Biochimica et Biophysica Acta, 379 (1975) 553-561

© Elsevier ScientificPublishing Company, Amsterdam -- Printed in The Netherlands BBA 36931 VITAMIN D-DEPENDENT KIDNEY

CALCIUM-BINDING PROTEIN

FROM

RAT

CAROL L. HERMSDORFa and FELIX BRONNERb aDepartment of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Conn. 06510 (U.S.A.) bDepartment of Oral Biology, The University of Connecticut Health Center, Farmington, Conn. 06032 (U.S.A.)

(Received July 25th, 1974)

SUMMARY A calcium-binding protein has been partially purified from rat kidney. It is found in the cortex, but not in the medulla. It is Vitamin D-dependent, as it occurs in normal, but not in Vitamin D-deficient rats. The molecular weight is 28 000, more than twice that of the Vitamin D-dependent calcium-binding proteins from rat intestinal mucosa. The apparent dissociation constant of the partially purified renal calcium-binding protein is approx. 10 -5 M.

INTRODUCTION Stimulated by the discovery of a calcium-binding protein in the intestinal mucosa of chicken [1, 2] and several mammals (man [3-6], cow [7], dog [8], pig [5], rat [5, 9-16, 29], for a review see [17]), several workers have looked for such a protein in kidney. Taylor and Wasserman [18] reported in 1967 that kidneys from rachitic chicks contained no calcium-binding protein and that Vitamin D administration to such animals led to the appearance of the protein. Recently, the same workers [19] expanded on their findings and have reported that small amounts of calcium-binding protein, measured by a specific antiserum to the chick binding protein [20] and a radial immunoassay [21], persist in frankly rachitic chicks. This persistence of calciumbinding protein was explained as having resulted from the relatively slow turnover of renal cells. Sands and Kessler [22] identified a calcium-binding component of dog kidney cortex in the 16 000 × g supernate, tested by a competitive binding assay (Chelex assay [1, 2, 23]), and related its binding capacity to renal calcium excretion. Piazolo et al. [4] isolated a highly purified calcium-binding protein from human kidney by means of repeated gel-filtration and ion exchange chromatography, estimating a yield of 2.8 ~ of the crude protein mixture. These workers report that human renal cortex contains 4 times more calcium-binding protein than medulla and estimate the molecular weight as slightly higher than that of human somatotropic hormone (21 500). This would place its molecular weight in the range of the chicken intestinal binding protein (25 000-28 000 [1 ]). This observation has very recently been confirmed in human renal calcium-binding protein fractionated from necropsy material [24].

554 Hurwich et al. [25] have reported the existence of a renal calcium-binding factor in the rat and, on the basis of the Chelex test on heated kidney supernates, claim higher activity in the renal medulla than cortex. A Vitamin D-dependency of the renal calcium-binding protein has been established only for the chick [I 8, 19]. We now report the isolation of a Vitamin D-dependent calcium-binding protein from the cortical region of rat kidneys. MATERIALS AND METHODS

Animals, diets and harvesting of tissues Two types of Vitamin D deficiency were studied, that associated with rickets and produced experimentally by feeding weanling rats a regimen (diet VII-D) devoid of Vitamin D, high in calcium and low in phosphorus, and another due to Vitamin D deficiency alone and produced by feeding weanling rats a regimen (diet II-D) containing normal amounts of calcium and phosphorus, but devoid of Vitamin D. Vitamin D deficiency was considered to have been attained when animals on the high calcium, low phosphorus regimen had significantly lower plasma phosphorus values as compared with their controls, fed identical diets, but containing 2200 I.U. Vitamin Dz/kg diet (VII). In the case of the animals fed normal mineral intakes, Vitamin D deficiency was characterized by a significantly lower plasma calcium level, as compared to controls fed in addition 2200 1.U. Vitamin D2/kg diet (II). For further description, see [26]. Table I shows a typical set of plasma and urine parameters of two groups of Vitamin D-deficient animals and their respective controls from whom kidneys had been harvested. All animals were male rats, purchased from Sprague-Dawley, Madison, Wisconsin. For the Vitamin D deficiency studies, animals were weanlings and were usually sacrificed 4 weeks later, when they were 7-8 weeks old. To get larger amounts, kidneys TABLE1 CORRELATION BETWEEN PHYSIOLOGICAL CHARACTERISTICS AND RENAL CALCIUM BINDING PROTEIN Diet make-up as follows:

