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1.
Using a rapid pH electrode, measurements were made of the flash-induced proton transport in isolated spinach chloroplasts. To calibrate the system, we assumed that in the presence of ferricyanide and in steady-state flashing light, each flash liberates from water one proton per reaction chain. We concluded that with both ferricyanide and methylviologen as acceptors two protons per electron are translocated by the electron transport chain connecting Photosystem II and I. With methyl viologen but not with ferricyanide as an acceptor, two additional protons per electron are taken up due to Photosystem I activity. One of these latter protons is translocated to the inside of the thylakoid while the other is taken up in H2O2 formation. Assuming that the proton released during water splitting remains inside the thylakoid, we compute ratios of 3 and 4 for ferricyanide and methyl viologen, respectively.In continuous light of low intensity, we obtained the same ratios. However, with higher intensities where electron transport becomes rate limited by the internal pH, the ratio approached 2 as a limit for both acceptors.A working model is presented which includes two sites of proton translocation, one between the photoacts, the other connected to Photosystem I, each of which translocates two protons per electron. Each site presents a ≈ 30 ms diffusion barrier to proton passage which can be lowered by uncouplers to 6–10 ms. 相似文献
2.
Human red cell and guinea pig kidney were phosphorylated at 0°C. Using concentrations of ATP ranging from 10?6 to 10?8 M, ATP-dependent regulation of reactivity is observed with red cell but not kidney at 0°C. In particular, with the red cell enzyme only, the following are observed: (i) the ratio of enzyme-bound ATP (E·ATP, measured by the pulse-chase method of Post, R.L., Kume, S., Tobin, T., Orcutt, B. and Sen, A.K. (1969) J. Gen. Physiol. 54, 306s-326s) to steady-state level of total phosphoenzyme (EP) decreases with decrease in ATP concentration and (ii) the apparent turnover of phosphoenzyme (ratio of Na+-stimulated ATP hydrolysis to level of total EP at steady state) also varies as a function of ATP concentration. In addition, when EP is formed at very low ATP (0.02 μM), and then EDTA is added, rapid disappearance of a fraction of EP occurs, presumably due to ATP resynthesis, only with the red cell enzyme. These differences in behaviour of the red cell and kidney enzymes are explained on the basis of the observed predominance of K+-insensitive EP in red cell, but K+-sensitive EP in kidney at 0°C. 相似文献
3.
Treatment by EDTA of purified plasma membranes from MF2S cells (a variant of the murine plasmacytoma MOPC 173) solubilized proteins and increased by a 1000-fold the sensitivity of to ouabain. When added back with Ca2+ to treated plasma membranes, these EDTA-solubilized proteins restored the initial sensitivity of the enzyme to its inhibitor. We report the purification of a protein of 32 000, isolated from the EDTA-treated membrane supernatant. This protein was purified by a one-step procedure involving a preparative polyacrylamide gel electrophoresis without detergent. In the presence of Ca2+ it was able to restore the original sensitivity to ouabain of from EDTA-treated membrane. This protein was shown to be similar to the β-actinin described by Maruyama by the following criteria: (1) molecular weight and amino acid composition; (2) cross-reactivity with their respective antisera; (3) in the presence of Ca2+ the same quantitative biological activity on ouabain sensitivity of the . A possible interaction between β-actinin, calmodulin and membrane-bound is discussed. 相似文献
4.
The tumor promoter 12-0-tetradecanoyl phorbol-13-acetate (TPA) stimulates hexose uptake into rat thymocytes. This study explores two possible messengers of this stimulation: changes in cytosolic [Ca2+], and activation of the Na+/H+ antiport. The cytosolic level of Ca2+, determined by the fluorescence of quin-2, was elevated by TPA, and this rise required extracellular Ca2+. In contrast, stimulation of hexose uptake was still observed in Ca2+ -free media even when cytoplasmic [Ca2+] was buffered with quin-2. TPA also raised the cytoplasmic pH, presumably through activation of the Na+/H+ exchange. However, replacement of extracellular Na+ by N-methylglucamine+ or choline+ which prevents the cytoplasmic alkanization did not prevent stimulation of hexose uptake by TPA. Moreover, amiloride, at concentrations that inhibit Na+/H+ exchange in these cells, did not interfere with stimulation of hexose uptake by TPA. In conclusion, stimulation of hexose uptake by phorbol ester in rat thymocytes does not appear to be mediated by changes in cytosolic free Ca2+ or in the activity of the Na+/H+ antiport. 相似文献
5.
