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91.
Summary pH gradient-dependent sodium transport in highly purified rat parotid basolateral membrane vesicles was studied under voltage-clamped conditions. In the presence of an outwardly directed H+ gradient (pHin=6.0, pHout=8.0)22Na uptake was approximately ten times greater than uptake measured at pH equilibrium (pHin=pHout=6.0). More than 90% of this sodium flux was inhibited by the potassium-sparing diuretic drug amiloride (K
1
=1.6 m) while the transport inhibitors furosemide (1mm), bumetanide (1mm) SITS (0.5mm) and DIDS (0.1mm) were without effect. This transport activity copurified with the basolateral membrane marker K+-stimulatedp-nitrophenyl phosphatase. In addition22Na uptake into the vesicles could be driven against a concentration gradient by an outwardly directed H+ gradient. pH gradient-dependent sodium flux exhibited a simple Michaelis-Menten-type dependence on sodium concentration cosistent with the existence of a single transport system withK
M
=8.0mm at 23°C. A component of pH gradient-dependent, amiloride-sensitive sodium flux was also observed in rabbit parotid basolateral membrane vesicles. These results provide strong evidence for the existence of a Na+/H+ antiport in rat and rabbit parotid acinar basolateral membranes and extend earlier less direct studies which suggested that such a transporter was present in salivary acinar cells and might play a significant role in salivary fluid secretion. 相似文献
92.
The kinetic parameters of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (EC 4.1.1.39) in wheat (Triticum aestivum L.) and rice (Oryza sativa L.) were determined by rapidly assaying the leaf extracts. The respective K
m and V
max values for carboxylase and oxygenase activities were significantly higher for wheat than for rice. In particular, the differences in the V
max values between the two species were greater. When the net activity of CO2 exchange was calculated at the physiological CO2-O2 concentration from these kinetic parameters, it was 22% greater in wheat than in rice. This difference in the in-vitro RuBP-carboxylase/oxygenase activity between the two species reflected a difference in the CO2-assimilation rate per unit of RuBP-carboxylase protein. However, there was no apparent difference in the CO2-assimilation rate for a given leaf-nitrogen content between the two species. When the RuBP-carboxylase/oxygenase activity was estimated at the intercellular CO2 pressure from the enzyme content and kinetic parameters, these estimated enzyme activities in wheat and rice were similar to each other for the same rate of CO2 assimilation. These results indicate that the difference in the kinetic parameters of RuBP carboxylase between the two species was offset by the differences in RuBP-carboxylase content and conductance for a given leaf-nitrogen content.Abbreviations DTT
dithiothreitol
- EDTA
ethylenediamine-tetraacetic
- PAR
photosynthetically active radiation
- RuBP
ribulose-1,5-bisphosphate 相似文献
93.
The short-term, in-vivo response to elevated CO2 of ribulose-1,5-bisphosphate carboxylase (RuBPCase, EC 4.1.1.39) activity, and the pool sizes of ribulose 1,5-bisphosphate, 3-phosphoglyceric acid, triose phosphates, fructose 1,6-bisphosphate, glucose 6-phosphate and fructose 6-phosphate in bean were studied. Increasing CO2 from an ambient partial pressure of 360–1600 bar induced a substantial deactivation of RuBPCase at both saturating and subsaturating photon flux densities. Activation of RuBPCase declined for 30 min following the CO2 increase. However, the rate of photosynthesis re-equilibrated within 6 min of the switch to high CO2, indicating that RuBPCase activity did not limit photosynthesis at high CO2. Following a return to low CO2, RuBPCase activation increased to control levels within 10 min. The photosynthetic rate fell immediately after the return to low CO2, and then increased in parallel with the increase in RuBPCase activation to the initial rate observed prior to the CO2 increase. This indicated that RuBPCase activity limited photosynthesis while RuBPCase activation increased. Metabolite pools were temporarily affected during the first 10 min after either a CO2 increase or decrease. However, they returned to their original level as the change in the activation state of RuBPCase neared completion. This result indicates that one role for changes in the activation state of RuBPCase is to regulate the pool sizes of photosynthetic intermediates.Abbreviations and symbols
A
net CO2 assimilation rate
- Ca
ambient CO2 partial pressure
- Ci
intercellular CO2 partial pressure
- CABP
2-carboxyarabinitol 1,5-bisphosphate
- kcat
catalytic turnover rate per RuBPCase molecule
- PFD
photon flux density (400 to 700 nm on an area basis)
- PGA
3-phosphoglyceric acid
- Pi
orthophosphate
- RuBP
ribulose 1,5-bisphosphate
- RuBPCase
ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) 相似文献
94.
Neurotransmitter receptors and ion channels play a critical role in the transduction of signals at chemical synapses. The modulation of neurotransmitter receptor and ion channel function by protein phosphorylation is one of the major regulatory mechanisms in the control of synaptic transmission. The nicotinic acetylcholine receptor (nAcChR) has provided an excellent model system in which to study the modulation of neurotransmitter receptors and ion channels by protein phosphorylation since the structure and function of this receptor have been so extensively characterized. In this article, the structure of the nAcChR from the electric organ of electric fish, skeletal muscle, and the central and peripheral nervous system will be briefly reviewed. Emphasis will be placed on the regulation of the phosphorylation of nAcChR by second messengers and by neurotransmitters and hormones. In addition, recent studies on the functional modulation of nicotinic receptors by protein phosphorylation will be reviewed. 相似文献
95.
