1,25-Dihydroxyvitamin D3-mediated intestinal calcium transport. Biochemical identification of lysosomes containing calcium and calcium-binding protein (calbindin-D28K)

A variety of intestinal cell organelles and proteins have been proposed to mediate 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-stimulated calcium absorption. In the present study biochemical analyses were undertaken to determine the subcellular localization of 45Ca after calcium transport in vivo in ligated duodenal loops of vitamin D-deficient chicks injected with 1.3 nmol of 1,25-(OH)2D3 or vehicle 15 h prior to experimentation. Separation of Golgi, mitochondria, basal lateral membrane, and lysosome fractions in the epithelial homogenates was achieved by differential sedimentation followed by centrifugation in Percoll gradients and evaluation of appropriate marker enzyme activities. Both vitamin D-deficient and 1,25-(OH)2D3-treated chicks had the highest levels of 45Ca-specific activity in lysosomal fractions. The lysosomes were also the only organelles to exhibit a 1,25-(OH)2D3-mediated difference in calcium content, increasing to 138% of controls. Lysosomes prepared from 1,25-(OH)2D3-treated chicks also contained the greatest levels of immunoreactive calbindin-D28k (calcium-binding protein). Chloroquine, a drug known to interfere with lysosomal function, was tested and found to inhibit 1,25-(OH)2D3-stimulated intestinal calcium absorption. Neither 1,25-(OH)2D3 nor chloroquine affected [3H]2O transport. In additional experiments, microsomal membranes (105,000 X g pellets) were subjected to gradient centrifugation. The highest levels of 45Ca-specific activity and calcium-binding protein in material from 1,25-(OH)2D3-treated chicks were found in fractions denser than endoplasmic reticulum and may represent endocytic vesicles. In studies on intestinal mucosa of 1,25-(OH)2D3-treated birds fractionated after 30 min of exposure to lumenal Ca2+ or Ca2+ plus chloroquine, 45Ca was found to accumulate in lysosomes and putative endocytic vesicles, relative to controls. A mechanism involving vesicular flow is proposed for 1,25-(OH)2D3-mediated intestinal calcium transport. Endocytic internalization of Ca2+, fusion of the vesicles with lysosomes, and exocytosis at the basal lateral membrane complete the transport process.     

Immunochemical studies on the putative plasmalemmal receptor for 1, 25-dihydroxyvitamin D(3). III. Vitamin D status

The effect of vitamin D status on levels of the putative 1, 25(OH)(2)D(3) membrane receptor (pmVDR) was studied in chick intestine, kidney, and brain. Western analyses and assays for specific [(3)H]1,25(OH)(2)D(3) binding indicated that, in intestine, pmVDR levels were greatest in -D chicks relative to +1,25D and +D animals (P < 0.05). In kidney, protein levels and specific binding followed the order +D > +1,25D, -D. In brain, vitamin D status did not affect protein levels or specific binding levels. In tissue from normal chicks, both protein and specific binding followed the order of intestine > kidney > brain membranes. Intestinal cells were further evaluated for the effect of 1,25(OH)(2)D(3) on selected "rapid responses." Extrusion of (45)Ca in response to 130 pM 1, 25(OH)(2)D(3) in vitro was greater in cells from -D chicks than from +1,25D or normal birds. Analyses of signal transduction events revealed diminished hormone-induced intracellular calcium oscillations (as assessed by fura-2 fluorescence), and lack of steroid-enhanced protein kinase (PK) A activity in intestinal epithelial cells from -D chicks relative to +D chicks. PK C activation by 130 pM 1,25(OH)(2)D(3) was approximately twofold in cells from +D or -D chicks. The combined results indicate that vitamin D status differentially affects the pmVDR in intestine, kidney, and brain. In intestine, vitamin D deficiency differentially affects (45)Ca handling, intracellular calcium oscillations, PK A and PK C activities in response to 1,25(OH)(2)D(3).     