Françoise Giraud Michel Claret K.Richard Bruckdorfer Bernadette Chailley 《生物化学与生物物理学报:生物膜》1981,647(2):249-258
Cholesterol depletion alters the apparent affinity of the internal cationic sites and the maximal translocation rate but not the affinity of the external cationic sites of the pump in human erythrocytes. To test whether these effects were mediated by a direct cholesterol-internal site interaction or by a change in membrane lipid order, the effects of five fluidizing amphiphiles (chlorpromazine, imipramine, benzyl alcohol, sodium oleate and sodium benzenesulphonate) on the kinetic parameters of the pump were determined. The cholesterol removal and all the agents used induced dose-response decreases in membrane lipid order as measured by fluorescence polarization or ESR. Positive and neutral amphiphiles mimicked the effects of cholesterol removal on the affinity of the internal sites of the pump and to a lesser extent on the maximal translocation rate. Anionic amphiphiles had no effect on internal sites, probably because they distributed preferentially within the outer leaflet on the membrane. These results indicate that cholesterol controls the affinity of the internal sites of the pump by altering the membrane lipid order. In contrast, neither cholesterol depletion nor the agents used altered the affinity of the external sites of the pump. This difference in sensitivity to membrane lipid order suggests that internal and external cationic sites, although borne by the same protein, are in different lipid environments. 相似文献
6.
The immunologic cross-reactivity of the α and α+ forms of the large subunit and the β subunit of the ( from brain and kidney preparations was examined using rabbit antiserum prepared against the purified holo lamb kidney enzyme. As previously reported by Sweadner ((1979) J. Biol. Chem. 254, 6060–6067) phosphorylation of the large subunit of the ( in the presence of Na+, Mg2+, and [ revealed that dog and, very likely, rat brain contain two forms of the large subunit (designated α and α+) while dog, rat, and lamb kidney contain only one form (α). The cross-reactivity of the α and α+ forms in these preparations was investigated by resolving the subunits by SDS-polyacrylamide gel electrophoresis. The separated polypeptides were transferred to unmodified nitrocellulose paper, and reacted with rabbit anti-lamb kidney serum, followed by detection of the antigen-antibody complex with 125I-labeled protein A and autoradiography. By this method, the α and α+ forms of rat and dog brain, as well as the α form found in kidney, were shown to cross-react. In addition, membranes from human cerebral cortex were shown to contain two immunoreactive bands corresponding to the α and α+ forms of dog brain. In contrast, the brain of the insect Manduca sexta contains only one immunoreactive polypeptide with a molecular weight intermediate to the α and α+ forms of dog brain. The β subunit from lamb, dog and rat kidney and from dog and rat brain cross-reacts with anti-lamb kidney ( serum. The mobility of the β subunit from dog and rat brain on SDS-polyacrylamide electrophoresis gels is greater than the mobility of the β subunit from lamb, rat or dog kidney. 相似文献
7.
Krister Gréen 《Prostaglandins & other lipid mediators》1978,15(5):813-821
This isomer of PGF2α is relatively resistant to metabolic degradation in the Cynomolgus monkey. Thus, 16–20 per cent of the amount injected was excreted unchanged in the urine. Five metabolites with 20, 18, 16 and 14 carbon atoms in the skeleton were identified. The data are similar to those earlier seen in the rat and further support the idea that this analogue of PGF2α could have a long half-life time in the mammalian body and thus a long duration of its pharmacological actions. 相似文献
8.