Allison A. Welder Tina Machu Steven W. Leslie Richard E. Wilcox June Bradlaw Daniel Acosta 《In vitro cellular & developmental biology. Plant》1988,24(8):771-777
Summary Primary mycolardial cell cultures and freshly isolated cardiac cells in suspension resprensent two isolated, whole cell models
for investigating cellular transsarcolemmal45Ca++ exchange in response to a receptor-coupled stimulus. Studies were performed to characterize beta-adrenergic receptor binding,
beta-adrenergic receptor mediated cellular calcium (45Ca++) exchange, and viability in purified primary myocardial cell cultures and freshly isolated cardiac cells in suspension obtained
from 3-to 3-d-old Sprague-Dawley rats. In addition, beta-adrenergic receptor binding was characterized in whole-heart crude
membrane preparations. All three preparations had saturable beta-adrenergic binding sites with the antagonist [125I]iodopindolol ([125I]IPIN). The suspensions had a significantly lower B
max
(42±6 fmol/mg protein) than the membranes and cultures (77±8 and 95±10 fmol/mg protein, respectively). The K
D
of the cultures (218±2.0 pM) was significantly higher than that for the suspensions (107 ±1.3 pM) and membranes (93±1.3 pM). Viability was significantly lower in the suspensions (57%) when compared to 94% viability in myocardial cell cultures after
3 h of incubation in Kreb's Henseleit buffer. Incubation of the cultures with 5.0×10−7
M isoproterenol resulted in a significant increase in45Ca++ exchange as early as 15 s. In contrast,45Ca++ exchange into the suspensions was not increased. Although both primary cell cultures and cardiac cells in suspension possess
saturable beta-adrenergic receptors, only the monolayer cultures exhibited functional beta-adrenergic receptor-mediated45Ca++ exchange. Of the two intact cell models investigated, these data suggest that primary myocardial cell cultures are more suitable
than cell suspensions for investigating beta-adrenergic receptor binding and functions in the postnatal rat heart.
This research was supported by The University of Texas Research Institute, a grant from the Texas Advanced Research Technology
Program awarded to S. W. Leslie and R. E. Wilcox, and contract 223-86-2109 from the Food and Drug Administration. 相似文献
96.
Simultaneous determination of ascorbic acid and dehydroascorbic acid in cultures of C3H/10T1/2 cells
Luminita L. V. Ibric William F. Benedict Andrew R. Peterson 《In vitro cellular & developmental biology. Plant》1988,24(7):669-676
Summary A reproducible method is described for the separation and quantification of ascorbic acid and dehydroascorbic acid by ion-pairing
reverse-phase high performance liquid chromatography and detection by absorbance at 232 nm. Lowest detectable concentrations
with a linear response of detection were 5 nmol for ascorbic acid and 50 nmol for dehydroascorbic acid. This method was applied
to the analysis of C3H/10T1/2 cells and culture medium after influx or efflux experiments and single or multiple treatments
with ascorbic acid. Subsequent measurement of the radioactivity in the eluted fractions increased the detectability of both
ascorbic acid and dehydroascorbic acid to 10 to 20 pmol.
This research was supported by grant CA 09320 and CA 31574 from the National Cancer Institute, Bethesda, MD, and grant BC441
from The American Cancer Society. 相似文献
97.
P Mentré F Escaig 《Biology of the cell / under the auspices of the European Cell Biology Organization》1992,74(1):119-126
Flotation on hot water (about 60 degrees C) which is frequently employed to stretch semithin sections on substrates for SIMS (secondary ion mass spectrometry) microscopy, is the cause of numerous artefacts. In the case of epoxy resin-embedded tissue, one observes loss of potassium and sodium and accumulation of calcium. The relative contrast of cell nuclei in the ionic images, is rapidly affected by these ion migrations. After prolonged contact with hot water, tissue becomes uniformly emissive. In the case of hydrosoluble resin-embedded tissue, potassium and sodium do not appear to be affected by the action of water, which suggests that they are covalently bound with chelating sites buried beneath the layer of water bound to the surface of the macromolecules. Calcium accumulates, probably on widely exposed anionic sites. Moreover, the domains observed in hydrosoluble resin-embedded tissue shrink differently according to the proportion of water removed by melamine; this can provide interesting information on the initial equilibrium between water, ion sand macromolecules. Our results seem to support the assumption that bound water should play an important role in the preservation of both macromolecular architecture and ion distributions. 相似文献
98.
T. J. Budd C. D. Dolman A. M. Lawson W. Chai J. Saxton F. W. Hemming 《Glycoconjugate journal》1992,9(5):274-278
N-Acetylneuraminic acid (Neu5Ac) andN-glycoloylneuraminic acid (Neu5Gc) are distributed widely in nature. Using a Carbopac PA-1 anion exchange column, we have determined the ratios of Neu5Ac and Neu5Gc in hydrolysates of platelets and their precursors: a rat promegakaryoblastic (RPM) cell line and a human megakaryoblastic leukemia cell line (MEG-01). The ratio of Neu5Gc:Neu5Ac in cultured RPM cells is 16:1, whereas in platelet rich plasma and cultured MEG-01 cells it is 1:38 and 1:28, respectively. The nature of these sialic acids from RPM cells was verified using thin layer chromatography and liquid secondary ion mass spectrometry. The relevance of increased Neu5Gc levels in early stages of development is discussed.Abbreviations Neu5Ac
N-acetylneuraminic acid
- Neu5Gc
N-glycoloylneuraminic acid
- RPM
rat promegakaryoblast
- MEG-01
human megakaryoblastic leukaemia cell line
- PAD
pulsed amperometric detection
- WGA
wheat germ agglutinin
- FCS
foetal calf serum
- PPEADF
phosphatidylethanolamine dipalmitoyl
- LSIMS
liquid secondary ion mass spectrometry
- HPAEC
high performance anion exchange chromatography
- TBA
thiobarbituric acid 相似文献
99.
100.