Localization of vitamin D3-responsive alkaline phosphatase in cultured chondrocytes

Alkaline phosphatase activity appears to be altered when chondrocyte cultures are incubated with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). This study examined whether the hormone-responsive enzyme activity is associated with alkaline phosphatase-enriched extracellular membrane organelles called matrix vesicles. Confluent, third passage cultures of rat costochondral growth cartilage (GC) or resting zone chondrocytes (RC) were incubated with 1,25-(OH)2D3 or 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) and enzyme specific activity was assayed in the cell layer or in isolated matrix vesicle and plasma membrane fractions. Alkaline phosphatase-specific activity in the matrix vesicles was enriched at least 2-fold over that of the plasma membrane and 10-fold over that of the cell layer. Matrix vesicle alkaline phosphatase was stimulated by 1,25-(OH)2D3 in GC cultures and by 24,25-(OH)2D3 in RC cultures. The cell layer failed to reveal these subtle differences. 1,25-(OH)2D3 increased GC enzyme activity but the effect was one-half that observed in the matrix vesicles alone. No effect of 1,25-(OH)2D3 on enzyme activity of the RC cell layer or of 24,25-(OH)2D3 on either GC or RC cell layers was detected. Thus, response to the metabolites is dependent on chondrocytic differentiation and is site specific: the matrix vesicle fraction is targeted and not the cells per se.     

1,25(OH)2D3 increases membrane associated protein kinase C in MDBK cells.

To determine whether 1,25-dihydroxycholecalciferol [1,25(OH)2D3] affects protein kinase C (PKC) activity in kidney, as has been demonstrated in HL-60 cells we measured 1,25(OH)2D3 binding, PKC activity and PKC immunoreactivity in Madin Darby bovine kidney (MDBK) cells, a normal renal epithelial cell line derived from bovine kidney. Our data demonstrate that MDBK cells exhibit specific high affinity binding for 1,25(OH)2D3, indicating the presence of the vitamin D receptor (VDR). Treatment of MDBK cells with 1,25(OH)2D3 for 24 h increased membrane PKC activity and immunoreactivity. The effect of 1,25(OH)2D3 was dose-dependent, with a peak effect observed at 10(-7)M 1,25(OH)2D3. The 1,25(OH)2D3 induced increase in membrane PKC was paralleled by a comparable decrease in cytosolic PKC activity and amount. Although time course studies were consistent with a VDR mediated effect of 1,25(OH)2D3 on PKC protein synthesis, total PKC activity was not increased by 1,25(OH)2D3, suggesting an effect on PKC translocation or localization. These results suggest that 1,25(OH)2D3 modulates PKC mediated events in kidney, a classic target for this steroid hormone.     

Vitamin D3 regulation of stromelysin-1 (MMP-3) in chondrocyte cultures is mediated by protein kinase C

Matrix metalloproteinases (MMPs) are a group of enzymes with the potential to degrade extracellular matrix proteins. One of the MMPs, stromelysin-1 (MMP-3) has been localized to extracellular matrix vesicles in growth plate chondrocyte cultures, suggesting involvement of this enzyme in remodeling of the extracellular matrix during endochondral development, a process which is regulated by the vitamin D metabolites, 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃. To determine whether stromelysin-1 is regulated by vitamin D as well, confluent cultures of cells derived from growth zone (GC) and resting zone (RC) rat costochondral cartilage were treated with 1α,25-(OH)₂D₃ (1,25) and 24R,25-(OH)₂D₃ (24,25), respectively, and the effect on stromelysin-1 assessed by casein gel zymography and Western blots. Although stromelysin-1 activity was enriched in the matrix vesicle fraction, only the plasma membrane enzyme was affected by the treatment; 1,25 and 24,25 caused a marked decrease in plasma membrane stromelysin-1 activity in their target cells. Since plasma membrane protein kinase C (PKC) activity is stimulated by 1,25 and 24,25, we hypothesized that stromelysin-1 activity was regulated by the vitamin D metabolites via PKC-dependent phosphorylation. To test this, membrane fractions (containing endogenous PKCα and ζ as well as stromelysin-1) were incubated in the presence of purified rat brain PKC and/or recombinant human (rh) stromelysin-1 and [γ³²P]-ATP and anti-stromelysin-1 immunoprecipitates were analyzed by autoradiography and Western blots. Immuno-phospho-stromelysin-1 was localized to a 52-kDa band in the plasma membrane fraction only; no phosphorylation was observed in the matrix vesicle fraction. Selective inhibitors of PKC activity demonstrated that phosphorylation was inhibited by H7 and low concentrations of H8, but not by HA1004, indicating that PKC, not PKA, was responsible. Protein phosphatase 2A, (PP2A), a serine/threonine-specific phosphatase, selectively removed the radiolabel in a time-dependent manner, providing further support for a PKC-dependent phosphorylation mechanism. Incubation of resting zone cell plasma membranes with 24,25, but not 1,25, resulted in phosphorylation of stromelysin-1, demonstrating that the nongenomic effect was metabolite-specific. This suggests that this may be one mechanism by which vitamin D metabolites regulate stromelysin-1 activity and that PKC-dependent phosphorylation inhibits the metalloproteinase. © 1996 Wiley-Liss, Inc.     

