Control of vitamin D-dependent calcium-binding protein in rat intestine by growth and fasting.

The vitamin D-dependent calcium binding protein (CaBP) content of the proximal small intestine of normal female rats was related to the animal’s growth rate and feeding pattern. CaBP in a low molecular weight fraction of 40,000 x g duodenal supernatant was quantitated by ljCa saturation analysis; changes in CaBP were confirmed by polyacrylamide disc gel electrophoresis with the use of a purified CaBP marker. The total amount of CaBP was unchanged between 4 and 9 weeks of age during the rapid phase of growth. By 15 and 20 weeks of age, when the growth rate had decreased to less than 10 g/week, CaBP levels were 46% and 30%, respectively, of values obtained in the younger animals. These decreases in CaBP with age were inversely related to changes in total mucosal protein content. The amount of CaBP/g of body weight decreased between 4 and 20 weeks of age in an exponential manner and paralleled the exponential decay in growth rate. At all ages, CaBP decreased by an average of 58% following a 22-h fast, whereas total mucosal protein decreased by only 16%. These alterations in CaBP are consistent with previously reported changes in calcium absorption observed in both well fed and undernourished animals. The data indicate that the vitamin D-dependent CaBP is affected by undefined factor(s) which condition the response to both growth and fasting. A rapid and simple method is described for isolating rat intestinal vitamin D-dependent CaBP in milligram quantities; the purified CaBP (M, = 10,000) has high affinity sites for Ca’+ binding, an apparent dissociation constant of 0.3 PM, with a maximum binding capacity of approximately 0.24 pmol of Ca”+/mg of protein (-2 mol of Ca’+/mol of CaBP).

Numerous studies suggest a close correlation between intestinal calcium absorption and a duodenal calcium-binding protein which is vitamin D '-dependent (1 (4, 5), egg laying (4, 6), dietary calcium and phosphorus (4, 7-9), strontium (lo), dilantin (111,and Solarium malacoxylin (12). In most of these situations, alterations in the levels of vitamin D metabolites can explain the changes in both CaBP and calcium absorption (13). An exception appears to be the effect of aging in which alterations of vitamin D metabolism have not yet been defined. The present report is the first characterization of the effects of fasting on CaBP, and of CaBP levels during the period of rapid growth. Earlier studies by others have related CaBP to dietary calcium intake. Under normal feeding conditions, low calcium intake results in increased levels of intestinal CaBP (4, 7-9). However, in the present study, acute fasting with a loss of 10 to 16% body weight produced a marked decrease in rat duodenal CaBP. Previous studies on aging and intestinal CaBP have compared only old animals to young animals, specifically young chicks to hens (4), and 100-g rats to animals weighing over 500 g (5). The results of this report indicate that the level of CaBP is tailored to the growth rate of the animal and is predictable for a given growth rate. These observations are consistent with the hypothesis that a vitamin D-dependent intestinal CaBP may prove essential to ensure maximal skeletal growth (1).  Fig. 3 illustrates the protein staining patterns of Fractions II and III after polyacrylamide disc gel electrophoresis. Measurements of CaBP activity of sequential segments of the slab gel ( Fig. 1) revealed that the CaBP activity of Frac- Vitamin D-dependent Calcium-binding Protein in Rat Intestine 4147 tion II resides primarily in a band with low mobility (Fraction III). The Ca*+ content in the concentrated Fraction III sample was approximately 45 FM, sufficient to saturate the high affinity Ca2+ binding sites which would neutralize amino acid groups involved in the binding process. In the presence of 0.1 mM EDTA, purified CaBP (Fraction III) migrated very rapidly with an R, of 0.78. This large increase in the anionic properties of rat intestinal CaBP by the addition of EDTA was similar to that previously reported for the intestinal porcine (24) and bovine CaBP (25). In the EDTA disc gel system, minor bands (~10%) were observed migrating slightly behind or in front of the major band (R, = 0.78). These minor bands may have calcium binding properties since their migration properties in disc gels were sensitive to the Ca2+ concentration. The arrow designates the CaBP band as determined from RE properties of purified CaBP in an EDTA buffer system (Fig.  3).

Effect of Age on Proximal Small Intestinal Proteins and
CaBP in Fed Rats - Table II summarizes the normal growth pattern of female rats fed ad libitum from 4 to 20 weeks of age. A rapid growth phase was noted between 4 and 9 weeks of age (81 to 201 g) followed by a slower rate of weight gain between 9 and 20 weeks of age (201 to 274 g). Although the total body weight greatly changed between 4 and 9 weeks of age, changes in total mucosal protein and Fraction I protein from the proximal small intestinal segment were insignificant.
Progressive maturation was associated with small increases in protein content. By 20 weeks of age, 30% and 33% increases in protein content were observed in total mucosal protein and Fraction I protein, respectively.
Age-related changes observed in the protein content of the low molecular weight Fraction II more closely paralleled the total body weight of the animals. Comparing all fed animals, a 2-fold increase in Fraction II was detected from 4 to 9 weeks of age, although no further increments were noted at 15 and 20 weeks of age.
The CaBP pattern was strikingly different from that of other intestinal protein fractions. Total CaBP activity in Fraction II was constant between 4 and 9 weeks of age. However, by 15 weeks of age there was a 54% decrease in CaBP activity observed at 4 to 9 weeks of age, and by 20 weeks of age the activity was only 30% of that observed in the youngest animals. The age-related quantitative decrease in CaBP was confirmed by polyacrylamide disc gel analysis (Fig. 4). When constant amounts of low molecular weight protein (Fraction II) were applied to each gel, the specific activity of CaBP in this fraction paralleled the percentage of the CaBP band in disc gel. At 4, 9, and 15 weeks of age, the mean (? S.E.) specific activity of CaBP (nmol of Ca*+ bound/mg of protein) of Fraction II (Table II) was 21.4 (? 1. 41, 11.8 (k 2.11, and 5.0 (f 0.61, respectively. When CaBP of intestinal segments, expressed in terms of body weight, was analyzed as a function of age (Fig. 51, an exponential decrease in CaBP was apparent between 4 and 20 weeks of age which was similar to the declining growth rate of normal rats (26). In fact, duodenal CaBP content per g of body weight proved to be a direct linear function of the rat's growth rate as shown in the inset of Fig. 5   intestinal parameters such as duodenal wet weight or protein content of the various fractions detailed in Table II Table II summarized the effect of fasting (22-h removal of food from cages) on the body weight and mucosal protein content of rats. At all ages, a consistent 10 to 16% weight loss was observed. Fasting caused small decreases in the amount of isolated protein in the various fractions; however, there was no consistent pattern of change in the total protein content when fed and fasted animals were compared. The mean percentage of decreases (-+-S.E.) in total mucosal protein, Fraction I, and Fraction II were 15.6 (? 5.61, 19.6 (+ 4.81, and 21 (2 7.31, respectively, for each of the five groups. In marked contrast, CaBP activity in each group of animals was consistently and markedly reduced by fasting. The decrease in CaBP with fasting ranged between 42% and 75%, and the mean percentage of decrease (+ S.E.) for all ages studied was 57.8% (* 5.8). Polyacrylamide disc gel analysis of Fraction II was used to evaluate the effect of fasting on intestinal CaBP. As illustrated in Fig. 6, the density of the CaBP band in the fed animals was approximately twice that observed in the fasted group. No similar decrease was observed in the other low molecular weight proteins. DISCUSSION Several groups have isolated the vitamin D-dependent intestinal CaBP from avian and mammalian intestines and have reported on the properties of these proteins (18,24,25,27,28).