluster from S. spiroverticillatus as the first step toward
engineering TTM biosynthesis for novel analogs
(26). We report here: (i)
cloning and sequencing of the complete ttm gene cluster, (ii)
determination of the ttm gene cluster boundaries, (iii)
bioinformatics analysis of the ttm cluster and a proposal for TTM
biosynthesis, and (iv) genetic characterization of the TTM pathway to support
the proposed pathway. Of particular interest has been the identification of
genes possibly related to dialkylmaleic anhydride biosynthesis, the unveiling
of the ttm polyketide synthase (PKS) genes predicted to select and
incorporate four different starter and extender units for TTM production, and
the apparent lack of candidate genes associated with spiroketal formation.
These findings now set the stage to engineer TTM analogs for novel PP-1- and
PP-2A-specific inhibitors by applying combinatorial biosynthetic methods to
the TTM biosynthetic machinery. 相似文献
14.
15.
Genetic Analysis of Glutamate Transport and Glutamate Decarboxylase in Escherichia coli 总被引:13,自引:10,他引:3 下载免费PDF全文
The location of the Escherichia coli K-12 genes determining or regulating glutamate transport, and the location of the gene determining glutamate decarboxylase synthesis, were established by conjugation. The ability to grow on glutamate as the sole source of carbon and energy was used to select for glutamate transport recombinants. Two genes determining the ability to grow on glutamate as the sole source of carbon and energy were mapped. One (gltC) is located near mtl (mannitol), and the other (gltH) appears to be located between the gal (galactose) and trp (tryptophan) loci. The glutamate decarboxylase gene (gad) is strongly linked to gltC. The gltC(+) recombinants grow on glutamate much faster and accumulate this amino acid to a greater extent than do the gltH(+) recombinants. The gltH(+) gene functioned only in one female strain (P678), whereas the gltC gene functioned in all the female strains tested (P678, C600, W1). 相似文献
16.
A major regulatory feature of brain glutamate decarboxylase (GAD) is a cyclic reaction that controls the relative amounts of holoenzyme and apoenzyme [active and inactive GAD with and without bound pyridoxal 5'-phosphate (pyridoxal-P, the cofactor), respectively]. Previous studies have indicated that progression of the enzyme around the cycle should be stimulated strongly by the substrate, glutamate. To test this prediction, the effect of glutamate on the incorporation of pyridoxal-P into rat-brain GAD was studied by incubating GAD with [32P]pyridoxal-P, followed by reduction with NaBH4 to link irreversibly the cofactor to the enzyme. Adding glutamate to the reaction mixture strongly stimulated labeling of GAD, as expected. 4-Deoxypyridoxine 5'-phosphate (deoxypyridoxine-P), a close structural analogue of pyridoxal-P, was a competitive inhibitor of the activation of glutamate apodecarboxylase by pyridoxal-P (Ki = 0.27 microM) and strongly inhibited glutamate-dependent labeling of GAD. Analysis of labeled GAD by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed two labeled proteins with apparent molecular masses of 59 and 63 kDa. Both proteins could be purified by immunoaffinity chromatography on a column prepared with a monoclonal antibody to GAD, and both were labeled in a glutamate-dependent, deoxypyridoxine-P-sensitive manner, indicating that both were GAD. Three peaks of GAD activity (termed peaks I, II, and III) were separated by chromatography on phenyl-Sepharose, labeled with [32P]pyridoxal-P, purified by immunoaffinity chromatography, and analyzed by SDS-polyacrylamide gel electrophoresis. Peak I contained only the 59-kDa labeled protein. Peaks II and III contained the both the 59- and 63-kDa proteins, but in differing proportions.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
17.
18.
Some biochemical characteristics of L-glutamate decarboxylase (GAD) were compared using crude homogenates of the rat oviduct, ovary, and hypothalamus. As estimated by the measurement of CO2 production, the specific activities of oviductal and ovarian GAD were about 10 and 3% of the hypothalamic value, respectively. Stoichiometric formation of gamma-aminobutyric acid (GABA) and CO2 from L-glutamate could be observed in oviduct and hypothalamus, while in ovarian homogenates the production of CO2 was more than nine times that of GABA. Hypothalamic and tubal GAD showed similar time course, temperature dependence, and pH dependence. The dependence on protein concentration and on exogenous cofactor supply was also similar in these two tissues. The kinetic constant for L-glutamate as a substrate was nearly the same in oviduct (1.30 mM) and hypothalamus (1.64 mM). The responsiveness of tubal and hypothalamic GAD to various inhibitors was also similar. The present findings indicate that the oviductal and the hypothalamic GAD may be identical, and they suggest a possible GABAergic innervation of the Fallopian tube. 相似文献
19.
Rapid Inactivation of Brain Glutamate Decarboxylase by Aspartate 总被引:2,自引:2,他引:0
In the absence of its cofactor, pyridoxal 5'-phosphate (pyridoxal-P), glutamate decarboxylase is rapidly inactivated by aspartate. Inactivation is a first-order process and the apparent rate constant is a simple saturation function of the concentration of aspartate. For the beta-form of the enzyme, the concentration of aspartate giving the half-maximal rate of inactivation is 6.1 +/- 1.3 mM and the maximal apparent rate constant is 1.02 +/- 0.09 min-1, which corresponds to a half-time of inactivation of 41 s. The rate of inactivation by aspartate is about 25 times faster than inactivation by glutamate or gamma-aminobutyric acid (GABA). Inactivation is accompanied by a rapid conversion of holoenzyme to apoenzyme and is opposed by pyridoxal-P, suggesting that inactivation results from an alternative transamination of aspartate catalyzed by the enzyme, as previously observed with glutamate and GABA. Consistent with this mechanism pyridoxamine 5'-phosphate, an expected transamination product, was formed when the enzyme was incubated with aspartate and pyridoxal-P. The rate of transamination relative to the rate of decarboxylation was much greater for aspartate than for glutamate. Apoenzyme formed by transamination of aspartate was reactivated with pyridoxal-P. In view of the high rate of inactivation, aspartate may affect the level of apoenzyme in brain. 相似文献
20.