Svein U. Toverud

Purification and partial characterization of two acid phosphatases from rat bone

SummaryAcid phosphatase activity in homogenized tibiae and femora of suckling rats was extracted with 0.3M KCl and 0.1% Triton X-100. A high-speed supernatant was treated with protamine sulfate, dialyzed, and chromatographed on CM-52 cellulose. All of the acid phosphatase activity was eluted with a sodium acetate buffer and combined ionic strength-pH gradient into two peaks (E1 and E2). Both enzyme peaks were further purified with Sephadex G-200, which resulted in 700- and 1000-fold purification for E2 and E1, respectively. A total of 220 units (µmoles substrate/min) of E2 with a specific activity of 160 units/mg protein has been obtained in one run by this procedure. E1 has a high molecular weight (>100,000) and shows preference for monophosphate ester substrates, is markedly inhibited by tartrate, and has a pH optimum near 5. E2 has a lower molecular weight (<40,000) and shows negligible activity with monophosphate esters [except withp-nitrophenyl phosphate (p-NPP)], but high activity with ADP and ATP. E2 is unaffected by tartrate and shows a pH optimum near 6. Both enzymes are competitively inhibited by inorganic phosphate, and E2, but not E1, is markedly inhibited byp-chloromercuribenzoate. Withp-NPP as substrate, E1 and E2 have distinctly different values for Km. E1 appears similar to the high molecular weight acid phosphatases of soft tissues. However, E2 appears to differ from the low molecular weight phosphatases in soft tissues with regard to substrate specificity.

Quantitative changes of bone collagen crosslinks and precursors in vitamin D deficiency

Abstract The crosslinks from NaB3H4-reduced bone collagen of 4-week-old rachitic and normal chicks were compared. The ratio of the reduced crosslink δ,δ′-dihydroxylysinonorleucine (DHLNL) to δ-hydroxylysinonorleucine (HLNL) was significantly increased in vitamin D deficiency. The ratio of ϵ-hydroxynorleucine (HNL) (reduced crosslink precursor of HLNL) to the reduced unknown peak 1 was decreased while that of HLNL to HNL was unaffected. The data indicate that vitamin D affects the quantitative relationships between crosslinks and aldehydic precursors that are present in mineralizing macromolecular matrices.

The effect of vitamin D on the structural crosslinks and maturation of chick bone collagen.

The quantitative relationships were determined between the structural crosslinks, dihydroxylysinonorleucine (Lys(OH)2-Nle) and hydroxylysinonorleucine (Lys (OH) -Nle) in NaB 3H4-reduced diaphyseal bone collagen from 1-, 2-, 3- and 4-week-old chicks fed either a vitamin D-deficient diet, a normal-vitamin D diet or a high-, but non toxic, vitamin D diet from time of hatching. Chicks fed the normal diet showed a progressive decrease in the ratio of Lys(OH)2-Nle/Lys(OH)-Nle with age. This decrease was accelerated in chicks receiving the High-vitamin D diet. In the vitamin D-deficient group, the ratio was higher than controls at 1 and 2 weeks and increased further at 3 and 4 weeks. Similar changes in Lys(OH)2-Nle/Lys(OH)-Nle ratio did not occur in skin collagen. Compared to Control-vitamin D animals, the increased crosslink ratios in the vitamin D-deficient bone collagen occurred prior to changes in growth rate and could not be correlated with lysine hydroxylation or the hypocalcemia seen in this group. These results suggest that the type of crosslink analysis used in this study provides one of the earliest and most sensitive indications of a bone disturbance due to vitamin D deficiency and that vitamin D specifically acts to increase the rate of maturation of bone collagen.

Purification and characterization of purple acid phosphatase from developing rat bone.

