Cytochemical studies suggest that Na+,K+-ATPase is localized to sinusoidal and lateral portions of the hepatocyte plasma membrane whereas Mg++-ATPase and alkaline phosphatase are luminal or canalicular membrane markers. To validate further these cytochemical findings, we have isolated from the nuclear pellet of rat liver homogenates a liver plasma membrane (LPM) fraction enriched in all three enzyme markers, as previously described (Biochimica et Biophysica Acta 1975; 401:59-52). Following tight Dounce homogenization, the vesiculated membrane preparation was further separated on a multiple-step discontinuous sucrose density gradient (d 1.12 to 1.22). Na+,K+-ATPase activity "dissociated" from Mg++-ATPase activity, sedimenting at densities of 1.14 and greater. Further studies were carried out in two-step discontinuous sucrose gradients (1.13 and d greater than 1.13), and a light density fraction (d 1.13) was further characterized in calcium-free media (since addition of calcium increased contamination with intracellular membranes). Electron microscopy demonstrated a homogeneous vesicular membrane population in contrast to the heavy density fraction (d greater than 1.13) which contained membrane sheets and junction complexes as well as vesicles. The light density fraction was highly enriched n Mg++-ATPase (42.1 x homogenate specific activity) and alkaline phosphatase (64.6 x homogenate), 3 to 4 times their activities in the original LPM. In contrast, Na+,K+-ATPase activity in the light density fraction, diminished 16-fold from values in the original unfractionated LPM. All but 15% of total Na+,K+-ATPase activity in the original LPM could be accounted for in unwashed preparations. Neither cholesterol/phospholipid ratios nor an analysis of peptides on sodium dodecyl sulfate gel electrophoresis demonstrated differences in the composition of the light vs. heavy density subfractions, although there were relative increases in several peptide bands in the light density subfraction. These studies provide further supporting biochemical evidence for the concept that Na+,K+-ATPase resides on different membrane domains than does Mg++-ATPase and alkaline phosphatase and further characterizes a vesiculated membrane preparation highly enriched in putative "canalicular" enzyme markers.

Adenosine Triphosphatases↗

PubMedDate not supplied

Jejunal ulceration and push enteroscopy.

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Adult↗

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The cesarean birth method.

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Adult↗

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Determination of serum progesterone levels using a direct 125I-radioimmunoassay.

A new conventional simple and direct 125I-radioimmunoassay (RIA) for serum progesterone is described. In comparison with the classical tritiated assays which are preceded by extraction, the assay is more simple, less time-consuming, less subject to error and hence less expensive. In normal cycling females the serum progesterone range during the follicular phase was 0.9 to 5.5 ng/ml (mean 2.5 ng/ml). Levels above 12 ng/ml occurring during the luteal phase are indicative of adequate luteal function. The actual progesterone levels assayed with this method are significantly greater than those found using the classical tritiated technique, and the reasons for this discrepancy are discussed. The assay is also capable of being used to assess the response to treatment for infertility, thus indicating that, in spite of the higher values obtained, there is no impairment in diagnostic sensitivity.

Female↗