George M. Lessard Department of Biochemlstry Lorna Lmda Unwers~tv Loma Linda. CA 92350
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The Determination of Vitamin D-Dependent Calcium Binding- Protein in Chick Intestine An undergraduate biochemistry laboratory experiment
A major challenge in the development of undergraduate biochemistry lahoratory experiments is t h a t of devising interesting experiments using only commonly available equipment which can he completed in a single 4-hr laboratory period. Additionally, the experiments must maximize the number of basic concepts and techniques to which the students are exposed. An experiment inducing rickets in chicks and correlating the disease t o a reduction in vitamin D-dependent calcium hinding protein affords a n exciting demonstration of hormone induction, protein isolation, and radioisotope methodology. T h e experiment requires only limited preparation all of which can he performed by lahoratory assistants. Only instruments normally found in a n undergraduate laboratory are needed for the experiment. T h e experiment has been successfully used a s a part of the junior year introductory hiochemistry lahoratory and gives consistently satisfactory results. T h e diversity of metabolic processes dependent on serum Ca2+ levels demands t h a t the uptake and excretion as well as deposition and resorption of calcium h e precisely regulated. T h e metabolism of Ca2+ and the regulation of its tissue content have been reviewed recently (1-3).The uptake of calcium by the intestinal mucosa is a function of the calcium transport system localized in the brush border memhrane of the small intestine. \\'bile the mechanism of this system is not fully understood, it is known that the transport of calcium is inducihle by 1,25-dihydroxycholecalciferol,the uptake-active metabolite of vitamin D3 (I).The synthesis of the metabolite is ultimately regulated by parathormone, t h e hormone responsible for t h e elevation of serum Ca2+ levels. Calcium transoort can he abolished hv inhibitors of transcrifltion and translation which suggests that protein synthesis is essential for the induction of calcium transoort. At least three proteins are induced in the intestinal muEosa by 1,z.F-dihydrixycholecalciferol (2). T h e maior has been identified a s a . protein . cytqdnsmic vitamin ])-dependent calcium hinding protein ( 7 ) .This orotein whirh is translated from indurihle messenger RNA (8j i s present in relatively high concentrations a n d is assayed easily by a competitive binding assay. Since the appearance of the vitamin D-dependent calcium binding protein, a s detected by immunological techniques paralleis-the increase in calcium transport, vitamin D-dependent calcium hinding protein is considered t o he a n integral part of the calcium &&port system by some reseachers (2).Other investigators place it in a permissive role, responsible for t h e maintenance of calcium transport h u t not a s a part of the primary transport system (6).Calcium hinding nrotein is a 28.000 dalton (. 7.). .heat-stable ~ r o r e i nwhirh hinds four moles of calcium per mole of protein (I). T h e concentration of the protein correlates well with t h e calcium transport activity df the tissue. ~
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Experlmental The only equipment necessary for this experiment is a PotterElvehjem homogenizer, a desk top centrifuge, a refrigerated highspeed centrifuge and a counter eapahle of deteetingd5Ca.in our lahoratory, aliquots are counted directly in Aquasol (New England Nuclear) wine a Beckman LS100C liquid scintillation counter. Placing the dliquutson plnnehets, drying the planchets, and countrng them in a thin-window Gerger-.Muller rcmnter will provide cqunllg ratisfactory results. 674 / Journal of Chemical Education
One day-old white Leghorn cockerels (generally available at no cost from local hatcheries) are divided into two groups. For a class of 1&20 students, 4-6 animals will suffice for each of the groups. Group I, supplemented with vitamin D (+Dl is fed a normal diet of Purina Chick Starter or equivalent. Group 11, deprived of vitamin D (-D) is fed a rachitogenicchick diet (ICN Nutritional Biochemicals). The groups are maintained for sir weeks on these diets in wire floored growth cages. Weight and appearance are recorded periodically. By five weeks of erowth.. svmotoms of erowth retardation and skeletal . . maltbrmation, characteristic of avian rickets. are ohvious. On the murningof the experiment, the chicks aresacrificed by decapitatiun, the peritoneal ra, itg is opened, end the duodenal loop of the small intestine is excised. One animal from each group is retained as a demonstration animal for use during the lahoratory period. Using a squeeze bottle, the intestinal segments are washed out with 0.85% NaCl and are opened along their entire length using a scalpel. The inner surface of the intestine is blotted drv. Usine" two "elass microscope slides as a working surface and scraper, the soft mucosal cells are scraped off the tough, muscular underlying tissue. The mucosal cell paste thus obtained is placed in one of two labelled (+D and -D) preweighed heakers, weighed and diluted 4:l (vlw) with 0.131 M Tris-HCI, 120mM NaC1,4.74 mM KC1, pH 7.4. The suspensions are then homoeenized bv 10 oasses with the Potter-Elvebeiem bomoeenizer usinga ~eflonGpesile.All steps to this point are carried outat temperatures near O°C. The two homogenates (+D and -D) are then centrifueed at 37.000 xG for 20 min. Each oost-mitochondrial supernatant irderanted csretull) intoseparate k t tubes (7l)and -1)) and i i incubated at 57°C f
