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1.
A major protein in the postsynaptic density fraction is -CAM kinase II, the -subunit of the Ca2+/calmodulin-dependent protein kinase. Autophosphorylation of the postsynaptic density-associated CaM kinase II is likely to be a crucial event in the induction of activity-dependent synaptic modification. This study focuses on the regulation and consequences of Ca2+-independent autophosphorylation of the enzyme. In isolated postsynaptic densities, a sub-stochiometric level of autophosphorylation in the presence of Ca2+ is sufficient to trigger maximal Ca2+-independent autophosphorylation of -CaM Kinase II. A major fraction of the sites phosphorylated in the absence of Ca2+ can be dephosphorylated by the endogenous phosphatase activity in the preparation. Ca2+-independent autophosphorylation is correlated with a drastic decrease in calmodulin binding to postsynaptic densities. This may represent a physiological mechanism that lowers the calmodulin trapping capacity of the organelle, thus increasing the availability of calmodulin to other elements within a spine. 相似文献
2.
Ogawa Noriyuki; Yabuta Naohiro; Ueno Yoshihisa; Izui Katsura 《Plant & cell physiology》1998,39(10):1010-1019
Phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31
[EC]
] of plantsundergoes regulatory phosphorylation in response to light ornutritional conditions. However, the nature of protein kinase(s)for this phosphorylation has not yet been fully elucidated.We separated a Ca2+-requiring protein kinase from Ca2+-independentone, both of which can phosphorylate maize leaf PEPC and characterizedthe former kinase after partial purification. Several linesof evidence indicated that the kinase is one of the characteristicCa2+-dependent but calmodulin-independent protein kinase (CDPK).Although the Mr, of native CDPK was estimated to be about 100kDa by gel permeation chromatography, in situ phosphorylationassay of CDPK in a SDS-polyacrylamide gel revealed that thesubunit has an Mr of about 50 kDa suggesting dimer formationor association with other protein(s). Several kinetic parameterswere also obtained using PEPC as a substrate. Although the CDPKshowed an ability of regulatory phosphorylation (Ser-15 in maizePEPC), no significant desensitization to feedback inhibitor,malate, could be observed presumably due to low extent of phosphorylation.The kinase was not specific to PEPC but phosphorylated a varietyof synthetic peptides. The possible physiological role of thiskinase was discussed.
1Present address: NEOS Central Research Laboratory, 1-1 Ohike-machi,Kosei-cho, Shiga, 520-3213 Japan.
2Present address: Chugai Pharmaceutical Co., Ltd., 1-135 Komakado,Gotemba, 412-0038 Japan.
4N.O. and N.Y. contributed equally to this work. 相似文献
3.
Expression and Localization of Phosphoenolpyruvate
Carboxylase in Developing and Germinating Wheat Grains 总被引:5,自引:0,他引:5 下载免费PDF全文
María-Cruz González Lidia Osuna Cristina Echevarría Jean Vidal Francisco J. Cejudo 《Plant physiology》1998,116(4):1249-1258
Phosphoenolpyruvate carboxylase (PEPC) activity and corresponding mRNA levels were investigated in developing and germinating wheat (Triticum aestivum) grains. During grain development PEPC activity increased to reach a maximum 15 d postanthesis. Western-blot experiments detected two main PEPC polypeptides with apparent molecular masses of 108 and 103 kD. The most abundant 103-kD PEPC subunit remained almost constant throughout the process of grain development and in the scutellum and aleurone layer of germinating grains. The less-abundant 108-kD polypeptide progressively disappeared during the second half of grain development and was newly synthesized in the scutellum and aleurone layer of germinating grains. PEPC mRNA was detected throughout the process of grain development; however, in germinating grains PEPC mRNA accumulated transiently in the scutellum and aleurone layer, showing a sharp maximum 24 h after imbibition. Immunolocalization studies revealed the presence of the enzyme in tissues with a high metabolic activity, as well as in the vascular tissue of the crease area of developing grains. A clear increase in PEPC was observed in the scutellar epithelium of grains 24 h after imbibition. The data suggest that the transiently formed PEPC mRNA in the scutellar epithelium encodes the 108-kD PEPC subunit. 相似文献
4.