Ca, ~ P, % vitamin D 2200 IU/kg

VII -- D

VII

II -- D

II

I

II1

1.5 0.2

1.5 0.2 ,

0.5 0.5

0.5 0.5 ~

0.06 0.2 ~

1.5 1.5 i

VII

li - D

II

1"

143 12.4 8.8 14.9 present

140 5.6 8.7 0.6 absent

Diet groups VII -- D Body weight (g) Plasma calcium (mg/100 ml) Plasma phosphate (mg/100 ml) Urinary calcium (mg/day) Renal calcium binding protein

135 I 1.1 4.8 19.7 absent

161 224 10.7 10.7 9.9 (10.0)** 1.0 0.3 present present

* Data for diet groups I and III, except renal calcium binding protein, from [40]. ** Not determined. The value given is typical for normal rats of this age.

III* 214 10.7 1.3 present

555 were also harvested from animals used in other studies. These were usually purchased when they weighed 120 g (typically 6-7 weeks old) and kept on a given regimen for 10-14 days, when they would weigh 150--170 g. Semi-synthetic diets, purchased from General Biochemicals, Chagrin Falls, Ohio, contained 28 ~o vitamin-flee test casein, 5 ~ corn oil, 1 ~o vitamin supplement (General Biochemicals Catalog No. 40060 with or without vitamin D2), 3 ~ salt mixture*, 56 ~o sucrose and an inert filler (cellophane spangles). The diets were formulated to contain 0.05~ Ca, 0.2~o P, l; 0 . 5 ~ Ca, 0 . 5 ~ P, II; 1.5~ Ca, 0.2~o P, VI1; and 1.5 ~o Ca, 1.5 ~ P, Ill. Calcium and phosphorus were added at the expense of the filler. Vitamin D-deficient diets II-D and VII-D were formulated like diet II or VII, respectively, but free from Vitamin D2. Analysis of the two Vitamin D-deficient diets by the line test (performed by Waft Institute, Inc., Madison, Wisc.) showed them to be free of detectable amounts of Vitamin D2. Calcium and phosphorus levels of the diets were verified by analysis of dry-ashed samples [26]. Complete formulations are available from General Biochemicals under the following codes: I, TD 67205A; III, TD 67207; II, TD 70387; II-D, TD 70388; VII, TD 70389; VII-D, TD 70390. Animals were killed by decapitation, the kidneys rapidly removed, decapsulated, weighed and processed. In some instances the kidneys were separated into three regions : the outer 2 mm cortical region, the inner medullary region, and the boundary region between cortex and medulla.

Purification The kidneys, or kidney regions, typically from 6 animals, were homogenized in iced Tris-HC1 buffer (0.013 M Tris-HCl, 0.12 M NaCI, 3 mM KCI, pH 7.4) with the aid of a Potter-Elvehjem Teflon homogenizer (tissue/buffer ratio: 1/4, w/v). The homogenate was centrifuged at 100 000 × g for 1 h. The supernate was decanted, lyophilized, aliquots were redissolved in a minimum volume (1-3 ml) and further fractionated by elution on Sephadex G-50 with 0.02 M ammonium acetate and 1 mM mercaptoethanol as elution buffer (pH 7.2). Runs were performed on Pharmacia K 16/100 columns (1.6 × 100 cm, maximum) equipped with two adaptable plungers. Fractions, typically 3 ml, were monitored for protein content (A2s0 nm) and assayed for calcium-binding activity.