A role for the cytoskeleton in renal vitamin D metabolism

Conversion of circulating 25-hydroxyvitamin D3 (25(OH)D3) to its active metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) occurs in the renal tubule mitochondrion. Recent reports have implicated the cytoskeleton in certain other steroid metabolizing cells as a mediator of a rate-limiting mitochondrial transport step. Whilst the activity of the renal converting enzyme, a typical steroid hydroxylase, is known to be regulated closely by a number of well studied factors, no information is available to indicate whether an analogous transport step is relevant to the regulation of vitamin D metabolism. Cytochalasin B and vinblastine were used as chemical antagonists of the microfilamentous and microtubular elements of the cytoskeleton. Both agents inhibited the conversion of 25(OH)D3 to 1,25(OH)2D3 by isolated vitamin D-deficient chick renal tubules in a dose-dependent manner. At the concentrations required to inhibit 25(OH)D3-1 alpha-hydroxylase activity in whole cells, these agents inhibited neither isolated mitochondrial 1,25(OH)2D3 production, nor 24,25(OH)2D3 synthesis by vitamin D-replete tubules. The cytoskeletal antagonists were found to increase the content of labelled 1,25(OH)2D3 and 25(OH)D3 in a mitochondrial fraction prepared by Percoll fractionation of tubule cells pre-exposed to the antagonists and labelled 25(OH)D3 substrate. The data suggest that disruption of the cytoskeleton may result in inhibition of transport of newly synthesised 1,25(OH)2D3 out of the mitochondrion and through the cell, and accumulating 1,25(OH)2D3 may oppose its further synthesis. This is consistent with a transport process mediated by the cytoskeleton being involved in the regulation of renal vitamin D metabolism.     

ISOLATION OF A GOLGI APPARATUS-RICH FRACTION FROM RAT LIVER : II. Enzymatic Characterization and Comparison with Other Cell Fractions

Enzymatic activities associated with Golgi apparatus-, endoplasmic reticulum-, plasma membrane-, mitochondria-, and microbody-rich cell fractions isolated from rat liver were determined and used as a basis for estimating fraction purity. Succinic dehydrogenase and cytochrome oxidase (mitochondria) activities were low in the Golgi apparatus-rich fraction. On the basis of glucose-6-phosphatase (endoplasmic reticulum) and 5'-nucleotidase (plasma membrane) activities, the Golgi apparatus-rich fraction obtained directly from sucrose gradients was estimated to contain no more than 10% endoplasmic reticulum- and 11% plasma membrane-derived material. Total protein contribution of endoplasmic reticulum, mitochondria, plasma membrane, microbodies (uric acid oxidase), and lysosomes (acid phosphatase) to the Golgi apparatus-rich fraction was estimated to be no more than 20–30% and decreased to less than 10% with further washing. The results show that purified Golgi apparatus fractions isolated routinely may exceed 80% Golgi apparatus-derived material. Nucleoside di- and triphosphatase activities were enriched 2–3-fold in the Golgi apparatus fraction relative to the total homogenate, and of a total of more than 25 enzyme-substrate combinations reported, only thiamine pyrophosphatase showed a significantly greater enrichment.     

