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Isoenzymes of lactate dehydrogenase in the enamel organ of the rat incisor
Archs
oral
Bid.
Vol.
15, pp.
1l-16,
1970.
Pergamon Press. Printed inGreat Britain.
ISOENZYMES OF LACTATE DEHYDROGENASE IN THE ENAMEL ORGAN OF THE RAT INCISOR H. FREDEN From the Department of Histology, University of Gateborg, Sweden Summary-The enamel organs from adult rats were dissected out and divided into four developmental zones. The samples were homogenized and analysed by acrylamide gel electrophoresis for lactate dehydrogenase activity. Five lactate dehydrogenase isoenzymes from enamel organ were demonstrated. During the formation of enamel there was a decrease in the relative amounts of LDH-1 and LDH-2 and in increase of LDH-4 and LDHJ. It is likely this change reflects a decrease in oxygen tension of the enamel organ. INVESTIGATIONS with
the light (MARSLAND, 1952) and electron (REITH, 1963) microscope have shown that ameloblasts in developing teeth from the rat change their intracellular morphology during the formation of tooth enamel. The studies of DEAKINS(1942) have shown that the percentages of water and organic matter in tooth enamel decrease after the enamel has achieved its complete thickness; that is when the synthesis of the organic enamel matrix has ceased. A difference in distribution of 45Ca and tetracycline in growing enamel and a subsequent resorption of tetracycline during maturation has been shown by HAMMARSTRGM (1967). Numerous histochemical studies of ameloblasts have also demonstrated changes in various enzymes during development (for review, see REITHand BUTCHER,1967). These studies indicate that during the life cycle of ameloblasts there are at least four morphologically and functionally distinct phases. A number of studies have been reported concerning changes in the isoenzyme pattern of lactate dehydrogenase during the development of different tissues (for review, see LATNER and SKILLEN, 1968). Lactate dehydrogenase exists as five isoenzymes of different sub-unit composition. They are each designated LDH-1, LDH-2, LDH-3, LDH-4 or LDH-5 in accordance with their electrophoretic mobility at basic pH. The five isoenzymes behave differently towards increasing pyruvate concentration and a possible relationship between substrate inhibition, metabolic role and sub-unit composition has been indicated by CAHN et al. (1963) and others. In anaerobically metabolizing tissues, LDH-5 is more prominent, whereas LDH-1 is more predominant in aerobically metabolizing tissues. ROBERTS and STRACHAN (1967) showed that the lactate dehydrogenase isoenzymes in tissue extracts of developing molars of mice changed with increasing age. After 10 days the two electrophoretically most fast-moving isoenzymes (LDH-1 and LDH-2) could not be demonstrated. 11
H.
12
FRED~N
The purpose of this investigation was to study the isoenzyme pattern of lactate dehydrogenase during the various developmental stages of the enamel organ of the continuously growing incisors of the adult rat. Twenty female white rats (Sprague-Dawley strain) weighing about 150 g each were killed by decapitation under ether anaesthesia. The two maxillary incisors were carefully dissected out with the whole enamel organ adhering to the enamel surface. Under a stereo microscope the overlying connective tissue was removed; the enamel organ was then divided into four segments, each representing one stage in enamel formation. Segment A denotes the functional stage of proliferation and differentiation of the inner dental epithelium to ameloblasts characterized by numerous mitoses. Segment B denotes the stage of matrix formation which includes the elaboration of the organic component of enamel and its almost immediate calcification. The ameloblasts are tall and closely packed with their nuclei at the basal end of the cells. At the distal ends are Tomes processes. Segment C denotes the functional stage of maturation when the enamel has reached its full thickness and becomes progressively harder. The ameloblasts are columnar, not as tall as the cells associated with matrix formation. The cells are not tightly packed and there are intercellular spaces between them. Tomes processes are no longer present. In the distal part of this segment the ameloblasts contain a yellow pigment which is characteristic for the rat incisor. Segment D denotes the functional stage of pigmentation of the enamel when calcification is finished. The ameloblasts are columnar and become shorter in the distal parts of this segment. A schematic drawing of the localization of the different segments is presented in Fig. 1. The lengths of the segments were determined from previously made histological slides, from rats of the same age and weight. The variation of the sizes of the samples was tested with protein estimation according to LOWRY(1951) and was too small to be of
FIG. 1.
The four segments of the enamel organ which were dissected out and analysed separately for LDH-isoenzymes. From the apex to the gingival margin the segments are called A, B, C, and D respectively.
