Phosphohydrolase Activities in Developing and Mature Dental Tissues Edda Warth* and Reinhard Jeck** * Department of Biochemistry, The University of Iowa, Iowa City, Iowa 52242 ** Klinikum der Johann Wolfgang Goethe-Universität, Gustav Embden-Zentrum der Biologischen Chemie, Abteilung für Enzymologie, Theodor-Stern-Kai 7, D-6000 Frankfurt am Main 70, Bundesrepublik Deutschland Z. Naturforsch. 45c, 280-292 (1990); received Juni 16/December 4, 1989 Phosphohydrolases, Dental Tissues, Tooth Development, Mineralization In the course of the odontogenesis of bovine incisors several clearly distinguishable phos­ phohydrolase activities are observed in the pulp and in dental hard tissues. Using various sub­ strates and inhibitors, unspecific alkaline phosphatase, two isoenzymes of acid phosphatase, Ca2+-activated ATPase and inorganic pyrophosphatase are characterized. The enzymatic ac­ tivity of alkaline phosphatase in pulp and hard tissues is significantly high at the beginning of dentine and enamel mineralization. The specific activity of this enzyme decreases quite fast with the beginning of root formation, then more slowly, until it reaches a constant final value. Histochemical studies show that during mineralization the maximum of alkaline phosphatase activity is in the subodontoblasts. Lower enzyme concentrations are found in the stratum intermedium and in the outer enamel epithelium during that process. The specific activities of ATPase, acid phosphatases and pyrophosphatase show little tem­ poral variation during tooth development, but they also appear in a characteristic spatial pattern in the dental tissues. Introduction donts and exhibit dental matrix proteins of similar The mineralization which causes the formation size and amino acid composition [26-29], Soft and of biological hard tissues is not a simple precipita­ hard tissues of every developmental stage from tion from an oversaturated solution. It rather pro­ toothgerm to completely mineralized teeth were ceeds on an extracellular organic network which studied enzymatically and histochemically. In this consists of phospho- and glycoproteins of unusual paper we report about the occurrence, localization composition [1-7]. The role of these matrix pro­ and properties of several phosphate releasing teins during hard tissue formation, especially du­ enzyme activities during odontogenesis. ring mineralization, is still obscure. Materials and Methods Two functional sets of enzymes are of primor­ dial importance for odontogenesis. The first one is Toothgerms and partially or completely mineral­ correlated with the formation and decomposition ized incisors were removed from the jaws of 18 to of the protein matrix, such as proteinkinases, gly- 36 month old cattle. Gum tissue was removed cosyltransferases and proteases. The second group carefully and the pulp with adhering odontoblasts affects the calcium and phosphate metabolism and was detached from the hard tissue. The separated is involved in the formation of hydroxylapatite, material was stored at -78 C unless it was used the main mineral of dental hard tissues. immediately. Phosphatases [1-18], ATPases [19-23] and pyrophosphatase [24, 25] are found in different Sample preparation of pulpal tissue soft and hard tissues of growing and mature teeth Enzyme activities in the pulp were measured in and first results show an increase of phosphatase freshly prepared crude extracts. For that purpose production during certain stages of odontogenesis. pulp tissue was weighed and homogenized in 3 to We have now studied in detail the temporal 20 volumes of distilled water with a Dounce homo- changes of phosphohydrolases in the course of genizer or an Ultraturrax at 0 °C. After 30 min of tooth development of one single mammal. Incisors incubation at 25 C collagen fibers and large of calves and young cattle were used, since the de­ organelles were removed by centrifugation at velopment of bovine incisors resembles the human 15,000 x g at 0 °C. The supernatant was stored at odontogenesis: Both species represent diphyo- 0 C during experiments. Reprint requests to Dr. R. Jeck. Specific activities are based on protein concen­ trations determined by the method of Bradford Verlag der Zeitschrift für Naturforschune. D-7400 Tübingen 0341-0382/90/0300-0280 $01.30/0 [30]. E. Warth and R. Jeck • Phosphohydrolases in Dental Tissues 281 Sample preparation of mineralized tissue Granström [9], A calibration curve was obtained Deep frozen dental hard tissues were pulverized with 0-0.1 m M KH2P04. For the blank values by means of a rotary beater mill (Pulverisette 14, 20% trichloroacetic acid was added prior to incu­ Fritsch GmbH, Idar-Oberstein) while cooling with bation with enzyme. dry ice. The cylindrical sieves and the rotor were cooled