Anne Nyholm Holdensen Jens Peter Andersen 《The Journal of biological chemistry》2009,284(18):12258-12265
Ion translocation by the sarcoplasmic reticulum Ca2+-ATPase
depends on large movements of the A-domain, but the driving forces have yet to
be defined. The A-domain is connected to the ion-binding membranous part of
the protein through linker regions. We have determined the functional
consequences of changing the length of the linker between the A-domain and
transmembrane helix M3 (“A-M3 linker”) by insertion and deletion
mutagenesis at two sites. It was feasible to insert as many as 41 residues
(polyglycine and glycine-proline loops) in the flexible region of the linker
without loss of the ability to react with Ca2+ and ATP and to form
the phosphorylated Ca2E1P intermediate, but the rate of
the energy-transducing conformational transition to E2P was reduced
by >80%. Insertion of a smaller number of residues gave effects gradually
increasing with the length of the insertion. Deletion of two residues at the
same site, but not replacement with glycine, gave a similar reduction as the
longest insertion. Insertion of one or three residues in another part of the
A-M3 linker that forms an α-helix (“A3 helix”) in
E2/E2P conformations had even more profound effects on the
ability of the enzyme to form E2P. These results demonstrate the
importance of the length of the A-M3 linker and of the position and integrity
of the A3 helix for stabilization of E2P and suggest that, during the
normal enzyme cycle, strain of the A-M3 linker could contribute to destabilize
the Ca2E1P state and thereby to drive the transition to
E2P.The sarco(endo)plasmic reticulum Ca2+-ATPase
(SERCA)2 is a
membrane-bound ion pump that transports Ca2+ against a steep
concentration gradient, utilizing the energy derived from ATP hydrolysis
(1–3).
It belongs to the family of P-type ATPases, in which the γ-phosphoryl
group of ATP is transferred to a conserved aspartic acid residue during the
reaction cycle. Both phospho and dephospho forms of the enzyme undergo
transitions between so-called E1 and E2 conformations
(Scheme 1). The E1 and
E1P states display specificity for reaction with ATP and ADP,
respectively (“kinase activity”), whereas E2P and
E2 react with water and Pi instead of nucleotide
(“phosphatase activity”). The E1 dephosphoenzyme of the
Ca2+-ATPase binds two Ca2+ ions with high affinity from
the cytoplasmic side, thereby triggering the phosphorylation from ATP. In
E1P, the Ca2+ ions are occluded with no access to either
side of the membrane, and Ca2+ is released to the luminal side
after the conformational transition to E2P, likely in exchange for
protons being countertransported. The structural organization and domain
movements leading to Ca2+ translocation have recently been
elucidated by crystallization of SERCA in various conformational states
thought to represent intermediates in the pump cycle
(4–7).
SERCA is made up of 10 membrane-spanning mostly helical segments, M1–M10
(numbered from the N terminus), of which M4–M6 and M8 contribute
liganding groups for Ca2+ binding, and a cytoplasmic headpiece
separated into three distinct domains, named A (“actuator”), P
(“phosphorylation”), and N (“nucleotide binding”). The
A-domain appears to undergo considerable movement during the functional cycle.
In the E1/E1P states, the highly conserved
TGE183S loop of the A-domain is at great distance from the
catalytic center containing nucleotide-binding residues and the phosphorylated
Asp351 of the P-domain, but during the Ca2E1P
→ E2P transition, the A-domain rotates ∼90° around an
axis perpendicular to the membrane, thereby moving the TGE183S loop
into close contact with the catalytic site such that Glu183 can
catalyze dephosphorylation of E2P
(8,
9). During the
dephosphorylation, Glu183 likely coordinates the water molecule
attacking the aspartyl phosphoryl bond and withdraws a hydrogen. Hence, the
movement of the A-domain during the Ca2E1P →
E2P transition is the event that changes the catalytic specificity
from kinase activity to phosphatase activity. During the dephosphorylation of
E2P → E2, there is only a slight change of the position
of the A-domain, and a large back-rotation is needed to reach the E1
form from E2; thus, the A-domain rotation defines the difference
between the E1/E1P class of conformations and the
E2/E2P class. Because the A-domain is physically connected
to transmembrane helices M1–M3 through the linker segments A-M1, A-M2,
and A-M3, the A-domain movement occurring during the
Ca2E1P → E2P transition may be a key event
in the opening of the Ca2+ sites toward the lumen, thus explaining
the coupling of ATP hydrolysis to Ca2+ translocation. An important
unanswered question is, however, how the movement of the A-domain is brought
about. Which are the driving forces that destabilize
Ca2E1P and/or stabilize E2P such that the
energy-transducing Ca2E1P → E2P transition
takes place? To answer this, it seems important to elucidate the exact roles
of the linkers. Intriguing results have been obtained by Suzuki and
co-workers, who demonstrated the importance of the A-M1 linker in connection
with luminal release of Ca2+ from E2P
(10). In this study, we have
addressed the role of the A-M3 linker. An alignment of two crystal structures
thought to resemble the Ca2E1P and
E2·Pi forms
(5), respectively, is shown in
Fig. 1. The A-domain rotation
is associated with formation of a helix (“A3 helix”) in the
N-terminal part of the A-M3 linker, and this helix seems to interact with a
helix bundle consisting of the P5–P7 helices of the P-domain, a feature
exhibited by all published crystal structures of the E2 type
(cf. supplemental Fig. S1 and Ref.
11). Moreover, when structures
of similar crystallographic resolution are compared (as in
Fig. 1), the non-helical part
of the A-M3 linker in E2-type structures has a higher relative
temperature factor (“B-factor”) than the corresponding
segment in Ca2E1P (Fig.
