With the aid of electrophoretic techniques it was shown that lactate dehydrogenase (LDH, EC. 1.1.1.27) of animal tissues and body fluids consisted of at least five isoenzymes. The properties of LDH isoenzymes of rat submandibular gland were reported [1]. In the present report, changes of LDH isoenzyme activities of submandibular gland were investigated during development in the rat.
Reduced nicotinamide-adenine dinucleotide (reduced NAD) was obtained from Boehringer Mannheim. Bovine albumin was obtained from Nutritional Biochemicals Corporation. Other materials used were also available commercially. Rats of Donryu strain were used. The animals were sacrificed by sharp blow on the head, and their submandibular glands were excised and placed in ice-cold 0.25 M sucrose solution. The tisses were homogenized with tephron homogenizer in 4 volumes of ice-cold 0.05 M barbital buffer (pH 8.6), and the supernatant fraction was obtained after centrifugation at 100, 000 × g for 1 hour. Electrophoretic separation of the isoenzymes was made with starch in a horizontal position. The supernatant fluid obtained from the homogenate was inserted into a slit in the starch with a supporting medium of starch granules. The separation was made in starch block of 30 × 1 × 1 cm. All of the runs were made for 21 hours at a field strength of 4 volts per 1 cm at 5°C. After 21 hours of electrophoresis, the starch was cut into 0.5 cm strips at right angles to the direction. These strips were extracted with 2.0 ml of 0.1 M phosphate buffer (pH 7.4) during 3 hours with occasional stirring, and the clear supernatant fluids were obtained by centrifugation at 800 x g for 10 minutes. The residues were extracted once more with 2.0 ml of same buffer during 1 hour and the supernatant fluids were added to the first extracts. The LDH activity of the supernatant fluid combined in each tube was determined. The enzyme was incubated with 0.33 μmole of reduced NAD, 2.3 μmoles of sodium pyruvate in phosphate buffer (680 pmoles, pH 7.4) in the final volume of 2.0 ml for 4 minutes at 25°C. The reduced NAD should be added last of all. Subsequently, the enzyme activity was determined by measuring the rate of optical density decrease at a wave length of 340 mμresulting from the oxidation of reduced NAD. One unit of LDH activity was defined as the amount of the enzyme that decreases 1.0 μmoles of reduced NAD per minute at 25°C. LDH isoenzymes were designated according to PLAGEMANN [2].
Total LDH activity of submandibular gland (expressed on a tissue weight basis) increased steadily throughout the early postnatal period, and thereafter it remained relatively constant throughout the 150-day experimental period and then decreased significantly by the postnatal day 200 (Table 1).
The data in Table 1 reveal that LDH isoenzyme patterns of submandibular gland were changed during development in the rat. Especially, LDH5 activity was decreased for the first 10 days after birth, then increased. However, LDH4 activity was increased for the 10 days after birth, then decreased. No activity of LDH1 and LDH2 was found immediately before birth, and no activity of LDH1, LDH2, LDH3 was also found in 120-200-day-old animals.
To clarify the physiological significance of developmental changes in the pattern of LDH isoenzymes of rat submandibular gland, further investigations are undergoing in our laboratory.
