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51.
The polyene antibiotic nystatin is used to reduce selectively to zero the apical membrane resistance of the rabbit descending colon, allowing the measurement of the current-voltage curve of the basolateral membrane. The I--V relationship is described by the Goldman-Hodgkin-Katz equations allowing calculation of PNa/PK, PCl/PK and PK for the basolateral membrane. Cs+ is found to block inward current (serosa to mucosa) in a manner similar to that found in excitable membranes. 相似文献
52.
Su-Chin C. Liu Mary J. Eaton Marvin A. Karasek 《In vitro cellular & developmental biology. Plant》1979,15(10):813-822
Summary Using gels of acid-soluble, collagen as a culture surface, trypsin-released keartinocytes from 0.1-mm, split-thickness sections
of newborn foreskin may be plated with high efficiency and subcultured at a 1∶5 split a 2- to 3-week intervals for three subpassages.
When plated at a density of 3.2×104 cells per cm2, keratinocytes attach to the gel with an efficiency of over 70%; after a lag phase of 3 days, the cells multiply exponentially
with a doubling time of 60 hr. Cultures reach a growth-plateau phase at a density of 47.7×104 cells per cm2. Both hydrocortisone and epidermal growth factor (EGF) stimulate slightly the growth of primary cultures; both factors are
required for proliferation of the 2nd and further passage of keratinocytes. As the cultures reach, confluence multilayers,
of stratified cells are formed and cells of squamous morphology are spontaneously released from the surface. When the released
cells and the attached cells are pulsed with [3H]-histidine and [14C]-leucine, a higher ratio of histidine to leucine is observed in the released cells indicating the biochemical onset of maturation.
Orange G-Aniline Blue staining of the released cells show some of the cells to be completely keratinized. Fibrous proteins
extracted from the cultured cells and analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis display the characteristic
stratum corneum proteins of 60,000 and 66,000 daltons.
Supported in part by Grants AM 14121 and AM 19595 of the U.S. Public Health Service. 相似文献
53.
The action of phospholipase C (Bacillus cereus) toward mixed micelles of phosphatidylcholine and the nonionic surfactant Triton X-100 is analyzed according to the “surfaceas-cofactor” kinetic scheme recently proposed for characterizing the action of lipolytic enzymes [Deems, R. A., Eaton, B. R., and Dennis, E. A. (1975) J. Biol. Chem.250, 9013–9020]. According to this scheme, the enzyme first associates with the surface or mixed micelles, where the dissociation constant is KsA. The enzyme, now part of the mixed micelle surface, then binds the substrate phospholipid molecule in its active site and this binding is related to the Michaelis constant, KmB. The surface, or mixed micelles in this scheme, behaves kinetically as a cofactor in that, under initial rate conditions, the surface properties of the mixed micelles are virtually unchanged after catalysis. For phospholipase C with egg phosphatidylcholine as substrate, it was found that at pH 6.4 (the pH optimum for the enzyme) and 40 °C, V is about 2 × 103 μmol min?1 (mg of protein)?1. KsA is about 2 mm and KmB is 1 to 2 × 10?10 mol cm?2. The kinetic constants for phospholipase C are compared with those previously reported for phospholipase A2 and the membrane-bound enzyme phosphatidylserine decarboxylase determined under similar conditions. 相似文献
54.
W A Eaton J Hofrichter P D Ross R G Tschudin E D Becker 《Biochemical and biophysical research communications》1976,69(2):538-547
We describe a technique for monitoring the kinetics of sickle cell hemoglobin gelation by observing the change in the amplitude and linewidth of the water proton magnetic resonance. The resulting kinetic progress curves are very similar to those obtained by optical birefringence and turbidity methods. The curves consist of a delay, followed by a rapidly accelerating signal change which terminates quickly. From a study of the temperature dependence of the delay time, it is shown that all three techniques see the onset of gelation simultaneously. The origin of the change in physical properties upon gelation is briefly discussed in relation to the component steps of the reaction. 相似文献
55.
The selective growth of malignant cells on non-adhesive Teflon substrates is reported. Neoplastically transformed cell lines of human and murine origin proliferated on Teflon whereas similarly seeded normal non-tumorigenic cells were unable to undergo cell division and subsequent growth. This substrate should be especially useful in establishing primary cultures from human tumor material. 相似文献
56.
A detailed kinetic scheme is proposed for the action of phospholipase A2 on mixed micelles of phospholipid and surfactant: see article. where E is the enzyme, A is the mixed micelle, and B is the phospholipid substrate in the mixed micelle. This scheme takes into account quantitatively the involvement of the lipid-water interface in the action of this enzyme toward substrate in macromolecular lipid complexes. The kinetic equation for this scheme is derived and four simplifying assumptions which are necessary for its practical application are described. Kinetic data are reported for the action of cobra venom phospholipase A2 (Naja naja naja) on 1,2-dipalmitosyl-sn-glycero-3-phosphorylcholine in mixed micelles with the nonionic surfactant Triton X-100, and these data are analyzed in terms of the kinetic equation presented. At 40 degrees, pH 8.0, and in the presence of 10 mM Ca2+, V was found to be about 4 X 10(3) mumol min(-1) mg of protein(-1). KsA, which is the dissociation constant for the enzyme-mixed micelle complex, is about 5 X 10(-4) M. KmB, the Michaelis constant for the catalytic step, which is (k-2 + k3)/k2, is 1 to 2 X 10(-10) mol cm-2. This kinetic treatment, together with the fact that the mixed micelle system allows the concentration of the substrate in the lipid-water interface to be varied, has made possible the quantitative separation of the association of a lipolytic enzyme with the lipid-water interface (expressed as KsA) and the binding to the substrate in the interface (reflected in the KmB term). The implications of this kinetic scheme for the analysis of phospholipase A2 from other sources acting on other aggregated forms of phospholipid and for the study of other phospholipases and lipases is considered. 相似文献
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A procedure for the separation, detection, and quantification of picomole levels of dansyl derivatives of the biogenic amines, dopamine, norepinephrine, octopamine, and serotonin, has been developed using high-performance thin-layer chromatography. The detection limit is 1 to 2 pmol. Each of the amine derivatives has been detected in insect brain tissue and a solvent system has been developed for the separation and quantification of octopamine in insect tissue samples. 相似文献