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81.
Tadao Hashimoto Yukuo Yoshida Kunio Tagawa 《Journal of bioenergetics and biomembranes》1990,22(1):27-38
An intrinsic ATPase inhibitor inhibits the ATP-hydrolyzing activity of mitochondrial F1F0-ATPase and is released from its binding site on the enzyme upon energization of mitochondrial membranes to allow phosphorylation of ADP. The mitochondrial activity to synthesize ATP is not influenced by the absence of the inhibitor protein. The enzyme activity to hydrolyze ATP is induced by dissipation of the membrane potential in the absence of the inhibitor. Thus, the inhibitor is not responsible for oxidative phosphorylation, but acts only to inhibit ATP hydrolysis by F1F0-ATPase upon deenergization of mitochondrial membranes. The inhibitor protein forms a regulatory complex with two stabilizing factors, 9K and 15K proteins, which facilitate the binding of the inhibitor to F1F0-ATPase and stabilize the resultant inactivated enzyme. The 9K protein, having a sequence very similar to the inhibitor, binds directly to F1 in a manner similar to the inhibitor. The 15K protein binds to the F0 part and holds the inhibitor and the 9K protein on F1F0-ATPase even when one of them is detached from the F1 part. 相似文献
82.
Techel D. Gebauer G. Kohler W. Braumann T. Jastorff B. Rensing L. 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1990,159(6):695-706
Summary Pulses of some Ca2+ channel blockers (dantrolene, Co2+, nifedipine) and calmodulin inhibitors (chlorpromazine) lead to medium (maximally 5–9 h) phase shifts of the circadian conidiation rhythm ofNeurospora crassa. Pulses of high Ca2+, or of low Ca2+, a Ca2+ ionophore (A23187) together with Ca2+, and other Ca2+ channel blockers (La3+, diltiazem), however, caused only minor phase shifts. The effect of these substances (A 23187) and of different temperatures on the Ca2+ release from isolated vacuoles was analyzed by using the fluorescent dye Fura-2. A 23187 and higher temperatures increased the release drastically, whereas dantrolene decreased the permeation of Ca2+ (Cornelius et al., 1989).Pulses of 8-PCTP-cAMP, IBMX and of the cAMP antagonist RP-cAMPS, also caused medium (maximally 6–9 h) phase shifts of the conidiation rhythm. The phase response curve of the agonist was almost 180° out of phase with the antagonist PRC. In spite of some variability in the PRCs of these series of experiments all showed maximal shifts during ct 0–12. The variability of the response may be due to circadian changes in the activity of phosphodiesterases: After adding cAMP to mycelial extracts HPLC analysis of cAMP metabolites showed significant differences during a circadian period with a maximum at ct 0.Protein phosphorylation was tested mainly in an in vitro phosphorylation system (with35S-thio -ATP). The results showed circadian rhythmic changes predominantly in proteins of 47/48 kDa. Substances and treatments causing phase-shifts of the conidiation rhythm also caused changes in the phosphorylation of these proteins: an increase was observed when Ca2+ or cAMP were added, whereas a decrease occurred upon addition of a calmodulin inhibitor (TFP) or pretreatment of the mycelia with higher (42° C) temperatures.Altogether, the results indicate that Ca2+-calmodulin-dependent and cAMP-dependent processes play an important, but perhaps not essential, role in the clock mechanism ofNeurospora. Ca2+ calmodulin and the phosphorylation state of the 47/48-kDa proteins may have controlling or essential functions for this mechanism. 相似文献
83.
Mark Phillippe Trevania Saunders Shrikar Bangalore 《In vitro cellular & developmental biology. Plant》1990,26(4):369-378
Summary The following studies were undertaken to develop a cultured uterine myocyte model which would allow further clarification
of the adrenergic signal transduction mechanisms utilized by these myocytes. After mechanical removal of the endometrium,
rabbit uterine myoctes were isolated by an overnight enzymatic disaggregation using collagenase and DNase I. The isolated
myocytes were maintained in culture in 75-cm2 flasks containing Waymouth's MB 751/1 medium-10% fetal bovine serum along with 10−8
M estradiol, penicillin, streptomycin, and Fungizone. The phase contrast and electron micrographic appearance of these cells
was consistent with that previously reported for smooth muscle myocytes in culture. Immunocytochemical studies utilizing monoclonal
anti-alpha-smooth muscle actin antibodies confirmed the presence of smooth muscle actin in these cultured myocytes. Western
blot studies similarly confirmed the presence of alpha-smooth muscle actin in rabbit myometrial tissue and the cultured myocytes,
both the primary and F1 generation. After prelabeling the myocytes with [3H]inositol, adrenergic stimulation experiments demonstrated alpha-1 receptor mediated stimulation of inositol phosphates.