1C, thick part colored orange-red for
high temperature factor), thus suggesting a higher degree of freedom of
movement relative to Ca2E1P. Hence, the A-M3 linker
appears more strained in Ca2E1P compared with E2
forms, and the greater flexibility of the linker in E2 forms may
promote the formation of the A3 helix.Open in a separate windowSCHEME 1.Ca2+-ATPase reaction cycle.Open in a separate windowFIGURE 1.A-M3 linker configuration in E1- and E2-type crystal
structures. Crystal structures with Protein Data Bank codes 2zbd
(Ca2E1P analog) and 1wpg (E2·Pi
analog) are shown aligned. A, overview of structure 2zbd in
bluish colors with green A-M3 linker and structure 1wpg in
reddish colors with wheat A-M3 linker. B,
magnification of the A-M3 linker (corresponding to the red box in
A) with arrows indicating site 1, between Glu243
and Gln244, and site 2, between Gly233 and
Lys234, in both conformations. The green A-M3 linker to
the right is structure 2zbd. The wheat A-M3 linker to the left is
structure 1wpg. Note the kinked A3 helix forming part of the latter structure.
C, same A-M3 linker structures as in B but with the
magnitude of the temperature factor (B-factor) indicated in colors
(red > orange > yellow > green
> blue) and by tube diameter. Because the two crystal structures
selected here as E1- and E2-type representatives have
similar crystallographic resolution (2.40 and 2.30 Å, respectively), the
differences in temperature factor in specific regions provide direct
information about chain flexibility.Here, we have determined the functional consequences of changing the length
(and thereby likely the strain) of the A-M3 linker. Polyglycine and
glycine-proline loops of varying lengths were inserted at two different sites
in the linker (Fig. 1), and
deletions were also studied. Rather unexpectedly, we were able to insert as
many as 41 residues in one of the sites without loss of expression or ability
to react with Ca2+ and ATP, forming Ca2E1P, but
the Ca2E1P → E2P transition was greatly
affected. 相似文献
5.
H.R. Matthews 《The Journal of general physiology》1997,109(2):141-146
To study the actions of Ca2+ on “early” stages of the transduction cascade, changes in cytoplasmic calcium concentration (Ca2+
i) were opposed by manipulating Ca2+ fluxes across the rod outer segment membrane
immediately following a bright flash. If the outer segment was exposed to 0 Ca2+/0 Na+ solution for a brief period
immediately after the flash, then the period of response saturation was prolonged in comparison with that in
Ringer solution. But if the exposure to 0 Ca2+/0 Na+ solution instead came before or was delayed until 1 s after
the flash then it had little effect. The degree of response prolongation increased with the duration of the exposure to 0 Ca2+/0 Na+ solution, revealing a time constant of 0.49 ± 0.03 s. By the time the response begins to recover from saturation, Ca2+
i seems likely to have fallen to a similar level in each case. Therefore the prolongation
of the response when Ca2+
i was prevented from changing immediately after the flash seems likely to reflect the abolition of actions of the usual dynamic fall in Ca2+
i on an early stage in the transduction cascade at a site which is
available for only a brief period after the flash. One possibility is that the observed time constant corresponds to
the phosphorylation of photoisomerized rhodopsin. 相似文献
6.
Mette Laursen Maike Bublitz Karine Moncoq Claus Olesen Jesper Vuust M?ller Howard S. Young Poul Nissen J. Preben Morth 《The Journal of biological chemistry》2009,284(20):13513-13518
We have determined the structure of the sarco(endo)plasmic reticulum
Ca2+-ATPase (SERCA) in an E2·Pi-like form
stabilized as a complex with , an
ATP analog, adenosine 5′-(β,γ-methylene)triphosphate
(AMPPCP), and cyclopiazonic acid (CPA). The structure determined at 2.5Å
resolution leads to a significantly revised model of CPA binding when compared
with earlier reports. It shows that a divalent metal ion is required for CPA
binding through coordination of the tetramic acid moiety at a characteristic
kink of the M1 helix found in all P-type ATPase structures, which is expected
to be part of the cytoplasmic cation access pathway. Our model is consistent
with the biochemical data on CPA function and provides new measures in
structure-based drug design targeting Ca2+-ATPases, e.g.
from pathogens. We also present an extended structural basis of ATP modulation
pinpointing key residues at or near the ATP binding site. A structural
comparison to the Na+,K+-ATPase reveals that the
Phe93 side chain occupies the equivalent binding pocket of the CPA
site in SERCA, suggesting an important role of this residue in stabilization
of the potassium-occluded E2 state of Na+,K+-ATPase.The Ca2+-ATPase from sarco(endo)plasmic reticulum of rabbit
skeletal muscle
(SERCA,5 isoform 1a)
is a thoroughly studied member of the P-type ATPase family
(1). SERCA possesses 10
transmembrane helices (M1 through M10) with both the N terminus and the C
terminus facing the cytoplasmic side and three cytoplasmic domains, inserted
in loops between M2 and M3 (A-domain) and between M4 and M5 (P- and N-domain)
(2). The enzyme mediates the
uptake of Ca2+ ions into the lumen of the sarcoplasmic reticulum
(SR) after their release into the cytoplasm through calcium release channels
during muscle contraction (3).