Localization of the membrane-associated CTP:phosphocholine cytidylyltransferase in Chinese hamster ovary cells with an altered membrane composition

The subcellular localization of the membrane-associated CTP:phosphocholine cytidylyltransferase was determined in Chinese hamster ovary cells in which the phospholipid composition had been altered by growth in the presence of N-methylethanolamine or treatment with phospholipase C. Cell homogenates were fractionated on Percoll density gradients, and marker enzyme activities were used to determine the location of the cellular membrane fractions. The peak of cytidylyltransferase activity occurred in the gradient at a density intermediate to that of the peaks of endoplasmic reticulum and plasma membrane markers. The profile of cytidylyltransferase activity most closely resembled that of the Golgi membrane marker; however, upon sucrose gradient centrifugation, the profile of the Golgi apparatus was very different from that of cytidylyltransferase. Differential centrifugation suggested a nuclear membrane association of the enzyme. Cytidylyltransferase was associated with a membrane fraction that sedimented when subjected to very low speed centrifugation (65 x g, 5 min). From Percoll gradient fractions, nuclei were identified by microscopy, and they migrated with cytidylyltransferase activity. The data are consistent with a localization of cytidylyltransferase in the nuclear membrane.     

Rapid Isolation of Rat Brain Nuclei on Percoll Gradients

A rapid isotonic method for fractionation of nuclei from rat brain is described. This procedure is based on the use of discontinuous colloidal silica gel (Percoll) gradients. We start from a 63,000-g purified nuclear pellet (fraction P3) isolated from gray matter and white matter separately. This is followed by fractionation of fraction P3 in an initial differential centrifugation step on five-step Percoll gradients producing six nuclear fractions designated 1, 2, 3 (gray matter) and 4, 5, 6 (white matter). Fractions 2, 4, and 5 obtained from this centrifugation are heterogeneous. These fractions are subfractionated further under isopycnic conditions using five-step Percoll gradients to yield subfractions 2b, 4b, and 5c. Three methods were used to characterize the nuclear types. First, light and electron microscopic examination was used to identify the nuclei in each preparation and to assess the purity of each preparation. Second, the activities of RNA polymerase I and II were monitored. Third, the protein/DNA ratios of the nuclear fractions were determined. Fraction 1 was enriched in neuronal nuclei; fractions 2b and 4b in astrocytic nuclei; and fractions 3, 5c, and 6 in nuclei of oligodendrocytes. RNA polymerase I and II activity was highest in fraction 1, which also displayed the highest protein/DNA ratio. Electron microscopy showed that the various classes of nuclei are congruent to 90% pure. Therefore, the procedure described here is suitable for obtaining highly purified neuronal and three types of glial nuclei from rat brain.     

Localization of 1,25-(OH)2D3-responsive alkaline phosphatase in osteoblast-like cells (ROS 17/2.8, MG 63, and MC 3T3) and growth cartilage cells in culture