beforehand with dry ice to minimize the in­ Coupled assays for phosphatase activity in activation of enzymes in the dental tissue. By the mineralized dental tissues with different substrates use of sieves with 80 |im, 200 pm, 500 (im, 1 mm The assay mix was stirred at room temperature and 4 mm nominal pore width material with dif­ in a cuvette: 5 mg pulverized hard tissue (particle ferent grain size distribution was obtained. The size = 80 mesh) was suspended in 2.0 ml of 0.5 m powder was dried over silica gel in a desiccator at diethanolamine/HCl pH 9.0, 0.5 mM MgCl2 and 4 °C and stored at -7 8 °C. 3 mM NAD+; 0.1 ml enzyme solution was added For enzyme assays samples of powdered hard first, then 100 [il 0.1 m substrate solution in buffer tissue were homogeneously suspended in test buf­ pH 9.0. The reaction was followed at 366 nm in a fer using an electrically driven stirring blade in the photometer “Eppendorf’. We used: fluorometer or a magnetic stirrer (Hellma cuv- A) glucose-6-phosphate, 1-a-phosphoglucose and O-stir model 333) in the case of absorbance meas­ 1-ß-phosphoglucose with glucose dehydro­ urements. The enzymatic test was started by addi­ genase from Bacillus megaterium (90 U/ml), tion of substrate containing solution. For blank B) L-a-glycerol-3-phosphate with glycerol de­ values pulverized dental hard tissue was heated to hydrogenase from Enterobacter aerogenes 140 °C for 10 min. (250 U/ml), C) ethylphosphate with alcohol dehydrogenase from yeast (400 U/ml) or Assays for alkaline phosphatase D) rc-butylphosphate with alcohol dehydrogenase from horse liver (45 U/ml). /j-Nitrophenylphosphate hydrolysis was meas­ ured in 0.5 m diethanolamine/HCl buffer pH 9.0, Assays for inorganic pyrophosphatase containing 0.5 mM MgCl2, following the in­ crease in absorbance at 405 nm at 25 °C (s The velocity of pyrophosphate hydrolysis was 18,800 M_ 1 - cm“1) [31, 32], In the blanks heat dena­ determined by measuring the amount of inorganic tured protein was used or levamisole was added to phosphate released [9] or by spectrofluorometry a final concentration of 3 mM. For the concentra­ using a modified assay of Cooperman et al. [35]: tion of different effectors used in the study of alka­ 3 m M inorganic pyrophosphate pH 8.0 in presence line phosphatase see Table II. of 2 m M Mg2+ ions and 1 (iM 4-methylumbelli­ Tests with 4-methylumbelliferylphosphate as ferone as pH-dependent fluorescence indicator. A substrate were carried out on a Perkin Elmer flu­ calibration curve was obtained from a gradient orescence spectrophotometer MPF 4 or a Beck­ generated from the magnesium pyrophosphate so­ man fluorometer 1076. Excitation and emission lution described above and a sample of this solu­ wavelengths were 362 nm and 445 nm, respectively tion which had been completely hydrolyzed. [33, 34], To estimate the activity per volume unit Hydrolysis was performed by the aid of inorganic and the specific activity standard curves of the pyrophosphatase until no further decrease in pH product 4-methylumbelliferone were generated in and fluorescence was observable, thereafter the so­ the concentration range from 5 nM to 25 |im. In ab­ lution was heated to 100 °C for 5 min in order to sorbance measurements of 4-methylumbelliferyl- destroy any enzyme activity. The calibration curve phosphate hydrolysis a molar extinction coeffi­ correlating pH- and fluorescence-decrease with cient of 17,300 at 360 nm was used. pyrophosphate hydrolysis is almost linear down­ In assays of alkaline phosphatase activity with ward to pH 7.6, a value indicating the release of pyrophosphate or other non-chromogenic or non- 0.6 m M of inorganic phosphate. Using a concen­ fluorogenic substrates the amount of released tration of 100 |j.M 4-methylumbelliferone the sys­ orr/zo-phosphate was determined according to tem could be used for spectrophotometric meas- 282 E. Warth and R. Jeck • Phosphohydrolases in Dental Tissues urements of pyrophosphatase activity at 362 or conditions. Thus the results of different measure­ 366 nm. ments are comparable. Characterization and dif­ The concentration of phosphatase inhibitors ferentiation of single enzyme/isoenzyme activities used to distinguish inorganic pyrophosphatase was attempted by the addition of various sub­ from other phosphohydrolases are included in stances, known to inhibit individual enzymes. A Table VIII. comparable procedure for the quantitation of phosphohydrolases in dental hard tissues is given Assays for acid phosphatase in the following paragraph. The activity was measured with /?-nitrophenyl- 1.1 Alkaline phosphatase phosphate as substrate in 0.1 m acetate buffer pH 4.2 [36]. When non-chromogenic substrates 1.1.1 Calibration experiments were used, the activity was determined on the basis Alkaline phosphatase