SERCA, plasma membrane Ca2+-ATPase, and a third, Golgi-located
secretory pathway Ca2+-ATPase are important factors in calcium and
manganese homeostasis, transport, signaling, and regulation
(4,
5).Crystal structures of all major states in the reaction cycle of SERCA have
been determined. These include the Ca2E1·ATP
state (6,
7) with high affinity
Ca2+ binding sites accessible from the cytoplasmic side of the SR
membrane, the calcium-occluded
transition state (6), the open
E2P state with luminal facing ion binding sites that have low affinity for
Ca2+ and high affinity for protons
(8) and the proton-occluded
H2–3E2[ATP] state with a bound modulatory ATP
(9). This considerable amount
of structural information has turned the Ca2+-ATPase into a
valuable model system for studies on structural rearrangements that take place
during the catalytic cycle of P-type ATPases. SERCA is considered a promising
drug target in medical research, with a particular focus on prostate cancer
and infectious diseases. Several compounds have already been shown to bind and
inhibit SERCA by stabilizing the enzyme in a particular conformational state.
Thapsigargin (TG), cyclopiazonic acid (CPA), and 2,5-di-(tert-butyl)
hydroquinone (BHQ) stabilize an E2-like state, and 1,3-dibromo-2,4,6-tri
(methylisothiouronium)benzene stabilizes an E1-P-like conformation
(10–13).
CPA is a toxic indole tetramic acid first isolated from Penicillium
cyclopium (14) and later
found to be produced by Aspergillus versicolor and Aspergillus
flavus. Like TG, CPA specifically binds to and inhibits SERCA with
nanomolar affinity (15).
Indeed, CPA is widely used in biochemical and physiological studies on
Ca2+ signaling and muscle function, where it causes Ca2+
store depletion due to specific inhibition of Ca2+ reuptake by
SERCA. CPA and TG were originally proposed to bind to similar sites on SERCA
(16), but recent crystal
structures have shown a distinct site of interaction
(17,
18). Despite these structural
insights, a previously demonstrated magnesium dependence of CPA binding
(19) remained unexplained, and
opposing CPA binding modes were observed (see below).Tetramic acids are synthesized naturally, and more than 150 natural
derivatives have been isolated from bacterial and fungal species (reviewed in
Ref. 20). Tetramic acids
possessing a 3-acyl group have the ability to chelate divalent metal ions. For
instance, tenuazonic acid from the fungus Phoma sorghina has been
shown to form complexes with Ca2+ and Mg2+
(21), as well as heavier
metals such as Cu(II), Ni(II), and Fe(III)
(22).Previously published crystallographic structures of the SERCA·CPA
complex (PDB ID 2O9J and 2EAS) demonstrated that CPA binds within the proposed
calcium access channel of SERCA. However, the structures did not reveal a role
for magnesium, and the orientation of CPA within this binding site differed in
the two studies (17,
18). To address these
ambiguities, we have determined the crystal structure of SERCA in complex with
, AMPPCP (an ATP analog), and
Mn2+·CPA. The structure reveals novel insight into CPA
binding, which we find to be mediated by a divalent cation, as demonstrated by
means of the anomalous scattering properties of Mn2+. Further and
improved refinement using previously deposited data (PDB ID 2O9J and 2OA0), in
light of our new findings, also revealed a strong plausibility for a magnesium
ion bound at this site. Furthermore, we find a new configuration of the bound
AMPPCP nucleotide, addressing the modulatory role of ATP binding to the
E2·Pi occluded conformation of SERCA. 相似文献
7.
Nhiri Mohamed; Bakrim Naima; Pacquit Valerie; Hachimi-Messouak Zakia El; Osuna Lidia; Vidal Jean 《Plant & cell physiology》1998,39(2):241-46
Calcium-dependent phosphoenolpyruvate carboxylase protein kinasewas copurified with C4 phosphoenolpyruvate carboxylase (C4 PEPC)from illuminated Sorghum leaves during purification by variousprocedures. Isolated mesophyll cell protoplasts contained bothcalcium-dependent and -independent protein kinases. The latterwas induced by light and weak bases and was found to be themajor protein kinase phosphorylating C4 PEPC in the mesophyll. (Received July 29, 1997; Accepted November 28, 1997) 相似文献
8.