Beta receptor stimulation experiments confirmed cAMP production in these cultured myocytes, and the ability of clonidine,
an alpha-2 agonist, to inhibit forskolin stimulated cAMP production confirmed the presence of functional alpha-2 adrenergic
receptors in these myocytes. In conclusion, these cultured rabbit uterine myocytes have provided an in vitro model which can
be utilized to further clarify the adrenergic receptor signal transduction mechanisms in genital tract smooth muscle.
This research was supported by grant HD-22063 from the National Institutes of Health, Bethesda, MD. 相似文献
84.
Time-Dependent Increase in Protein Phosphorylation Following One-Trial Enhancement in Hermissenda 总被引:1,自引:1,他引:0
Abstract: One-trial conditioning of the nudibranch mollusk Hermissenda produces short- and long-term changes in excitability (enhancement) of identified sensory neurons. To investigate the biochemical mechanisms underlying this example of plasticity, we have examined changes in protein phosphorylation at different times following the in vitro conditioning trial. Changes in the incorporation of 32 PO4 into proteins were determined using two-dimensional polyacrylamide gel electrophoresis, autoradiography, and densitometry. Conditioning resulted in increases in levels of several phosphoproteins, five of which, ranging in apparent molecular mass from 22 to 55 kDa, were chosen for analysis. The increased phosphorylation of the 46- and 55-kDa phosphoproteins detected 2 h postconditioning was significantly greater than the level of phosphorylation detected in an unpaired control group, indicating that long-term enhancement is pairing specific. Statistically significant increases in phosphorylation as compared with the control group that received only light were detected immediately after conditioning (5 min) for the 55-, 46-, and 22-kDa phosphoproteins, at 1 h for the 55- and 46-kDa phosphoproteins, and at 2 h for the 55-, 46-, and 22-kDa phosphoproteins. The 46- and 55-kDa phosphoproteins are putative structural proteins, and the 22-kDa phosphoprotein is proposed to be a protein kinase C substrate previously identified in Hermissenda following multitrial classical conditioning. Time-dependent increases in protein phosphorylation may contribute to the induction and maintenance of different memory stages expressed in sensory neurons after one-trial conditioning. 相似文献
85.
86.
Abstract: Antibody Ab262 was raised against a synthetic τ peptide (SKIGSTENLK, amino acids 258–267 of τ, termed Ser262 peptide). The antibody was more reactive with Ser262 peptide and unphosphorylated τ than a related phosphopeptide [SKIGS(P)TENLK, termed P-Ser262 peptide] and τ phosphorylated by a partially purified kinase, glycogen synthase kinase (GSK) 3β. Ab262 reacted poorly with a peptide having the sequence DRVQSKIGSLD (amino acids 348–358). Treatment of P-Ser262 peptide or GSK 3β phosphorylated τ with alkaline phosphatase increased Ab262 immunoreactivity, indicating that Ab262 is a reagent useful for studying τ phosphorylation at the Ser262 residue. The Ab262 immunoreactivity was detected in τ from normal brains and Alzheimer paired helical filament (PHF-τ) and in PHFs. Alkaline phosphatase treatment had no effect on the Ab262 immunoreactivity of normal τ and PHF-τ but altered the Tau-1 and PHF-1 immunoreactivities. τ proteins from rat brains at 3 and 8 h postmortem exhibited 5 and 19%, respectively, more Ab262 immunoreactivity than τ from fresh tissues. In comparison, rat τ at 8 h postmortem was 40% more immunoreactive with Tau-1. The results suggest that Ser262 is not a major phosphorylation site in vivo. Moreover, there is little or no difference between PHF-τ and normal τ in the extent of phosphorylation at Ser262 . 相似文献
87.