LDH LSOENZYMES IN DEVELOPING
ENAMEL ORGAN
13
any significance. Material from the two incisors from one animal was pooled. The samples, each representing one developmental stage, were briefly homogenized in 0 -5 ml ice-cold 0.1 M Tris-buffer, pH 7 ‘4, in a motorized glass-glass homogenizer 20 times at 3000 rev/min. A volume of O-1 ml of the homogenate was applied on an acrylamide gel for electrophoresis according to ORNSTEIN(1964) and DAVIES(1965). No sample gel was used. The pH of the spacer and the running gel were 6.7 and 8 *9 respectively. The pH of the electrode buffer was 8 *3. The concentration of acrylamide used was 2.4 per cent in the spacer gel and 5 per cent in the running gel. The electrophoresis was carried out at + 10°C with a voltage of 150 V and a current of 1mA and was stopped after 2 hr when the fluorescein, which was used as a marker, had reached 5 cm from the beginning of the running gel. The gels were taken out of the glass tubes and stained histochemically for lactate dehydrogenase activity by a nitroblue-tetrazolium reaction (DEWEYand CONKLIN,1960). The staining reacton was completed after 10 min at 37°C in the dark. After fixation in 7 *5 per cent acetic acid in 25 per cent ethyl alcohol, the gels were photographed and the photographic negatives were scanned in a Joyce, Loebl densitometer. The area under each peak representing one isoenzyme was calculated and the per cent distribution of the isoenzymes in each gel was calculated. The mean values for the five isoenzymes were calculated in each of the four stages of development and the significance of the differences was tested by Student’s r-test.
lsoenzyme
lsoenzyme
pottern
pattern
segment A
segment D
FIG.2. Typical examples of LDH-isoenzymes patterns after densitometric scanning from segments A and D.
H. FREDBN
14
TABLE1. DIS-~RIBUTION OF LDH-ISOENZYMES IN THEFOURDEVELOPMENTAL ZONES 0F THE ENAMELORGAN (n = 20) Zone per cent distribution
A B C D
1 10*9&1.1 7*5*0.8 5*7*0*6 2.9kO.6
of LDH-isoenzymes
3 16*6$1.0 17.5*1.0 14.7+0.7 15.510-6
2 12.710-8 8.9kl.O 9*1+0* 1 5.9hO.6
4 26.411.9 27.81t1.5 28*9+0*8 35.951.1
5 33.5kl.O 38.4*1.0 41.7k1.7 39.950.9
Significance of LDH isoenzyme changes between zone A and zone D A:l--D:l : P
m
LDH I
El LDH
qLDH2
•nllLDH
3
LbH 4
•$j
5
i
A
B
C
D
FIG. 3. The per cent distribution of LDH-isoenzymes in the different segments. If LDH-1 and LDH-2 and LDH-4 and LDH-5 are pooled, the developmental pattern is even more striking, as can be seen from the thin lines which represent the sum of the figures for LDH-1 and LDH-2 and LDH4 and LDH-5 respectively.
LDH
ISOENZYMES
IN DEVELOPING
ENAMEL
ORGAN
15
In every developmental stage five isoenzymes of lactate dehydrogenase were clearly demonstrable. The five isoenzymes were numbered according to general practice, LDH-1, LDH-2, LDH-3, LDH4, and LDH-5 from anode to cathode. The isoenzyme pattern of lactate dehydrogenase in the four segments of the enamel organ is characterized by a decrease in relative amounts of the two fast moving isoenzymes (LDH-1 and LDH-2) during the maturation of the enamel (Fig. 2). The mean values for the per cent distribution of the five isoenzymes in the four segments are presented in Table 1. The difference between segment A and segment D is highly significant for LDH-1, LDH-2, LDH-4 and LDH-5. If the combined values for each of the two pairs (LDH-1 and LDH-2) and (LDH4 and LDH-5) are calculated, this change in the isoenzymatic pattern is even more striking (Fig. 3). The present findings demonstrate a decrease in the relative amounts of the two anodal isoenzymes and an increase of the two cathodal isoenzymes during the development of the enamel. CAHN et al. (1962) have pointed out that tissues containing a preponderance of LDH-1 and LDH-2 allow pyruvate to accumulate and activate the citric acid cycle, whereas tissues containing a preponderance of LDH4 and LDH-5 do not allow pyruvate to accumulate but respire anaerobically. This explains why LDH-5 