A naly t ical procedures Calcium-binding to the protein was determined by a modification of the competitive Chelex assay [1, 4, 23, 27, 29]. 45Ca2+, specific activity 10-15 Ci/g was made into a stock solution with Tris-HC1 buffer (pH 7.4) to approx. 107 cpm/ml. Chelex resin (Bio-Rad Laboratories, Richmond, Calif., U.S.A.), washed and equilibrated with Tris-HC1 buffer, was kept as an 80~o resin slurry. Column profiles were monitored by adding 50 ,ul 45Ca2+ and 50/~1 resin slurry to 250 #1 of an eluate fraction, and by counting 50 #1 aliquots of the resin supernate, * Magnesiumoxide, 2.82533 g/kg diet; potassium chloride 5.9097 g/kg; potassium citrate 6.2099 g/kg; sodium chloride 2.4599 g/kg; potassium sulfate 0.62996 g/kg; ferric citrate (16.7~ Fe) 0.48 g/kg; potassium iodide 0.012 g/kg; sodium fluoride 0.012 g/kg; manganese sulfate (MnSO4.H20) 0.18458 g/kg; aluminum potassium sulfate (AIK(SO,h- 12 H20) 0.0024 g/kg; cupric sulfate 0.037678 g/kg; zinc sulfate (ZnSO4.7 H20) 0.21992 g/kg. To obtain 0.06 ~ calcium add CaCO3 at the expense of the filler (cellophane spangles). To increase both calcium and/or P, add CaCO3 and/or Ca(H2PO4)2"H20 at the expense of the filler.

556 obtained by centrifuging 2 min at 8000 × g. A stable baseline value of 500-800 cpm was typical for samples eluted with ammonium acetate buffer (pH 7.2). A typical free Ca 2+ concentration in the column assay was less than 1 /zg/ml. Quantitative binding assays (as a function of calcium concentration) were performed by equilibrating 250/A sample, 250/~1 Tris-HC1 buffer containing 0-400 mg CaZ+/ml, 100 #1 45Ca2+ and 100 ~1 resin slurry. After centrifugation, 100 #1 of the resin supernate was added to 4 ml Bray's scintillator [28] and counted. The specific radioactivity of the calcium was calculated by taking into account the 4°CaZ+ content of the sample, of the 45Ca2+ solution, and the amount of added 4°Ca2+. From experiments with blanks run for each calcium concentration the free Ca 2+ concentration was calculated for each experimental condition. Calcium bound to the protein was calculated from the difference between calcium in the supernate in the presence and absence of the binding protein, and the total calcium in the assay system. Dipicolinic acid was used as binding assay control. When the binding control gave a result that differed by more than 10~o from the established mean, the assay series was rejected. For a more complete description of binding assays, see [291. Calibration of a Sephadex G-100 column for molecular weight determination was done with the aid of blue dextran, ovalbumin (Mr = 45 000), c~-chymotrypsinogen (Mr = 23 200), and RNAase (Mr = 13 683). Protein content was determined spectrophotometrically [30]. Calcium analysis was by atomic absorption spectrophotometry [26] and phosphorus by a modification [31] of the Fiske and Subbarow procedure [32] as adapted to the Technicon AutoAnalyzer [33]. Fractions from Peak B (see below) were subjected to analytical gel electrophoresis (7.5 % acrylamide gel, Tris-glycine buffer, pH 8.9) performed by a modification [34] of the method of Davis [35]. Gels were stained for protein. Duplicate gels were frozen, cut into slices by hand and the slices placed into binding assay tubes, 0.25 ml ammonium acetate buffer was added, the gels disrupted with a glass rod, 0.250 ml 45Ca and 0.050 ml resin suspension added and shaken for 45 rain. After centrifugation 0.050 ml aliquots were removed and counted. Some extracts were also subjected to repeated electrophoresis. RESULTS

Vitamin D-dependence The absorbance and calcium binding protein (solid line) shown in Fig. l are typical of those obtained with kidney material from animals fed adequate amounts of Vitamin D (diets VII, I1, 1 and III). Of the three peaks of binding activity shown, peaks A, B, and C, that in the peak C region was insensitive to variations in calcium assay content. Moreover, it persisted when Tris buffer alone was chromatographed on Sephadex G-100 equilibrated with ammonium acetate buffer. Therefore peak C binding was due to the salt contained in the sample, particularly as its salt concentration had been raised over that of the Tris buffer by lyophilization and redissolving in a smaller volume. Peak A, the void volume peak, was similar in all materials studied. Peak B, however, was absent in material derived from Vitamin D-deficient animals,

557

3.5-

'\L~'~~k

PEAK A

PEAK

T

2000 I t~

PEAK C

'

I000