Muthuswamy Balasubramanyam Ramalingham A. Balaji Balakrishnan Subashini Viswanathan Mohan 《Experimental diabetes research》2000,1(4):275-287
Altered cytosolic Ca2+ is implicated in the aetiology
of many diseases including diabetes but there are
few studies on the mechanism(s) of the altered Ca2+
regulation. Using human lymphocytes, we studied
cytosolic calcium (Cai) and various Ca2+ transport
mechanisms in subjects with Type 2 diabetes
mellitus and control subjects. Ca2+-specific fluorescent
probes (Fura-2 and Fluo-3) were used to
monitor the Ca2+ signals. Thapsigargin, a potent and
specific inhibitor of the sarco(endo)plasmic reticulum
Ca2+-ATPase (SERCA), was used to study Ca2+-
store dependent Ca2+ fluxes. Significant (P < 0.05)
elevation of basal Cai levels was observed in
lymphocytes from diabetic subjects. Cai levels were
positively correlated with fasting, plasma glucose
and HbAlc. There was also a significant (P < 0.05)
reduction in plasma membrane calcium (PMCA)
ATPase activity in diabetic subjects compared to
controls. Cells from Type 2 diabetics exhibited an
increased Ca2+ influx (as measured both by Fluo-3
fliorescence and C45a assays) as a consequence of
of thapsigargin-mediated Ca2+ store depletion. Upon
addition of Mn2+ (a surrogate of Ca2+), the fura-2
fluorescence decayed in an exponential fashion and
the rate and extent of this decline was steeper and
greater in cells from type 2 diabetic patients. There
was also a significant (P < 0.05) difference in the
Na+/Ca2+ exchange activity in Type 2 diabetic
patients, both under resting conditions and after challenging the cells with thapsigargin, when the
internal store Ca2+ sequestration was circumvented.
Pharmacological activation of protein kinase C
(PKC) in cells from patients resulted in only partial
inhibition of Ca2+ entry. We conclude that cellular
Ca2+ accumulation in cells from Type 2 diabetes
results from (a) reduction in PMCA ATPase activity,
(b) modulation of Na+/Ca2+ exchange and (3)
increased Ca2+ influx across the plasma membrane. 相似文献
9.
Pathogen-Induced Changes in the Antioxidant Status of the
Apoplast in Barley Leaves 总被引:24,自引:0,他引:24 下载免费PDF全文
Leaves of two barley (Hordeum vulgare L.) isolines, Alg-R, which has the dominant Mla1 allele conferring hypersensitive race-specific resistance to avirulent races of Blumeria graminis, and Alg-S, which has the recessive mla1 allele for susceptibility to attack, were inoculated with B. graminis f. sp. hordei. Total leaf and apoplastic antioxidants were measured 24 h after inoculation when maximum numbers of attacked cells showed hypersensitive death in Alg-R. Cytoplasmic contamination of the apoplastic extracts, judged by the marker enzyme glucose-6-phosphate dehydrogenase, was very low (less than 2%) even in inoculated plants. Dehydroascorbate, glutathione, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase were present in the apoplast. Inoculation had no effect on the total foliar ascorbate pool size or the redox state. The glutathione content of Alg-S leaves and apoplast decreased, whereas that of Alg-R leaves and apoplast increased after pathogen attack, but the redox state was unchanged in both cases. Large increases in foliar catalase activity were observed in Alg-S but not in Alg-R leaves. Pathogen-induced increases in the apoplastic antioxidant enzyme activities were observed. We conclude that sustained oxidation does not occur and that differential strategies of antioxidant response in Alg-S and Alg-R may contribute to pathogen sensitivity. 相似文献
10.
Satoshi Matsuoka Debora A. Nicoll Zhaoping He Kenneth D. Philipson 《The Journal of general physiology》1997,109(2):273-286
The cardiac sarcolemmal Na+-Ca2+ exchanger is modulated by intrinsic regulatory mechanisms. A
large intracellular loop of the exchanger participates in the regulatory responses. We have proposed (Li, Z., D.A.
Nicoll, A. Collins, D.W. Hilgemann, A.G. Filoteo, J.T. Penniston, J.N. Weiss, J.M. Tomich, and K.D. Philipson.
1991. J. Biol. Chem. 266:1014–1020) that a segment of the large intracellular loop, the endogenous XIP region, has
an autoregulatory role in exchanger function. We now test this hypothesis by mutational analysis of the XIP region. Nine XIP-region mutants were expressed in Xenopus oocytes and all displayed altered regulatory properties.
The major alteration was in a regulatory mechanism known as Na+-dependent inactivation. This inactivation is
manifested as a partial decay in outward Na+-Ca2+ exchange current after application of Na+ to the intracellular
surface of a giant excised patch. Two mutant phenotypes were observed. In group 1 mutants, inactivation was
markedly accelerated; in group 2 mutants, inactivation was completely eliminated. All mutants had normal Na+ affinities. Regulation of the exchanger by nontransported, intracellular Ca2+ was also modified by the XIP-region
mutations. Binding of Ca2+ to the intracellular loop activates exchange activity and also decreases Na+-dependent
inactivation. XIP-region mutants were all still regulated by Ca2+. However, the apparent affinity of the group 1 mutants for regulatory Ca2+ was decreased. The responses of all mutant exchangers to Ca2+ application or removal
were markedly accelerated. Na+-dependent inactivation and regulation by Ca2+ are interrelated and are not completely independent processes. We conclude that the endogenous XIP region is primarily involved in movement
of the exchanger into and out of the Na+-induced inactivated state, but that the XIP region is also involved in regulation by Ca2+. 相似文献
11.