Theodore G. Gabig Bruce A. Lefker 《Biochemical and biophysical research communications》1984,118(2):430-436
The resolved flavoprotein and cytochrome b559 components of the NADPH dependent generating oxidase from human neutrophils were the subject of further study. The resolved flavoprotein, depleted of cytochrome b559, was reduced by NADPH under anaerobic conditions and reoxidized by oxygen. NADPH dependent generation by the resolved flavoprotein fraction was not detectable, however it was competent in the transfer of electrons from NADPH to artificial electron acceptors. The resolved cytochrome b559, depleted of flavoprotein, demonstrated no measureable NADPH dependent generating activity and was not reduced by NADPH under anaerobic conditions. The dithionite reduced form of the resolved cytochrome b559 was rapidly oxidized by oxygen, as was the cytochrome b559 in the intact oxidase. 相似文献
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Respiration-driven proton translocation has been studied with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing H2 during reduction of O2, NO?3, NO?2 or N2O. A simplified scheme of anaerobic electron transport and associated proton translocation is shown that is consistent with the measured . Furthermore, the kinetics and energetics of NO?3 uptake in whole cells of P. denitrificans were studied. For this purpose, we measured H2 consumption or N2O production after addition of NO?3 to a cell suspension, which indirectly gave information about uptake (and reduction) of NO?3. It was found that a lag phase in H2 consumption or N2O production appeared whenever the membrane potential was dissipated by addition of thiocyanate, carbonyl cyanide m-chlorophenylhydrazone or triphenyl-methylphosphonium bromide. However, these lag phases were not observed when NO?2 was present at the moment of introduction of NO?3. On the basis of these findings we conclude that there are two uptake systems for NO?3. One system is dependent on the proton-motive force and is probably used for initiation of NO?3 uptake. The other is an antiport and its function is to take over NO?3 uptake from the first system. 相似文献
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Samuel A. Sholl 《Steroids》1983,41(6):769-776
3β-Hydroxysteroid activity (3Δ-HSDH) was examined in the rhesus monkey () placenta and fetal adrenal at 135 and 155–162 days of gestation. Activity was evaluated in microsomes by the conversion of [3H]pregnenolone to [3H]progesterone. There was a 7-fold increase in enzyme activity in the whole adrenal (minus medulla) between the two stages of development. Combining data from both periods, enzyme activity was greater in the outer than in the inner region of the adrenal. No stage-dependent change in placental activity was evident. The temporal patterns in 3β-HSDH activity are consistent with corticoid and progesterone patterns in the circulation. Thus, the level of 3β-HSDH activity may be rate limiting in both the fetal adrenal and placenta.Enzyme activity was assessed in incubations which included unex-tracted, heat-treated, 100,000 g tissue supernatants. In both placental and adrenal incubations, competitive inhibition was noted. Ethyl ether extracts of 100,000 g tissue supernatants also inhibited 3β-HSDH in the respective tissues. GLC analysis of these extracts revealed the presence of putative dehydroepiandrosterone. Hormone levels and the nature of the inhibition that were observed are compatible with the conclusion that dehydroepiandrosterone can inhibit the conversion of pregnenolone to progesterone . The physiological importance of this remains to be determined. 相似文献
13.
The thermal transitions of rat liver microsomes and isolated lipids were investigated by using differential scanning calorimetry. Endothermic transitions at ≈?5°C and between ≈18° and 40°C were detected in the membranes and at ≈?10°C and between ≈10° and 20°C in the extracted lipids.Interaction with of microsomal membranes and of extracted lipids influences the thermotrophic behaviour as revealed by differential scanning calorimetry and eliminates the break in the Arrhenius plot of the enzymic activity of . 相似文献
14.
Hans G. Förster Detlef Müller Hans R. Kricheldorf 《International journal of biological macromolecules》1983,5(2):101-105
30.5 MHz 15N m.m.r. (CP/MAS) spectra of various solid polypeptides were measured using the cross-polarization/magic angle spinning technique. In order to obtain optimum signal-to-noise ratios, relatively short contact times (1 ± 0.5 ms) are required, because the cross-polarization times (TNH) are short and because the proton rotating-frame relaxation times (T1p) are in the order of 20 ms. The 15N n.m.r. signals of copolypeptides may be sensitive to sequence effects; yet they are in most cases more sensitive to the nature of the secondary structure. The signals of α-helices absorb ca. 8–10 ppm upfield of β-sheet structures, whereas the polyglycine II helix absorbs downfield. The natural abundance spectrum of crystalline gramicidin-S exhibits a signal at ?247 ppm, a characteristic chemical shift of the antiparallel pleated sheet structure. 相似文献
15.