Previous studies have shown 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-responsive alkaline phosphatase in cultured growth zone cartilage chondrocytes is localized in extracellular matrix vesicles (MV). Since osteoblast-like cells also have 1,25-(OH)2D3-responsive alkaline phosphatase, this study determined whether the 1,25-(OH)2D3-responsive enzyme activity is localized to MV produced by these cells as well. Osteoblast-like cells from rat (ROS 17/2.8), mouse (MC 3T3), human (MG 63), and rat growth zone cartilage were cultured in Dulbecco's modified Eagle's medium containing 10(-7)-10(-12) M 1,25-(OH)2D3. Alkaline phosphatase total activity and specific activity were measured in the cell layer, MV, and plasma membrane (PM) fractions. MV and PM purity were verified by electron microscopy and MV alkaline phosphatase specific activity compared to PM (MV versus PM: ROS 17/2.8 6 x; MG 63, 5.5 x; MC 3T3, 33 x; GC, 2 x). There was a dose-dependent stimulation of MV alkaline phosphatase (5- to 15-fold increase at 10(-7)-10(-9) M) in all cell types in response to the 1,25-(OH)2D3. The PM enzyme was stimulated in a parallel fashion in the osteoblast cultures. No effect of 1,25-(OH)2D3 was observed in growth cartilage PM. Although MV accounted for less than 20% of the total activity they contributed 50% of the increase in alkaline phosphatase activity in the cell layer in response to 1,25-(OH)2D3 and MV specific activity was enriched 10 times over that of the cell layer. These are common features of MV produced by cells which calcify their matrix and suggest that hormonal regulation of MV enzymes may be important in primary calcification.     

Analytical study of microsomes and isolated subcellular membranes from rat liver. VII. Distribution of protein-bound sialic acid

Detailed investigations by quantitative centrifugal fractionation were conducted to determine the subcellular distribution of protein-bound sialic acid in rat liver. Homogenates obtained from perfused livers were fractionated by differential centrifugation into nuclear fraction, large granules, microsomes, and final supernate fraction, or were used to isolate membrane preparations enriched in either plasma membranes or Golgi complex elements. Large granule fractions, microsome fractions, and plasma membrane preparations were subfractionated by density equilibration in linear gradients of sucrose. In some experiments, microsomes or plasma membrane preparations were treated with digitonin before isopycnic centrifugation to better distinguish subcellular elements related to the plasma membrane or the Golgi complex from the other cell components; in other experiments, large granule fractions were obtained from Triton WR-1339-loaded livers, which effectively resolve lysosomes from mitochondria and peroxisomes in density gradient analysis. Protein-bound sialic acid and marker enzymes were assayed in the various subcellular fractions. The distributions obtained show that sialoglycoprotein is restricted to some particular domains of the cell, which include the plasma membrane, phagolysosomes, and possibly the Golgi complex. Although sialoglycoprotein is largely recovered in the microsome fraction, it has not been detected in the endoplasmic reticulum-derived elements of this subcellular fraction. In addition, it has not been detected either in mitochondria or in peroxisomes. Because the sialyltransferase activities are associated with the Golgi complex, the cytoplasm appears compartmentalized into components which biogenetically involve the Golgi apparatus and components which do not.     

Immunochemical studies on the putative plasmalemmal receptor for 1, 25(OH)(2)D(3). I. Chick intestine

Antisera were raised against the NH(2)-terminus of the putative basal lateral membrane (BLM) receptor for 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3); BLM-VDR]. In Western analyses of BLM proteins, antibody (Ab) 099 was monospecific for a 64.5-kDa band. A protein of 64.5 kDa was also labeled by the affinity ligand [(14)C]1, 25(OH)(2)D(3)-bromoacetate; label was diminished in the presence of excess unlabeled secosteroid. The monoclonal antibody against the nuclear VDR (9A7) failed to detect an appropriate band in BLM fractions. Preincubation of isolated intestinal cells with Ab 099, but not 9A7, affected the following two 1,25(OH)(2)D(3)-mediated signal transduction events: augmented intracellular calcium and protein kinase C activity. Subcellular distribution of Ab 099 reactivity by Western analyses and fluorescence microscopy revealed the highest concentrations in BLM followed by the endoplasmic reticulum. Exposure of isolated intestinal cells to 1,25(OH)(2)D(3) for 10 s or vascular perfusion of duodena for 5 min resulted in a time-dependent increase in nuclear localization of the BLM-VDR antigen, as judged by electron microscopy, whereas 24, 25-dihydroxyvitamin D(3) failed to increase antigenic labeling in nuclei. Densitometric quantitation of Western blots of subcellular fractions prepared from isolated intestinal cells treated with vehicle or 1,25(OH)(2)D(3) confirmed a hormone-induced increase of putative BLM-VDR in the nucleus. It is concluded that a novel cell surface binding protein for 1,25(OH)(2)D(3) has been identified.     