Tartrate-resistant acid phosphatase active on nucleoside di- and triphosphate substrates was isolated from developing rat bone and purified 2500-fold. The enzyme concentration had a purple coloration and activity that was sensitive to reducing agents. Mild reducing agents such as ferrous ion and ascorbic acid caused loss of purple color and increased activity toward substrates severalfold; however, a strong reductant such as dithionite caused loss of both color and activity which were partially restored by addition of ferrous ion and ascorbic acid. Enzyme activity was homogeneous with protein during the final gel permeation steps of chromatography and gave an apparent molecular size of about 40,000 Da. Determination of iron in the most pure preparation revealed the presence of 1.3 atoms of iron per molecule of the tartrate-resistant enzyme E2. Other properties of the purified enzyme include a pI of approximately 9.5 and sensitivity to inhibition by ions of copper, zinc, fluoride, and molybdate. Antibody prepared to the pre-concanavalin A (Con A)-Sepharose purified enzyme reacted with all protein from the Con A step, but it did not react with tartrate-sensitive acid phosphatase from rat bone or with potato acid phosphatase. Purple acid phosphatase from rat bone has many properties that parallel the iron-containing purple acid phosphatases from rat spleen, bovine spleen, and pig uterine secretions.

Hormonal control of calcium metabolism in lactation.

Publisher Summary This chapter discusses the hormonal control of calcium metabolism in lactation. In lactating animals that secrete relatively large amounts of calcium in the milk, a number of adaptive changes in calcium metabolism occur. The efficiency of calcium absorption from the intestine increases twofold or more, and, in the rat, absorption may remain elevated for some time after weaning if dietary calcium intake is limited so that skeletal calcium stores are mobilized to meet the demands of milk secretion. Vitamin D and parathyroid hormone act in concert to mediate these changes. Vitamin D through its conversion to 1,25-(OH) 2 D 3 stimulates calcium absorption, and both parathyroid hormone and the vitamin D metabolite enhance skeletal calcium mobilization. Both hormones are also closely related to the serum calcium level. An elevated blood level of parathyroid hormone, in lactating cows, is stimulated and maintained by a low serum calcium level, but may be required to prevent an excessive drop in serum calcium in response to the calcium drain into the milk. High circulating levels of calcitonin are observed in lactating rats and sheep and in some lactating women. The urinary excretion of calcium is reduced during lactation in several species.

The ovariectomized, lactating rat as an experimental model for osteopenia: calcium metabolism and bone changes.

The ovariectomized, lactating rat (Sprague-Dawley) is proposed as an experimental model for the rapid development of osteopenia which may be used to test the effectiveness of bone-retentive drugs potentially useful in treating osteoporotic women. Rats were ovariectomized (OVX) on day 2 postpartum and were kept on a low-calcium diet (0.1%). Measurements of serum total calcium, ionic calcium, albumin and parathyroid hormone were conducted between days 4 and 21 of lactation. Serum total and ionic calcium and albumin were significantly lower and serum parathyroid hormone was significantly higher in all lactating rats at 16 days postpartum compared to nonlactating controls. Mean bone mass of the femurs of OVX lactating rats measured at day 21 was approximately 50% of that of non-lactating intact controls. The enhanced duodenal calcium absorption (in everted gut sacs) associated with lactation was not affected by OVX and neither was the average litter weight gain between 2 and 14 days of lactation. In conclusion, lactation coupled with a low-calcium diet resulted in marked osteopenia, depressed serum calcium (both total and ionic) and significantly elevated serum parathyroid hormone concentration. The rapid and extensive bone loss of this model makes it appropriate for the study of therapeutic agents designed to retain bone mass.

Diet and vitamin D: A review with an emphasis on human function

Abstract Vitamin D, either derived from the diet or skin production, has become an increasingly important molecule in human function because of the extended longevity of human populations in many nations. The actual vitamin D requirements of elderly individuals remain difficult to determine because environmental conditions, especially sunlight exposure, vary so greatly throughout the world. Data are limited on the potential prophylactic benefit of vitamin D or 1,25(OH)2D in metabolic bone diseases, such as osteoporosis, in the absence of vitamin D deficiency. Possible roles of vitamin D or its metabolites in the etiology or treatment of diabetes mellitus, cancer, and hypertension have been reviewed recently by Bikle. New information on the functional roles of 1,25(OH)2D on target tissues and the mechanism of action of this key molecule continue to emerge.