is more predominant in anaerobically metabolizing tissue whereas LDH-1 is more prominent in aerobically metabolizing tissues. A developmental pattern of LDH isoenzymes similar to the one demonstrated in the enamel organ in the present study has been reported by ROBERTS and STRACHAN (1967) in whole molar tooth buds during the early postnatal period in mice. These authors suggest that the isoenzyme pattern of developing teeth reflects a decreasing oxygen tension in these cells during ageing. It seems likely that the isoenzyme pattern of the enamel organ reported by the present author reflects a decreasing activity of the citric acid cycle and an increasing activity of anaerobically glycolysis during the formation of enamel. Quantitative assays of the enzymes of the citric acid cycle and the glycolytic pathway which have been performed in our laboratory support this hypothesis. Acknowledgements-This work has been supported by grants from the Swedish Medical Research Council (B69-24X-2385-03A) and Gijteborgs TandlBkarsHllskap, Gothenburg, Sweden. R&urn&--Les organes de l’email de rats ad&es sont preleves par dissection et divises en quatre zones de developpement. Ces Cchantillons sont homogeneists et analyses par Clectrophorese sur gel d’acrylamyde pour l’activite en lactate deshydrogenase. Cinq isoenzymes de lactate deshydrogenase ont et6 isolts dans l’organe de l’tmail. Pendant la formation de l’bmail, on observe une diminution dans les proportions relatives de LDH-1 et LDH-2 et une augmentation de LDH-4 et LDH-5. Ce changement semble traduire une diminution de la tension en oxygene de l’organe de l’tmail. Zusammenfassung-Die SChmelzorgane erwachsener Ratten wurden herausprlpariert und in vier Entwicklungsz onen aufgeteilt. Die Gewebsproben wurden homogenisiert und mit Hilfe der Acrylamid-Gel-Elektrophorese auf Laktatdehydrogenase Aktivitat untersucht. Ftinf Isoenzyme der Laktatdehydrogenase konnten im Schmelzorgan nachgewiesen werden. W1 hrend der Schmelzbildung beobachtet man eine Abnahme
16
H. FREDBN im relativen Gehalt von LDH-1 und LDH-2 und eine Zunahme von LDH4 und LDHJ. Es scheint so, daB diese Verlnderungen ein Ausdruck fiir die Abnahme der Sauerstoffspannung im Schmelzorgan sind. REFERENCES
CAHN, R. D., KAPLAN, N. D., LEVINE,L. and ZWILLING,E. 1962. Nature and development of lactic dehydrogenases. Science, Lond. 136,962-969. DAVIS, B. J. 1964. Disc electrophoresis II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121, 404-427. DEAKINS,M. 1942. Changes in the ash, water and organic content of pig enamel during calcification. J. dent. Res. 21, 429-435. DEWEY,M. M. and CONCLIN,J. L. 1960. Starch gel electrophoresis of lactic dehydrogenase from rat kidney. Proc. Sot. exp. Biol. 105,492-494. HAMMARSTRGM, L. 1967. Different localization of tetracycline and simultaneously injected radiocalcium in developing enamel. Calc. Tiss. Res. 1, 229-242. LATNER,A. L. and SKILLEN,A. W. 1968. Isoenzymes in Biology andMedicine. Academic Press, London. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L. and RANDALL,R. S. 1951. Protein measurement with the folin phenol reagent. Sot. Biol. Chem. 193,265-275. MARSLAND,E. A. 1952. A histological investigation of amelogenesis in rats. 11. Maturation. Br. dent. J. 92, 109119. ORNSTEIN,L. 1964. Disc electrophoresis. I. Background and theory. Ann NY. Acad. Sci. 121,321-349. REITH, E. J. 1963. The ultrastructure of ameloblasts during early stages of maturation of enamel. J. Cell Biol. 18, 691-696. REITH, E. J. and BUTCHER,E. 0. 1967. Microanatomy and histochemistry of amelogenesis. In: Structural and Chemical Organization of Teeth (edited by MILES, A. E.W.) Chapter I, pp. 371394, Academic Press, New York. REITH, E. J. and Corn, U. F. 1962. Autoradiographic studies on calcification of enamel. Archs oral Biol. 7, 365-372. ROBERTS,R. W. and STRACHAN,D. S. 1967. Quantitation of lactate dehydrogenase of developing molar teeth of the mouse. J. dent. Res. 46, 522-526.
oral
Bid.