Isolation and identification of 1,25-dihydroxy-24-oxo-vitamin D3 and 1,23,25-trihydroxy-24-oxo-vitamin D3. New metabolites of vitamin D3 produced by a C-24 oxidation pathway of metabolism for 1,25-dihydroxyvitamin D3 present in intestine and kidney

Two new vitamin D metabolites were isolated in pure form from separate incubations of homogenates of chick small intestinal mucosa or rat kidney employing either 1 alpha,25-dihydroxyvitamin D3 (28 microM) or 1 alpha,24R,25-trihydroxyvitamin D3 as substrate (0.17-1.3 microM). The newly characterized compounds and the amounts isolated in pure form from separate isolations are, respectively: 1 alpha,25-dihydroxy-24-oxo-vitamin D3 (1,25(OH)2-24-oxo-D3), 147 micrograms from kidney and 4.2 and 40 micrograms from intestine; 1 alpha,23,25-trihydroxy-24-oxo-vitamin D3 (1,23,25(OH)3-24-oxo-D3), 155 micrograms from kidney and 5.9 and 34 micrograms from intestine. Their structures were identified after extensive high pressure liquid chromatography by means of ultraviolet absorption spectrometry, mass spectrometry of the free compounds and their trimethylsilylated derivatives, proton nuclear magnetic resonance spectrometry, specific chemical reduction of the 24-oxo functionality with sodium borohydride, as well as direct comparison with synthetic 1,25(OH)2-24-oxo-D3. These structural assignments for both compounds correct previous determinations which had been proposed (Ohnuma, N., Kruse, J. R., Popjak, G., and Norman, A. W. (1982) J. Biol. Chem. 257, 5097-5102). The activity of the C-24 oxidation pathway used for the production of the 1,25(OH)2-24-oxo-D3 and 1,23,25(OH)3-24-oxo-D3 can be enhanced 10-fold by prior priming of the chicks or rats with a single intravenous dose of 1,25(OH)2D3 (1-12 nmol/100 g body weight); the induction of the enzyme activity is maximal by 3-6 h and returns to basal levels within 12 h. Further, 1,25(OH)2D3, 1,24,25(OH)3D3, and 1,25(OH)2-24-oxo-D3 all were found to be capable of serving as a precursor with chick intestine and rat kidney homogenates of 1,23,25(OH)3-24-oxo-D3. Collectively these results suggest the existence of a C-24 oxidation pathway for metabolism of 1,25(OH)2D3 by the target intestinal mucosa and kidney to 1,23,25(OH)3-24-oxo-D3. The pathway may play an important role in controlling the tissue levels of this hormonally active form of vitamin D3.     

Effect of three A-ring analogs of 1 alpha,25-dihydroxyvitamin D3 on 25-OH-D3-1 alpha-hydroxylase in isolated mitochondria and on 25-hydroxyvitamin D3 metabolism in cultured kidney cells

Three A-ring analogs of 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3)--2-nor-1,3-seco-1,25(OH)2D3 (2-nor analog), 2-oxa-3-deoxy-25-OH-D3 (2-oxa analog), and A-homo-3-deoxy-3,3-dimethyl-2,4-dioxa-25-OH-D3 (A-homo analog)--were tested for their ability to inhibit 25-OH-D3-1 alpha-hydroxylase (1 alpha-hydroxylase) in isolated mitochondria and to alter 25-OH-D3 metabolism in cultured chick kidney cells. The 2-nor and 2-oxa analogs were relatively potent (Kis of 60 and 30 nM, respectively, compared with 170 nM for 1,25(OH)2D3), whereas the A-homo analog was completely ineffective in inhibiting 1 alpha-hydroxylase activity. In contrast, all three analogs were able to repress 1 alpha-hydroxylase and induce 24-hydroxylase activity in cultured chick kidney cells, suggesting that this process is not one of direct action in the mitochondria, but is more likely to be a receptor-mediated one.     