Regulation of Serum Calcitriol by Serum Ionized Calcium in Rats During Pregnancy and Lactation

Serum calcitriol concentrations in rats follow a biphasic pattern during reproduction, with elevated levels during late pregnancy, a decline after parturition, and a rise to even higher levels during peak lactation. We have previously shown that serum calcitriol in rats at peak lactation correlates significantly with, and appears to be regulated by, serum ionized Ca (Ca2+), with parathyroid hormone (PTH) serving a permissive role. We have extended this study by determining if serum calcitriol also correlates with serum Ca2+ during late pregnancy, when calcitriol levels are clearly elevated, and during early lactation, when only modest increases in serum calcitriol are observed. Analyses of data combined from nonmated, 21‐day pregnant (P), and 1‐day lactating rats (L) revealed a significant regression (p < 0.001) of calcitriol on Ca2+, but a nonsignificant regression (p = 0.34) of calcitriol on serum PTH. An even stronger correlation (p < 0.001) between calcitriol and Ca2+ was found for the combined data for 5−, 8−, and 14‐day L rats. The partial correlation coefficient for calcitriol versus Ca2+, with PTH as the independent variable, was highly significant (p < 0.01) for the data from both combined groups. However, the coefficient for calcitriol versus PTH, with Ca2+ as the independent variable, was not significant (p > 0.05). Fetal weights (uterus and contents) correlated significantly with both maternal calcitriol and Ca2+ concentrations (p < 0.01), but not with maternal PTH levels. Litter weights for 14‐day‐old pups likewise correlated significantly with maternal calcitriol and Ca2+ (p < 0.001). We conclude that hypocalcemia, induced by the demands for Ca for fetal calcification and milk production, appears to be a controlling factor in serum calcitriol elevation in late pregnancy and throughout lactation, whereas PTH may be important for calcitriol synthesis without playing a direct regulatory role.

Further studies on the separation and identification of two phosphatases with acid optima from rat bone

SummaryStudies were designed to examine the effects of isotonic sucrose and hypertonic KCl on the extraction and properties of phosphatases (acid optima) from long bones of suckling rats. Low-speed supernatants (S) of homogenates in sucrose were dialyzed against sucrose or deionized H2O. Free enzyme was recovered from both retentates by ultracentrifugation before (S1) or after sequential treatment with KCl and Triton X-100 (S2), protamine (S3), and a final dialysis (S4). Activity was measured withp-nitrophenylphosphate (p-NPP), β-glycerophosphate (β-GP), ATP, and casein. S was evaluated for latent enzyme with hypertonic KCl and classic lysing methods. Fractionation of activity in final extracts was accomplished with CM-52 cellulose or electrophoresis. Results showed that: (a) sucrose did not alter the types or properties of enzymes extracted, but did decrease yield of activities; (b) activity in S was increased approximately 20% by lysing methods, 80% by hypertonic KCl, and was unstable unless salt and detergent were present; and (c) chromatography or electrophoresis of S4 resolved only two enzymes: a tartrate-sensitive phosphomonoesterase (E1) responsible for 15% of the total activity in S, and a tartrate-insensitive enzyme (E2) which accounted for 85% of the activity, was unstable in isotonic medium, had high affinity for ATP andp-NPP, and had low affinity for casein and β-GP. It is concluded that sucrose is not necessary for the isolation of total bone acid phosphatase activity, that hypertonic KCl does not negatively affect the properties of the enzymes isolated, and that E1 and E2 show different latencies.

Chromatographic separation of two acid phosphatases from rat bone.

SummaryExtracts of tibiae of suckling rats were prepared with 0.3 M KCl containing 0.1% Triton X-100 and were chromatographed with CM-52 cellulose. Most of the acid phosphatase activity determined with p-nitrophenylphosphate (p-NPP) was bound to the cellulose and could be eluted with a sodium acetate buffer gradient in 2 distinct peaks. The major peak, E2, was bound strongly to the cellulose and showed high activity with p-NPP and inorganic pyrophosphate (P-Pi), but only slight activity withβ-glycerophosphate (β-GP) and was unaffected by tartrate. The minor peak, E1, was weakly bound to the adsorbent, showed equal activity with p-NPP andβ-GP, but negligible activity with P-Pi and was completely inhibited by tartrate. These results support earlier evidence suggesting that bone contains at least 2 different acid phosphatases and that the more abundant enzyme may function as a pyrophosphatase.