Vol.
15, pp.
1l-16,
1970.
Pergamon Press. Printed inGreat Britain.
ISOENZYMES OF LACTATE DEHYDROGENASE IN THE ENAMEL ORGAN OF THE RAT INCISOR H. FREDEN From the Department of Histology, University of Gateborg, Sweden Summary-The enamel organs from adult rats were dissected out and divided into four developmental zones. The samples were homogenized and analysed by acrylamide gel electrophoresis for lactate dehydrogenase activity. Five lactate dehydrogenase isoenzymes from enamel organ were demonstrated. During the formation of enamel there was a decrease in the relative amounts of LDH-1 and LDH-2 and in increase of LDH-4 and LDHJ. It is likely this change reflects a decrease in oxygen tension of the enamel organ. INVESTIGATIONS with
the light (MARSLAND, 1952) and electron (REITH, 1963) microscope have shown that ameloblasts in developing teeth from the rat change their intracellular morphology during the formation of tooth enamel. The studies of DEAKINS(1942) have shown that the percentages of water and organic matter in tooth enamel decrease after the enamel has achieved its complete thickness; that is when the synthesis of the organic enamel matrix has ceased. A difference in distribution of 45Ca and tetracycline in growing enamel and a subsequent resorption of tetracycline during maturation has been shown by HAMMARSTRGM (1967). Numerous histochemical studies of ameloblasts have also demonstrated changes in various enzymes during development (for review, see REITHand BUTCHER,1967). These studies indicate that during the life cycle of ameloblasts there are at least four morphologically and functionally distinct phases. A number of studies have been reported concerning changes in the isoenzyme pattern of lactate dehydrogenase during the development of different tissues (for review, see LATNER and SKILLEN, 1968). Lactate dehydrogenase exists as five isoenzymes of different sub-unit composition. They are each designated LDH-1, LDH-2, LDH-3, LDH-4 or LDH-5 in accordance with their electrophoretic mobility at basic pH. The five isoenzymes behave differently towards increasing pyruvate concentration and a possible relationship between substrate inhibition, metabolic role and sub-unit composition has been indicated by CAHN et al. (1963) and others. In anaerobically metabolizing tissues, LDH-5 is more prominent, whereas LDH-1 is more predominant in aerobically metabolizing tissues. ROBERTS and STRACHAN (1967) showed that the lactate dehydrogenase isoenzymes in tissue extracts of developing molars of mice changed with increasing age. After 10 days the two electrophoretically most fast-moving isoenzymes (LDH-1 and LDH-2) could not be demonstrated. 11
H.
12
FRED~N
The purpose of this investigation was to study the isoenzyme pattern of lactate dehydrogenase during the various developmental stages of the enamel organ of the continuously growing incisors of the adult rat. Twenty female white rats (Sprague-Dawley strain) weighing about 150 g each were killed by decapitation under ether anaesthesia. The two maxillary incisors were carefully dissected out with the whole enamel organ adhering to the enamel surface. Under a stereo microscope the overlying connective tissue was removed; the enamel organ was then divided into four segments, each representing one stage in enamel formation. Segment A denotes the functional stage of proliferation and differentiation of the inner dental epithelium to ameloblasts characterized by numerous mitoses. Segment B denotes the stage of matrix formation which includes the elaboration of the organic component of enamel and its almost immediate calcification. The ameloblasts are tall and closely packed with their nuclei at the basal end of the cells. At the distal ends are Tomes processes. Segment C denotes the functional stage of maturation when the enamel has reached its full thickness and becomes progressively harder. The ameloblasts are columnar, not as tall as the cells associated with matrix formation. The cells are not tightly packed and there are intercellular spaces between them. Tomes processes are no longer present. In the distal part of this segment the ameloblasts contain a yellow pigment which is characteristic for the rat incisor. Segment D denotes the functional stage of pigmentation of the enamel when calcification is finished. The ameloblasts are columnar and become shorter in the distal parts of this segment. A schematic drawing of the localization of the different segments is presented in Fig. 1. The lengths of the segments were determined from previously made histological slides, from rats of the same age and weight. The variation of the sizes of the samples was tested with protein estimation according to LOWRY(1951) and was too small to be of
FIG. 1.
The four segments of the enamel organ which were dissected out and analysed separately for LDH-isoenzymes. From the apex to the gingival margin the segments are called A, B, C, and D respectively.