3-Methylcrotonyl-Coenzyme A Carboxylase Is a Component of the
Mitochondrial Leucine Catabolic Pathway in Plants 总被引:3,自引:1,他引:2
Marc D. Anderson Ping Che Jianping Song Basil J. Nikolau Eve Syrkin Wurtele 《Plant physiology》1998,118(4):1127-1138
3-Methylcrotonyl-coenzyme A carboxylase (MCCase) is a mitochondrial biotin-containing enzyme whose metabolic function is not well understood in plants. In soybean (Glycine max) seedlings the organ-specific and developmentally induced changes in MCCase expression are regulated by mechanisms that control the accumulation of MCCase mRNA and the activity of the enzyme. During soybean cotyledon development, when seed-storage proteins are degraded, leucine (Leu) accumulation peaks transiently at 8 d after planting. The coincidence between peak MCCase expression and the decline in Leu content provides correlative evidence that MCCase is involved in the mitochondrial catabolism of Leu. Direct evidence for this conclusion was obtained from radiotracer metabolic studies using extracts from isolated mitochondria. These experiments traced the metabolic fate of [U-14C]Leu and NaH14CO3, the latter of which was incorporated into methylglutaconyl-coenzyme A (CoA) via MCCase. These studies directly demonstrate that plant mitochondria can catabolize Leu via the following scheme: Leu → α-ketoisocaproate → isovaleryl-CoA → 3-methylcrotonyl-CoA → 3-methylglutaconyl-CoA → 3-hydroxy-3-methylglutaryl-CoA → acetoacetate + acetyl-CoA. These findings demonstrate for the first time, to our knowledge, that the enzymes responsible for Leu catabolism are present in plant mitochondria. We conclude that a primary metabolic role of MCCase in plants is the catabolism of Leu. 相似文献
12.
13.
Vincent Chaptal Michela Ottolia Gabriel Mercado-Besserer Debora A. Nicoll Kenneth D. Philipson Jeff Abramson 《The Journal of biological chemistry》2009,284(22):14688-14692
The mammalian Na+/Ca2+ exchanger, NCX1.1, serves as
the main mechanism for Ca2+ efflux across the sarcolemma following
cardiac contraction. In addition to transporting Ca2+, NCX1.1
activity is also strongly regulated by Ca2+ binding to two
intracellular regulatory domains, CBD1 and CBD2. The structures of both of
these domains have been solved by NMR spectroscopy and x-ray crystallography,
greatly enhancing our understanding of Ca2+ regulation.
Nevertheless, the mechanisms by which Ca2+ regulates the exchanger
remain incompletely understood. The initial NMR study showed that the first
regulatory domain, CBD1, unfolds in the absence of regulatory Ca2+.
It was further demonstrated that a mutation of an acidic residue involved in
Ca2+ binding, E454K, prevents this structural unfolding. A
contradictory result was recently obtained in a second NMR study in which
Ca2+ removal merely triggered local rearrangements of CBD1. To
address this issue, we solved the crystal structure of the E454K-CBD1 mutant
and performed electrophysiological analyses of the full-length exchanger with
mutations at position 454. We show that the lysine substitution replaces the
Ca2+ ion at position 1 of the CBD1 Ca2+ binding site and
participates in a charge compensation mechanism. Electrophysiological analyses
show that mutations of residue Glu-454 have no impact on Ca2+
regulation of NCX1.1. Together, structural and mutational analyses indicate
that only two of the four Ca2+ ions that bind to CBD1 are important
for regulating exchanger activity.Cardiac contraction/relaxation relies upon Ca2+ fluxes across
the plasma membrane (sarcolemma) of cardiomyocytes. Rapid Ca2+
influx (primarily through L-type Ca2+ channels) triggers the
release of additional Ca2+ from the sarcoplasmic reticulum
(SR),4 resulting in
cardiomyocyte contraction. Removal of cytosolic Ca2+ by reuptake
into the SR (through the SR Ca2+-ATPase) and expulsion from the
cell (primarily through the Na+/Ca2+ exchanger, NCX1.1)
results in relaxation (1).
