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1.
A method for the detection of the specific binding of 3-methylcholanthrene to rat liver cytosolic proteins is described. The separation of the protein-bound 3-methylcholanthrene from the free 3-methylcholanthrene was achieved using a batch DEAE-cellulose technique. Extraction of the DEAE-cellulose with 0.3 M KCl allowed the selective release and measurement of the amount of protein-bound 3-methylcholanthrene. The assay was optimized for the following parameters: time of incubation with DEAE-cellulose, time required for salt extraction, protein concentration, the concentration of KCl required to elute the specific binding proteins, the amount of DEAE-cellulose required to bind the specific binding proteins, and ligand specificity. The sedimentation properties of those 3-methylcholanthrene-binding proteins which were extracted with salt from DEAE-cellulose were examined on 5 to 20% sucrose gradients; the major binding species sedimented as a broad peak at 4.5 S. 相似文献
2.
The endogenous phosphorylation of human erythrocyte cytosolic proteins is markedly increased when the crude cytosol, prior to incubation in the presence of [y-32P] ATP, is submitted to DEAE-cellulose chromatography. Some proteins, including 22 and 23 kDa proteins, are preferentially phosphorylated by cytosolic casein kinase CS, whereas other proteins, including 42 kDa protein, are preferentially phosphorylated by casein kinase CTS. The CS-catalyzed phosphorylation is strongly inhibited by physiological ionic strength (150 mM KCl or NaCl) and by physiological levels (3 mM) of 2,3-bisphosphoglycerate, while CTS-catalyzed phosphorylation is unaffected. The very poor endogenous phosphorylation of these proteins in the crude cytosol may be due to the presence of other cytosolic inhibitors which are removed by DEAE-cellulose chromatography. 相似文献
3.
Summary Mitochondrial creatine kinase (CK) purified from canine myocardium showed a single protein band on SDS-PAGE and was free of
MMCK. Its amino acid composition was different than MMCK or BBCK and did not react to antiserum to MMCK or BBCK. Using purified
mitochondrial, MM and BBCK, the velocity of reaction (V) was estimated for creatine phosphate (CP), creatine (C), adenosine
triphosphate (ATP) and adenosine diphosphate (ADP) over a wide range of concentrations including those at Vmax. The values for Km (mM/L) derived from Lineweaver-Burke plots are shown:
The affinity of mitochondrial CK for C is much greater than MMCK which is compatible with the energy shuttle hypothesis, namely
ATP is converted by mitochondrial CK to CP, and then diffuses to the myofibril for conversion to ATP for utilization. 相似文献
4.
Summary The anastomosing ER system of epidermal cells of onion bulb scales is composed of three modifications: lamellar and tubular elements, located in the cell periphery, and long tubular stands located deeper in the cytoplasm. Cytoplasmic acidification of epidermal cells by loading with weak organic acids like acetic or propionic acid causes the decay of the lamellar elements and the disappearance of long tubular strands. Organelle movement is also inhibited. The effects depend on the pH of the incubation medium and on the administered acid concentration, and are characterized by a distinct lag phase of about 7 min. The induced ER changes are transient with adaptation starting after about 50min. Buffer components alone have little influence on the cellular ER organization within a pH-range of 4.0–8.0. However, the pH of the medium strongly affects the time course of the effects as well as recovery after omitting the administered acid. Both modulation and recovery occur more rapidly at neutral or slightly alkaline pH. Actin filaments, which play a major role in ER organization and organelle movement, are not affected by cytosolic acidification.Dedicated to the memory of Professor Oswald Kiermayer 相似文献
5.
6.