A specific binding protein for 1 alpha,25-dihydroxyvitamin D in the chick embryo chorioallantoic membrane

A 3.7 S binding protein for the steroid hormone and vitamin D metabolite 1 alpha-25-dihydroxyvitamin D (1,25-(OH)2-D) was observed in high salt cytosol extracts of chick embryo chorioallantoic membrane. The binding protein was characterized after partial purification of cytosol extracts by ammonium sulfate fractionation. The binding of 1,25-(OH)2-D was saturable, had a high affinity (Kd = 0.16 nM), and was specific for hormonally active vitamin D metabolites. Analysis of the displacement of [3H]1,25-(OH)2-D by unlabeled analogues showed the affinities of vitamin D metabolites to be in the order of 1,25-(OH)2-D = 1,24R,25-(OH)3-D much greater than 25-OH-D = 1-OH-D greater than 24R,25-(OH)2-D. Hormone binding was sensitive to pretreatment with sulfhydryl-blocking reagents. The chorioallantoic membrane 1,25-(OH)2-D-binding protein associated with the chromatin fraction after homogenization of membranes in low salt buffer, and bound to DNA-cellulose columns, eluting as a single peak at 0.215 M KCl. These findings support identification of this 1,25-(OH)2-D-binding protein as a steroid hormone receptor, with properties indistinguishable from 1,25-(OH)2-D receptors in other chick tissues. The chorioallantoic membrane functions in the last third of embryonic development to reabsorb calcium from the eff shell for deposition in embryonic bone. 1,25-(OH)2-D binding activity in the chorioallantoic membrane increased 4- to 5-fold from day 12 to day 16 of incubation, immediately preceding the onset of shell reabsorption. This finding suggests that 1,25-(OH)2-D may act to regulate shell mobilization and transepithelial calcium transport by the chorioallantoic membrane. Finally, the similarity of shell mobilization to bone resorption, which is also stimulated by 1,25-(OH)2-D, suggests that the chorioallantoic membrane is a useful alternate model for the study of 1,25-(OH)2-D action on bone mineral metabolism.     

Synthesis of plasmalemmal glycoproteins in intestinal epithelial cells. Separation of Golgi membranes from villus and crypt cell surface membranes; glycosyltransferase activity of surface membrane

The relationship between Golgi and cell surface membranes of intestinal cells was studied. These membranes were isolated from intestinal crypt cells and villus cells. The villus cell membranes consisted of microvillus membrane, a Golgi-rich fraction, and two membrane fractions interpreted as representing lateral-basal membranes. The villus cell microvillus membrane was purified by previously published techniques while the other membranes were obtained from isolated cells by differential centrifugation and density gradient velocity sedimentation. The two membrane fractions obtained from villus cells and considered to be lateral-basal membranes were enriched for Na+,K+-ATPase activity, but one also showed enrichment in glycosyltransferase activity. The Golgi membrane fraction was enriched for glycosyltransferase activity and had low to absent Na+,K+-ATPase activity. Adenylate cyclase activity was present in all membrane fractions except the microvillus membrane but co-purified with Golgi rather than lateral-basal membranes. Electron microscopy showed that the Golgi fraction consisted of variably sized vesicles and cisternalike structures. The two lateral-basal membrane fractions showed only vesicles of smaller, more uniform size. After 125I labeling of isolated intact cells, radioactivity was found associated with the lateral-basal and microvillus membrane fractions and not with the Golgi fraction. Antibody prepared against lateral-basal membrane fractions reacted with the surface membrane of isolated villus cells. The membrane fractions from isolated crypt cells demonstrated that all had high glycosyltransferase activity. The data show that glycosyltransferase activity, in addition to its Golgi location, may be a significant property of the lateral-basal portion of the intestinal villus cell plasma membrane. Data obtained with crypt cells support earlier data and show that the crypt cell surface membrane possesses glycosyltransferase activity.