Signalling by protein kinase C isoforms in the heart 总被引:11,自引:0,他引:11
Understanding transmembrane signalling process is one of the major challenge of the decade. In most tissues, since Fisher and Krebs's discovery in the 1950's, protein phosphorylation has been widely recognized as a key event of this cellular function. Indeed, binding of hormones or neurotransmitters to specific membrane receptors leads to the generation of cytosoluble second messengers which in turn activate a specific protein kinase. Numerous protein kinases have been so far identified and roughly classified into two groups, namely serine/threonine and tyrosine kinases on the basis of the target amino acid although some more recently discovered kinases like MEK (or MAP kinase kinase) phosphorylate both serine and tyrosine residues.Protein kinase C is a serine/threonine kinase that was first described by Takai et al. [1] as a Ca- and phospholipid-dependent protein kinase. Later on, Kuo et al. [2] found that PKC was expressed in most tissues including the heart. The field of investigation became more complicated when it was found that the kinase is not a single molecular entity and that several isoforms exist. At present, 12 PKC isoforms and other PKC-related kinases [3] were identified in mammalian tissues. These are classified into three groups. (1) the Ca-activated -, -,and -PKCs which display a Ca-binding site (C2); (2) the Ca-insensitive -, -, -, -, and -PKCs. The kinases that belong to both of these groups display two cystein-rich domains (C1) which bind phorbol esters (for recent review on PKC structure, see [4]). (3) The third group was named atypical PKCs and include , , and -PKCs that lack both the C2 and one cystein-rich domain. Consequently, these isoforms are Ca-insensitive and cannot be activated by phorbol esters [5]. In the heart. evidence that multiple PKC isoforms exist was first provided by Kosaka et al. [6] who identified by chromatography at least two PKC-related isoenzymes. Numerous studies were thus devoted to the biochemical characterization of these isoenzymes (see [7] for review on cardiac PKCs) as well as to the identification of their substrates.This overview aims at updating the present knowledge on the expression, activation and functions of PKC isoforms in cardiac cells. (Mol Cell Biochem 157: 65–72, 1996) 相似文献
88.
Phosphorylation of cardiac junctional and free sarcoplasmic reticulum (SR) by protein kinase C (PKC) isoforms and was investigated. Both SR and PKC were isolated from canine heart. Junctional and free SR vesicles were prepared by calcium-phosphate-loading. The substrate specificities of PKC and PKC were found to be similar in both SR fractions. A high molecular weight junctionally-associated protein was phosphorylated by PKA, PKC and an endogenous Ca2+/calmodulin-dependent protein kinase activity: the highest levels of phosphate incorporation being catalysed by the latter kinase. In addition to this high molecular weight junctionally-associated protein, PKC induced phosphorylation of 45, 96 kDa and several proteins of greater than 200 kDa in junctional SR. A protein of 96 kDa was phosphorylated by both isoforms in junctional and free SR. The major substrate for PKA, PKC, PKC and the Ca2+/calmodulin-dependent protein kinase, in both junctional and free SR, was phospholamban. Although the phosphorylation of phospholamban by PKC was activated by Ca2+, a component of this activity appeared to be independent of Ca2+. PKC-mediated phosphorylation of phospholamban was fully activated by 1 M Ca2+ whereas the Ca2+/calmodulin dependent kinase required concentrations in excess of 5 M Ca2+. In the in vitro system employed in these studies, the concentrations of either PKC or the catalytic subunit of PKA required to phosphorylate phospholamban were found to be similar. In addition, in the presence of a 15 kDa sarcolemmal-associated protein, which becomes phosphorylated upon activation of PKC in vivo, phosphorylation of phospholamban by PKC was unaffected. These results demonstrate that, although substrates for both subtypes are found in both junctional and free SR, PKC and PKC do not show differences in selectivity towards these substrates.Abbreviations Ca2+
free calcium
- CaM kinase
Ca2+/calmodulin-dependent protein kinase
- DTT
dithiothreitol
- EDTA
ethylenediaminetetraacetic acid
- EGTA
ethylene glycol bis(b-aminoethylether)-N,N,N,N-tetraacetic acid
- FSR
free sarcoplasmic reticulum
- JSR
junctional sarcoplasmic reticulum
- PKC
protein kinase C
- PS
phosphatidylserine
- SDS
sodium dodecyl sulfate
- SAG
1-stearoyl-2-arachidonylglycerol
- TPCK
L-1-tosylamido-2-phenylethyl chloromethyl ketone
- Tris/HCI
tris(hydroxymethyl)aminomethane hydrochloride
This work was supported by a grant (to S.K.) from the Heart and Stroke Foundation of B.C. and Yukon. The costs of publication of this article were defrayed in part by the payment of page charges This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.Recipient of a Studentship form the Heart and Stroke Foundation of Canada. 相似文献
89.