Enhancement of NMDA-mediated responses by cyanide 总被引:2,自引:0,他引:2
Manisha N. Patel Robert W. Peoples George K. W. Yim Gary E. Isom 《Neurochemical research》1994,19(10):1319-1323
The effect of cyanide on NMDA-activated ion current and MK801 binding was studied in cultured rat hippocampal neurons. In microfluorometric analysis using fura-2, removal of extracellular Mg2+ resulted in a five-fold increase in NMDA-induced peak of [Ca2+]i. One mM NaCN enhanced the peak NMDA responses in the presence, but not in the absence of extracellular Mg2+. Cyanide enhanced the immediate rise in [Ca2+]i produced by NMDA, followed over a 1–5 min period by a gradual increase of [Ca2+]i. Similar results were obtained in whole-cell patch clamp recordings from hippocampal neurons. One mM KCN enhanced the NMDA-activated current in the presence, but not in the absence of extracellular Mg2+. This effect was independent of cyanide-mediated metabolic inhibition since the recording pipette contained ATP (2 mM). In binding assays NaCN (1 mM) increased the binding affinity of [3H]MK-801 to rat forebrain membranes in the presence of Mg2+, whereas in the absence of Mg2+, NaCN did not influence binding. These results indicate that cyanide enhances NMDA-mediated Ca2+ influx and inward current by interacting with the Mg2+ block of the NMDA receptor. The effect of cyanide can be explained by an initial interaction with the Mg2+ block of the NMDA receptor/ionophore which appears to be energy-independent, followed by a gradual increase in Ca2+ influx resulting from cellular energy reserve depletion.Abbreviations NMDA
N-Methyl-D-Aspartate
- EAA
excitatory amino acid
- MK-801
(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohept-5,10-imine maleate 相似文献
7.
8.
Christopher M. R. Bax Vijai S. Shankar A. S. M. Towhidul Alam Bridget E. Bax Baljit S. Moonga Christopher L. -H. Huang Mone Zaidi Barry R. Rifkin 《Bioscience reports》1993,13(3):169-174
We report the effects of tetracycline analogues on cytosolic Ca2+ transients resulting from application of ionic nickel (Ni2+), a potent surrogate agonist of the osteoclast Ca2+ receptor. Preincubation with minocycline (1 mg/l) or a chemically modified tetracycline, 4-dedimethyl-aminotetracycline (CMT-1) (1 or 10 mg/l), resulted in a significant attenuation of the magnitude of the cytosolic [Ca2+] response to an application of 5 mM-[Ni2+]. Preincubation with doxycycline (1 or 10 mg/l) failed to produce similar results. In addition, application of minocycline alone (0.1–100 mg/l) resulted in a 3.5-fold elevation of cytosolic [Ca2+]. The results suggest a novel action of tetracyclines on the osteoclast Ca2+ receptor. 相似文献
9.
H. H. Felle 《Plant biology (Stuttgart, Germany)》1993,106(1):5-12
The exact ion gradients across cellular membranes and their changes due to metabolic or transport processes can be best studied with the use of ion-selective microelectrodes. The last decade of research using ion-selective microelectrodes in intact cells has proven this technique to be indispensable for the investigation of a variety of physiological questions of regulatory processes, membrane transport, cellular signalling, developmental biology and plant nutrition. Their application to selected problems has led to numerous exciting observations, many of which have changed our view concerning cellular responses to environmental stimuli and in many instances have led to a new understanding of plant cell physiology. Since, with these electrodes, intracellular as well as extracellular free ion concentrations can be simultaneously detected with electrical transport parameters such as membrane potential and membrane conductance, they can be powerful tools in the hands of many plant cell biologists. 相似文献
10.
The C2 domain calcium-binding motif: structural and functional diversity. 总被引:21,自引:0,他引:21
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E. A. Nalefski J. J. Falke 《Protein science : a publication of the Protein Society》1996,5(12):2375-2390
The C2 domain is a Ca(2+)-binding motif of approximately 130 residues in length originally identified in the Ca(2+)-dependent isoforms of protein kinase C. Single and multiple copies of C2 domains have been identified in a growing number of eukaryotic signalling proteins that interact with cellular membranes and mediate a broad array of critical intracellular processes, including membrane trafficking, the generation of lipid-second messengers, activation of GTPases, and the control of protein phosphorylation. As a group, C2 domains display the remarkable property of binding a variety of different ligands and substrates, including Ca2+, phospholipids, inositol polyphosphates, and intracellular proteins. Expanding this functional diversity is the fact that not all proteins containing C2 domains are regulated by Ca2+, suggesting that some C2 domains may play a purely structural role or may have lost the ability to bind Ca2+. The present review summarizes the information currently available regarding the structure and function of the C2 domain and provides a novel sequence alignment of 65 C2 domain primary structures. This alignment predicts that C2 domains form two distinct topological folds, illustrated by the recent crystal structures of C2 domains from synaptotagmin 1 and phosphoinositide-specific phospholipase C-delta 1, respectively. The alignment highlights residues that may be critical to the C2 domain fold or required for Ca2+ binding and regulation. 相似文献