Ilkka K. Paunio

Exploitation of Blue Dextran as a dextranase substrate

Abstract A spectrophotometric method for the determination of dextranase activity with the use of Blue Dextran is described. Due to its rapidity the new method is especially recommended for the determination of the dextranase activity in column chromatography.

Evidence on certain common properties of alkaline phosphatase-like enzymes derived from human foetal mineralizing tissues.

The substrate specificity of alkaline phosphatases derived from different types of tissue is still open to investigation. In this laboratory investigations have been carried out on the human foetal alkaline phosphatase activity in order to elucidate, above all, the substrate specificity of the enzymes obtained from cartilaginous epiphysis, parietal bone and liver. A preliminary report on the results obtained has been given earlier [ 1 ] . Various chromatographic experiments revealed that parietal bone contains an alkaline phosphatase which is not very active or is present only in small amounts in the other tissues. In spite of the location of the different enzymes, the alkaline phosphatases found were considered to be similar in many respects, displaying perhaps only one main type of enzyme. This was evidenced in this paper by 1) essentially similar kinetic behaviour of most of the enzymes identified under the test conditions employed, and 2) similar activating effect of various divalent metal cations tested.

Effect of Sulfate Ions on the Solubility of Human Enamel and Dentin

The effect of ammonium sulfate on the rate of dissolution of human enamel, dentin, and synthetic hydroxylapatite at different pH values was studied. Ammonium sulfate enhanced the dissolution of all the tested materials. The sulfate ion was responsible for the increase in the solubility of powdered dentin at all tested pH values between 4.2 and 7.4.

Properties of alkaline phosphatase-like enzymes derived from mineralizing tissues of human foetuses

The occurrence and properties of enzymes possessing a lkallne phosphatGse-like activity was studied in human foetal epiphyseal cartilage from long mineralizing bones and mineralizing parietal bones. The alkaline phosphatase activity of human foetal liver was also studied in order to have a non-mineralizing reference tissue. Sodium a-naphthyl phosphate, p-nitrophenyl phosphate and several sugar phosphates were used as model substrates. When phosphatase activity was measured per mg weight of tissue, it could be noted that enzymes derived from parietal bones had hydrolyzed the substrates at a rate of 20-40 times that of epiphyseal cartilage and even of liver. These results did lead us to purify the alkaline phosphatase-llke enzymes from the three tissues given. The purification procedure involved a precipitation of the contaminating materials at pH 5.0, gel filtration and DEAE-cellulose chromatography. Two main alkaline phosphatase-like enzymes were separated by this procedure, a-Naphthyl phosphate and p-nitrophenyl phosphate were hydrolyzed most rapidly at pH 9.2 in glyclne-NaOH buffer by all enzyme preparations obtained. The activating effect of various divalent metal cations in the hydrolysis of these substrates increased in the following order: Mn, Ca, Sr, Ba, Mg. The results were same with all enzyme preparations obtained. Out of 13 different sugar phosphates stud ied, fructose-1, 6-diphosphate and a-D-ga lactose1-phosphate were most rapidly hydrolyzed by all enzyme preparations.

Kinetics of alkaline phosphatase-like enzymes derived from foetal mineralizing bone tissue

The purpose of this investigation was to elucidate the substrate specificity of alkaline phosphatase-like enzymes derived from human foetal mineralizing tissue. This was accomplished b y determining the dissociation constant, K~, (substrate constant) of the enzyme-substrate complex (ES) into enzyme (E) and substrate (S). The enzymes were obtained from cartilaginous epiphysis, parietal bones and liver. The alkaline phosphatase-like enzymes studied were obtained by part ial purification from the tissues mentioned, employing mainly DEAE cellulose chromatography. Three main enzymes were obtained from the mineralizing tissues and two from liver (3). The substrates included p-ni t rophenyl phosphate, a-naphthyl phosphate, several sugar phosphates and some nueleoside phosphates. The calculations were based on Hanes-equat ion and its extension (1) and were performed by giving each point a weight proport ional to vd/[S] (5). The programme applies the method of least squares and calculates a.o. Vm~x~ Ks, Kt (substrate inhibition constant (4) and their s tandard errors. The results obtained ra ther closely resembled those earlier obtained by various graphical methods, al though it was also clearly shown tha t the subject ivi ty of the last ment ioned method has led to too extensive generalizations in the case of some substrates (3, 2). One typical result is shown in Fig. 1. With each of the enzyme preparations the dissociation constant of the ES complex was of the same order, regardless of the origin of the enzyme preparation. With some phosphate esters, however, the values of K , differed from each others to a greater extent .

Fractionation of Enzymes from Human Dental Plaque Hydrolyzing α-Naphthyl Phosphate

Enzymes of human dental plaque that are capable of hydrolyzing α-naphthyl phosphate were fractioned by means of Sephadex G-100 gel filtration and DEAE-ion exchange chromatography. The results demonstrated the existence of several enzymes in human dental plaque that possessed this hydrolyzing activity.