Kaumann Alberto J. Sanders Louise Lynham James A. Bartel Sabine Kuschel Meike Karczewski Peter Krause Ernst-Georg 《Molecular and cellular biochemistry》1996,163(1):113-123
Evidence from ventricular preparations of cat, sheep, rat and dog suggests that both 1-adrenoceptors (1AR) and 2-adrenoceptors (2AR) mediate positive inotropic effects but that only 1AR do it through activation of a cAMP pathway. On the other hand, our evidence has shown that both 1 AR and 2 AR hasten relaxation of isolated human myocardium consistent with a common cAMP pathway. We have now investigated in the isolated human right atrial appendage, a tissue whose -AR comprise around 2/3 of 1AR and 1/3 of 2AR, whether or not 2AR-mediated effects occur via activation of a cAMP pathway. We carried out experiments on atria obtained from patients without advanced heart failure undergoing open heart surgery. To activate 2AR, we used the 2AR-selective ligand zinterol. Experiments were carried out on paced atrial strips (1 Hz) and tissue homogenates and membrane particles. Zinterol caused positive inotropic and lusitropic (i.e. reduction of t1:2 of relaxation) effects with EC50 values of 3 and 2 nM, respectively. The zinterol-evoked effects were unaffected by the AR-selective antagonist CGP 20712A (300 nM) but blocked surmountably by the 2AR-selective antagonist ICI 118551 (50 nM) which reduced both EC50 values to 1 M. Zinterol stimulated adenylyl cyclase activity with an EC50 of 30 nM and intrinsic activity of 0.75 with respect to (–)-isoprenaline (600 M); the effects were resistant to blockade by CGP 20712A (300 nM) but antagonised surmountably by ICI 118551 (50 nM). Zinterol bound to membrane PAR labelled with (–)-[125I] cyanopindolol with higher affinity for 2AR than for - 1 AR; the binding to 2AR but not to - BAR was reduced by GTPyS (10 M). In the presence of CGP 20712A (300 nM) (–)-isoprenaline (400 M); (to activate both 1AR and 2AR maximally) and zinterol (10 M); increased contractile force 3.4-fold and 2.5-fold respectively and reduced relaxation tut by 32% and 18% respectively. These effects of (–)-isoprenaline and zinterol were associated (5 min incubation) with phosphorylation (pmol P/mg supernatant protein) of troponin I and C-protein to values of 8.4 ± 2.0 vs 12.4 ± 2.3 and 10.1 ± 2.5 vs 8.6 ± 1.6 respectively. (–)-Isoprenaline and zinterol also caused phosphorylation of phospholamban (1.8 ± 0.3 vs 0.4 ± 0.1 pmol P/mg respectively) specifically at serine residues. We conclude that in human atrial myocardium activation of both 1AR and 2AR leads to cAMP-dependent phosphorylation of proteins involved in augmenting both contractility and relaxation. 相似文献
90.
Cathrine Lillo Lucy H. Smith Hugh G. Nimmo Malcolm B. Wilkins 《Physiologia plantarum》1996,98(1):140-146
Nitrate reductase (NR, EC 1.6.6.1) was tested in crude extracts of leaves from Bryophyllum fedtschenkoi plants growing under alternating light/darkness as well as in excised leaves kept in continuous light or darkness. In most extracts NR activity was inhibited 20–80% by 5 m M Mg2+ A light or darkness shift (30 min darkness) during the first part of the photoperiod gave an increase in the Mg2+ inhibition and a decrease in NR activity. Magnesium ion inhibition of NR also showed diurnal variations. Strongest inhibition was found in extracts made during the latter part of the photoperiod and start of the dark period. Pre-incubation of crude extracts with ATP increased Mg2+ inhibition, indicating that phosphorylation of NR is involved in regulation of NR in Crassulacean acid metabolism (CAM) plants. In continuous light an increase in Mg2+ inhibition occurred after 20 h and 40 h, indicating a rhythm in the phosphorylation of NR. A delay in the production of nitrite in the assay (hysteresis) was generally seen in extracts susceptible to Mg2+ inhibition. The rhythms related to NR activity showed the same period length (20 h) as the rhythm in CO2 exchange. However, in contrast to the rhythm in CO2 exchange, NR rhythms were strongly damped in continuous light. In constant darkness the rhythms were even more damped. The results show that post-translational modification of CAM NR is influenced by light/darkness and by an endogenous rhythm. 相似文献