LDH LSOENZYMES IN DEVELOPING
ENAMEL ORGAN
13
any significance. Material from the two incisors from one animal was pooled. The samples, each representing one developmental stage, were briefly homogenized in 0 -5 ml ice-cold 0.1 M Tris-buffer, pH 7 ‘4, in a motorized glass-glass homogenizer 20 times at 3000 rev/min. A volume of O-1 ml of the homogenate was applied on an acrylamide gel for electrophoresis according to ORNSTEIN(1964) and DAVIES(1965). No sample gel was used. The pH of the spacer and the running gel were 6.7 and 8 *9 respectively. The pH of the electrode buffer was 8 *3. The concentration of acrylamide used was 2.4 per cent in the spacer gel and 5 per cent in the running gel. The electrophoresis was carried out at + 10°C with a voltage of 150 V and a current of 1mA and was stopped after 2 hr when the fluorescein, which was used as a marker, had reached 5 cm from the beginning of the running gel. The gels were taken out of the glass tubes and stained histochemically for lactate dehydrogenase activity by a nitroblue-tetrazolium reaction (DEWEYand CONKLIN,1960). The staining reacton was completed after 10 min at 37°C in the dark. After fixation in 7 *5 per cent acetic acid in 25 per cent ethyl alcohol, the gels were photographed and the photographic negatives were scanned in a Joyce, Loebl densitometer. The area under each peak representing one isoenzyme was calculated and the per cent distribution of the isoenzymes in each gel was calculated. The mean values for the five isoenzymes were calculated in each of the four stages of development and the significance of the differences was tested by Student’s r-test.
lsoenzyme
lsoenzyme
pottern
pattern
segment A
segment D
FIG.2. Typical examples of LDH-isoenzymes patterns after densitometric scanning from segments A and D.
H. FREDBN
14
TABLE1. DIS-~RIBUTION OF LDH-ISOENZYMES IN THEFOURDEVELOPMENTAL ZONES 0F THE ENAMELORGAN (n = 20) Zone per cent distribution
A B C D
1 10*9&1.1 7*5*0.8 5*7*0*6 2.9kO.6
of LDH-isoenzymes
3 16*6$1.0 17.5*1.0 14.7+0.7 15.510-6
2 12.710-8 8.9kl.O 9*1+0* 1 5.9hO.6
4 26.411.9 27.81t1.5 28*9+0*8 35.951.1
5 33.5kl.O 38.4*1.0 41.7k1.7 39.950.9
Significance of LDH isoenzyme changes between zone A and zone D A:l--D:l : P
m
LDH I
El LDH
qLDH2
•nllLDH
3
LbH 4
•$j
5
i
A
B
C
D
FIG. 3. The per cent distribution of LDH-isoenzymes in the different segments. If LDH-1 and LDH-2 and LDH-4 and LDH-5 are pooled, the developmental pattern is even more striking, as can be seen from the thin lines which represent the sum of the figures for LDH-1 and LDH-2 and LDH4 and LDH-5 respectively.
LDH
ISOENZYMES
IN DEVELOPING
ENAMEL
ORGAN
15
In every developmental stage five isoenzymes of lactate dehydrogenase were clearly demonstrable. The five isoenzymes were numbered according to general practice, LDH-1, LDH-2, LDH-3, LDH4, and LDH-5 from anode to cathode. The isoenzyme pattern of lactate dehydrogenase in the four segments of the enamel organ is characterized by a decrease in relative amounts of the two fast moving isoenzymes (LDH-1 and LDH-2) during the maturation of the enamel (Fig. 2). The mean values for the per cent distribution of the five isoenzymes in the four segments are presented in Table 1. The difference between segment A and segment D is highly significant for LDH-1, LDH-2, LDH-4 and LDH-5. If the combined values for each of the two pairs (LDH-1 and LDH-2) and (LDH4 and LDH-5) are calculated, this change in the isoenzymatic pattern is even more striking (Fig. 3). The present findings demonstrate a decrease in the relative amounts of the two anodal isoenzymes and an increase of the two cathodal isoenzymes during the development of the enamel. CAHN et al. (1962) have pointed out that tissues containing a preponderance of LDH-1 and LDH-2 allow pyruvate to accumulate and activate the citric acid cycle, whereas tissues containing a preponderance of LDH4 and LDH-5 do not allow pyruvate to accumulate but respire anaerobically. This explains why