Samuel A. Sholl 《Steroids》1983,41(6):757-768
3β-Hydroxysteroid (3β-HSDH) was measured in the rhesus monkey () placenta, fetal adrenal (whole organ minus medulla), testis and ovary during late gestation (Days 145–162). Activities were evaluated from the conversion of [3H]-pregnenolone to [3H]progesterone. The maximum enzyme velocity (Vm) in adrenal microsomes (100,000 g pellet) was significantly higher (146 nmoles progesterone/h x mg?1protein) than in microsomes from the other tissues. Testicular Vm was greater than either ovarian or placental Vm which were not different from one another (11.5 versus 1.9, 1.2 nmoles progesterone/h x mg?1protein, respectively). Apparent Michaelis-Menten constants in the adrenal, placenta, testis and ovary averaged 1.8,2.5,0.27 and 0.16 μM, respectively. In some cases, substrate inhibition was noted. Estimated dissociation constants for pregnenolone were 2.3 μM (adrenal), 2.1 μM (placenta), 0.74 μM (testis) and 0.13 μM (ovary). 3β-HSDH was less active in a crude mitochondrial preparation from the fetal adrenal (10,000 g pellet) than in microsomes, whereas activity in the placenta and testis appeared to be equally distributed between mitochrondria and microsomes.Rate measurements were consistent with the apparent potentials of these organs to synthesize their characteristic hormones. Thus, 3β-HSDH activity may be an important rate determining step in hormone synthesis. The importance of substrate inhibition in progesterone formation remains to be assessed. 相似文献
16.
Yoshimitsu Kuroyanagi Hiroyuki Kobayashi Manabu Senō Masao Ishida Naoki Tominaga Toshihiro Akaike Munenori Sakamoto Gotthold Ebert 《International journal of biological macromolecules》1984,6(5):266-272
Copoly(α-amino acid)s consisting of γ-benzyl-l-glutamate and were prepared by the reaction of copoly(l-glutamate) containing succinimide ester, which served as active site for the coupling reaction with β-d-glucopyranosylamine. The α-helical conformation of these copolymers became unstable in DMF as the content of glutamine derivative increased. A dry film made from this copolymer could take a full α-helical conformation even at such a high content as 80% of the glutamine derivative, but in a wet film this ordered structure was partially disrupted by hydration. The hydraulic permeability of this copoly(α-amino acid) was clearly dependent on the molar content of glucopyranosyl groups. The attachment of fibroblast cells to these hydrated copolymer films was effectively depressed in the presence of a serum-free medium. The cells attached to the substrate were spherical in shape. 相似文献
17.
Jose C. Gan 《International journal of biological macromolecules》1980,2(2):97-104
An material has been purified to homogeneity from the soluble fraction of normal human liver by procedures adapted from those employed for plasma . The liver material, in contrast to a previous report1 has the same molecular weight as the corresponding normal plasma . The subunit structure, immunoelectrophoretic and immunological properties of the liver glycoprotein are identical to those of normal plasma . Amino acid and carbohydrate compositions of the liver material are similar to those of obtained from the plasma. The material has also been isolated and purified from the microsomal fraction of liver It has the same molecular weight and immunological properties as glycoprotein obtained from the cytosol. Although inhibitors of lysosmal proteases were added during the homogenization of the liver, the purified glycoprotein is devoid of trypsin-inhibitory capacity. The loss of inhibitory activity could be due to extensive cellular autolysis before autopsy. 相似文献
18.
(Na+/K+)-ATPase研究概况 总被引:7,自引:0,他引:7
谢静平 《生物化学与生物物理进展》1989,16(4):256-260
本文概述(Na+/K+)-ATPase的一般分子性质。介绍神经元和脂肪细胞中两种不同分子形式(Na+/K+)-ATPase的分离鉴定和功能性质,以及(Na+/K+)-ATPase主要功能亚基一级序列和高级结构研究所取得的一些进展。 相似文献
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