Altered Ca2+ cycling is observed in a number of pathophysiological
situations including ischemia, hypertrophy, and heart failure
(2). Understanding the function
and regulation of NCX1.1 is thus of fundamental importance to understand
cardiac physiology.NCX1.1 utilizes the electrochemical potential of the Na+
gradient to extrude Ca2+ in a ratio of three Na+ ions to
one Ca2+ ion (3). In
addition to transporting both Na+ and Ca2+, NCX1.1 is
also strongly regulated by these two ions. Intracellular Na+ can
induce NCX1.1 to enter an inactivated state, whereas Ca2+ bound to
regulatory sites removes Na+-dependent inactivation and also
activates Na+/Ca2+ exchange
(3). These regulatory sites are
located on a large cytoplasmic loop (∼500 residues located between
transmembrane helices V and VI) containing two calcium binding domains (CBD1
and CBD2), which sense cytosolic Ca2+ levels. We have previously
shown that Ca2+ binding to the primary site in CBD2 is required for
full exchange regulation (4);
CBD1, however, is a site of higher affinity and appears to dominate the
activation of exchange activity by Ca2+.Both CBDs have an immunoglobulin fold formed from two antiparallel β
sheets generating a β sandwich with a differing number of Ca2+
ions coordinated at the tip of the domain
(4,
5). CBD1 binds four
Ca2+ ions, whereas CBD2 binds only two Ca2+ ions. An
initial NMR study revealed a local unfolding of the upper portion of CBD1 upon
release of Ca2+ (6).
In contrast, CBD2 did not display an unfolding response upon Ca2+
removal. A comparative analysis between CBDs revealed a difference in charge
at residues in equivalent positions near the Ca2+ coordination
site; Glu-454 in CBD1 is replaced by Lys-585 in CBD2. The unstructuring of
CBD1 upon Ca2+ removal was alleviated by reversing the charge of
the acidic residue (E454K) involved in Ca2+ coordination
(6). Previously, we solved the
structures of the Ca2+-bound and -free conformations of CBD2 and
revealed a charge compensation mechanism involving Lys-585
(4). The positively charged
lysine residue assumes the position of one of the Ca2+ ions upon
Ca2+ depletion, permitting CBD2 to retain its overall fold
(4). A similar phenomenon is
predicted to take place in E454K-CBD1 mutant. In addition, Hilge et
al. (6) showed that the
E454K mutation of CBD1 decreases Ca2+ affinity to a level similar
to that of CBD2 and suggested that the E454K mutation would cause the loss of
primary regulation of NCX1.1 by CBD1.The significance of some of these observations is unclear as a recent NMR
study (7) of CBD1 under more
physiologically relevant conditions revealed no significant alteration in
tertiary structure in the absence of Ca2+. It was hypothesized that
Ca2+ binding induces localized conformational and dynamic changes
involving several of the binding site residues. To clarify this issue, we
solved the crystal structure of the E454K-CBD1 mutant and examined the
functional effects of different CBD1 mutations in the full-length NCX1.1. The
results indicate that charge compensation is indeed provided by the residue
Lys-454 to replace one Ca2+, whereas the overall E454K-CBD1
structure is only slightly perturbed. The charge compensation, however, has no
impact on Ca2+ regulation of NCX1.1. 相似文献
14.
15.
Di Paola M Zaccagnino P Montedoro G Cocco T Lorusso M 《Journal of bioenergetics and biomembranes》2004,36(2):165-170
Several observations have been reported in the last years indicating that ceramide may activate the mitochondrial route of apoptosis. We show here that on addition of either C2- or C16-ceramide to mitochondria isolated from rat heart and suspended in a saline medium, release of cytochrome c and apoptosis-inducing factor (AIF) from the intermembrane space takes place. The release process is Ca2+ -independent and is not inhibited by Cyclosporin A (CsA). For the protein release process to occur, the presence of an oxidizable substrate is required. When mitochondria are suspended in sucrose instead of potassium medium, only short chain C2-ceramide causes cytochrome c release through a Ca2+ -dependent and CsA sensitive mitochondrial permeability transition (MPT) mechanism. The latter effect appears to be related to the membrane potential dissipating ability exhibited by short chain C2-ceramide. 相似文献
16.
Abstract: A new protein kinase modulated by S-100 (tentatively referred to as protein kinase X) was partially purified from pig brain extracts. The activity of protein kinase X, which was independent of Ca2+ , was demonstrated when protamine (free base), but not protamine sulfate and other proteins (including histone), was used as substrate. The enzyme activity, found to distribute in both soluble and particulate fractions and to occur at the highest level in brain compared with other tissues (heart, kidney, liver, skeletal muscle, spleen, and testis) of rats, was also modulated by other acidic proteins (calmodulin, troponin C, and stimulatory modulator) in a Ca2+ -independent manner. S-100 and other acidic proteins appeared to function as "substrate modifiers" by interacting with protamine (a highly basic protein), but not with the enzyme, thus rendering protamine in the complex a superior phosphate acceptor. The two isoforms of S-100 (i.e., a and b) were equally effective. Although the enzyme was not inhibited by many agents (trifluoperazine, melittin, cytotoxin I, polymyxin B, and spermine) shown to inhibit markedly phospholipid/Ca2+ - or calmodulin/Ca2+ -stimulated protein kinase, gossypol was found to inhibit specifically protein kinase X. The present findings suggest that S-100, a major acidic protein specific to nervous system, may promote phosphorylation by protein kinase X of certain neural proteins resembling protamine or containing protamine-like domains, in addition to its presumed role of a low-affinity Ca2+ -binding protein. 相似文献
17.