LDH-5 is more predominant in anaerobically metabolizing tissue whereas LDH-1 is more prominent in aerobically metabolizing tissues. A developmental pattern of LDH isoenzymes similar to the one demonstrated in the enamel organ in the present study has been reported by ROBERTS and STRACHAN (1967) in whole molar tooth buds during the early postnatal period in mice. These authors suggest that the isoenzyme pattern of developing teeth reflects a decreasing oxygen tension in these cells during ageing. It seems likely that the isoenzyme pattern of the enamel organ reported by the present author reflects a decreasing activity of the citric acid cycle and an increasing activity of anaerobically glycolysis during the formation of enamel. Quantitative assays of the enzymes of the citric acid cycle and the glycolytic pathway which have been performed in our laboratory support this hypothesis. Acknowledgements-This work has been supported by grants from the Swedish Medical Research Council (B69-24X-2385-03A) and Gijteborgs TandlBkarsHllskap, Gothenburg, Sweden. R&urn&--Les organes de l’email de rats ad&es sont preleves par dissection et divises en quatre zones de developpement. Ces Cchantillons sont homogeneists et analyses par Clectrophorese sur gel d’acrylamyde pour l’activite en lactate deshydrogenase. Cinq isoenzymes de lactate deshydrogenase ont et6 isolts dans l’organe de l’tmail. Pendant la formation de l’bmail, on observe une diminution dans les proportions relatives de LDH-1 et LDH-2 et une augmentation de LDH-4 et LDH-5. Ce changement semble traduire une diminution de la tension en oxygene de l’organe de l’tmail. Zusammenfassung-Die SChmelzorgane erwachsener Ratten wurden herausprlpariert und in vier Entwicklungsz onen aufgeteilt. Die Gewebsproben wurden homogenisiert und mit Hilfe der Acrylamid-Gel-Elektrophorese auf Laktatdehydrogenase Aktivitat untersucht. Ftinf Isoenzyme der Laktatdehydrogenase konnten im Schmelzorgan nachgewiesen werden. W1 hrend der Schmelzbildung beobachtet man eine Abnahme
16
H. FREDBN im relativen Gehalt von LDH-1 und LDH-2 und eine Zunahme von LDH4 und LDHJ. Es scheint so, daB diese Verlnderungen ein Ausdruck fiir die Abnahme der Sauerstoffspannung im Schmelzorgan sind. REFERENCES
CAHN, R. D., KAPLAN, N. D., LEVINE,L. and ZWILLING,E. 1962. Nature and development of lactic dehydrogenases. Science, Lond. 136,962-969. DAVIS, B. J. 1964. Disc electrophoresis II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121, 404-427. DEAKINS,M. 1942. Changes in the ash, water and organic content of pig enamel during calcification. J. dent. Res. 21, 429-435. DEWEY,M. M. and CONCLIN,J. L. 1960. Starch gel electrophoresis of lactic dehydrogenase from rat kidney. Proc. Sot. exp. Biol. 105,492-494. HAMMARSTRGM, L. 1967. Different localization of tetracycline and simultaneously injected radiocalcium in developing enamel. Calc. Tiss. Res. 1, 229-242. LATNER,A. L. and SKILLEN,A. W. 1968. Isoenzymes in Biology andMedicine. Academic Press, London. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L. and RANDALL,R. S. 1951. Protein measurement with the folin phenol reagent. Sot. Biol. Chem. 193,265-275. MARSLAND,E. A. 1952. A histological investigation of amelogenesis in rats. 11. Maturation. Br. dent. J. 92, 109119. ORNSTEIN,L. 1964. Disc electrophoresis. I. Background and theory. Ann NY. Acad. Sci. 121,321-349. REITH, E. J. 1963. The ultrastructure of ameloblasts during early stages of maturation of enamel. J. Cell Biol. 18, 691-696. REITH, E. J. and BUTCHER,E. 0. 1967. Microanatomy and histochemistry of amelogenesis. In: Structural and Chemical Organization of Teeth (edited by MILES, A. E.W.) Chapter I, pp. 371394, Academic Press, New York. REITH, E. J. and Corn, U. F. 1962. Autoradiographic studies on calcification of enamel. Archs oral Biol. 7, 365-372. ROBERTS,R. W. and STRACHAN,D. S. 1967. Quantitation of lactate dehydrogenase of developing molar teeth of the mouse. J. dent. Res. 46, 522-526.