Arturo Hernández-Cruz Ariel L. Escobar Nicolás Jiménez 《The Journal of general physiology》1997,109(2):147-167
The role of ryanodine-sensitive intracellular Ca2+ stores present in nonmuscular cells is not yet completely understood. Here we examine the physiological parameters determining the dynamics of caffeine-induced
Ca2+ release in individual fura-2–loaded sympathetic neurons. Two ryanodine-sensitive release components were
distinguished: an early, transient release (TR) and a delayed, persistent release (PR). The TR component shows
refractoriness, depends on the filling status of the store, and requires caffeine concentrations ≥10 mM. Furthermore, it is selectively suppressed by tetracaine and intracellular BAPTA, which interfere with Ca2+-mediated feedback loops, suggesting that it constitutes a Ca2+-induced Ca2+-release phenomenon. The dynamics of release is
markedly affected when Sr2+ substitutes for Ca2+, indicating that Sr2+ release may operate with lower feedback
gain than Ca2+ release. Our data indicate that when the initial release occurs at an adequately fast rate, Ca2+ triggers further release, producing a regenerative response, which is interrupted by depletion of releasable Ca2+ and
Ca2+-dependent inactivation. A compartmentalized linear diffusion model can reproduce caffeine responses:
When the Ca2+ reservoir is full, the rapid initial Ca2+ rise determines a faster occupation of the ryanodine receptor Ca2+ activation site giving rise to a regenerative release. With the store only partially loaded, the slower initial
Ca2+ rise allows the inactivating site of the release channel to become occupied nearly as quickly as the activating
site, thereby suppressing the initial fast release. The PR component is less dependent on the store''s Ca2+ content.
This study suggests that transmembrane Ca2+ influx in rat sympathetic neurons does not evoke widespread amplification by CICR because of its inability to raise [Ca2+] near the Ca2+ release channels sufficiently fast to overcome
their Ca2+-dependent inactivation. Conversely, caffeine-induced Ca2+ release can undergo considerable amplification especially when Ca2+ stores are full. We propose that the primary function of ryanodine-sensitive stores in
neurons and perhaps in other nonmuscular cells, is to emphasize subcellular Ca2+ gradients resulting from agonist-induced intracellular release. The amplification gain is dependent both on the agonist concentration and on
the filling status of intracellular Ca2+ stores. 相似文献
18.
Mi?a Mojca Cajnko Maja Maru?i? Matic Kisovec Nejc Rojko Mojca Ben?ina Simon Caserman Gregor Anderluh 《PloS one》2015,10(6)
Listeria monocytogenes is a food and soil-borne pathogen that secretes a pore-forming toxin listeriolysin O (LLO) as its major virulence factor. We tested the effects of LLO on an intestinal epithelial cell line Caco-2 and compared them to an unrelated pore-forming toxin equinatoxin II (EqtII). Results showed that apical application of both toxins causes a significant drop in transepithelial electrical resistance (TEER), with higher LLO concentrations or prolonged exposure time needed to achieve the same magnitude of response than with EqtII. The drop in TEER was due to pore formation and coincided with rearrangement of claudin-1 within tight junctions and associated actin cytoskeleton; however, no significant increase in permeability to fluorescein or 3 kDa FITC-dextran was observed. Influx of calcium after pore formation affected the magnitude of the drop in TEER. Both toxins exhibit similar effects on epithelium morphology and physiology. Importantly, LLO action upon the membrane is much slower and results in compromised epithelium on a longer time scale at lower concentrations than EqtII. This could favor listerial invasion in hosts resistant to E-cadherin related infection. 相似文献
19.
Calcium-Dependent Protein Phosphorylation May Mediate the
Gibberellic Acid Response in Barley Aleurone 总被引:6,自引:0,他引:6 下载免费PDF全文
Peptide substrates of well-defined protein kinases were microinjected into aleurone protoplasts of barley (Hordeum vulgare L. cv Himalaya) to inhibit, and therefore identify, protein kinase-regulated events in the transduction of the gibberellin (GA) and abscisic acid signals. Syntide-2, a substrate designed for Ca2+- and calmodulin (CaM)-dependent kinases, selectively inhibited the GA response, leaving constitutive and abscisic acid-regulated events unaffected. Microinjection of syntide did not affect the GA-induced increase in cytosolic [Ca2+], suggesting that it inhibited GA action downstream of the Ca2+ signal. When photoaffinity-labeled syntide-2 was electroporated into protoplasts and cross-linked to interacting proteins in situ, it selectively labeled proteins of approximately 30 and 55 kD. A 54-kD, soluble syntide-2 phosphorylating protein kinase was detected in aleurone cells. This kinase was activated by Ca2+ and was CaM independent, but was inhibited by the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (250 μm), suggesting that it was a CaM-domain protein kinase-like activity. These results suggest that syntide-2 inhibits the GA response of the aleurone via an interaction with this kinase, implicating the 54-kD kinase as a Ca2+-dependent regulator of the GA response in these cells. 相似文献