Brain Research, 153 (1978) 79-85 ~ Elsevier/North-Holland Biomedical Press
79
PROLIDASE ACTIVITY IN RAT BRAIN; DEVELOPMENTAL, R E G I O N A L AND SUBCELLULAR DISTRIBUTION
KOON-SEA HUI and ABEL LAJTHA hlstitute of Neurochemistry, Rockland Research Institute, Ward's Island, New York, N. Y. 10035 ( U.S.A.)
(Accepted January 4th, 1978)
SUMMARY We determined the regional and subcellular distribution of prolidase in rat brain and its changes with development. The most rapid changes in enzyme activity occurred perinatally, with a maximum level of activity 2 days before birth and a minimum 1 day after birth. Of the 7 regions examined, cerebellum had the highest enzyme level, followed by medulla. The lowest levels were found in the hypothalamus in the adult and in the midbrain in the young. Prolidase was mainly soluble; over 55% was recovered in the $2 fraction, and the rest was released from the particulate fractions by hypotonic shock. Brains of male rats contained a slightly higher level of the enzyme.
INTRODUCTION Protein turnover is highly active in the brain, and most brain proteins are in a dynamic state14,1.~.. Rates of turnover, synthesis and breakdown change during development 4,13. Proteinase levels also change during development 19, but the relationship of enzyme levels to turnover and to the mechanism and control of development needs further clarification. The role of peptidases in development is not known; however, it is likely that they regulate the rate of a number of the metabolic reactions. We report here our studies on prolidase (imidodipeptidase, EC 3.4.3.7), the only enzyme that splits the peptide bonds of X-Pro peptides el. This enzyme is greatly in excess (100-1000-fold) of that needed to account for the turnover of cerebral proteins 11,r'. Since a high level of the enzyme would result in low levels of proline peptides, it is likely that this enzyme regulates the level of a number of hormone-releasing factors and neurotransmitters containing proline peptide bonds. To throw some light on the function of prolidase in the brain, we studied the regional and subcellular distribution of cerebral prolidase activity and its pre- and postnatal changes. Regional and subcellular distribution of prolidase activity in young (28 days old) and adult rats was also compared.
80 MATERIALS AND METHODS Fetal, neonatal and adult Wistar rats (250-300 g) bred in our colony were used in this investigation. To determine fetal age, we kept one male and 6 female adult rats in a cage overnight and then removed the male. This was taken as the beginning of the first day of pregnancy. The animals were killed 15 or 19 days later, and the fetuses were removed. Newborn rats (3 and 24 h after birth) were compared with the same litter. Other rats were male, unless otherwise stated. The tissues were homogenized with 9 parts of ice-cold phosphate buffer, 0.1 M, pH 6.8, with a glass pestle homogemzer (Dounce homogenizer; Kontes Products, Vineland, New Jersey) in a cold room (4 °C).
Isolation of brain regions and subcellularfractions Brains were removed as quickly as possible after killing the rats by decapitation. The brains were dissected on a cold glass plate (0 °C) according to the procedure outlined by Glowinski and Iversen 6 into regions listed in legends to Table I. The subcellular fractions were prepared by the procedure of Whittaker and Barker~L The rat brains were chilled, and homogenized in 0.32 M sucrose (1:9 w/v). The homogenate was centrifuged at 800 × g for 10 min to obtain the crude nuclear fraction (P1) and then at 12,000 × g for 20 min to separate the crude mitochondrial fractions (P~). The supernatant was the Sz fraction. For preparation of subfractions, the P2 fraction was resuspended in 0.32 M sucrose, and this suspension was layered over a discontinuous density gradient consisting of 10 ml each of 0.8 M and 1.2 M sucrose. The gradient was centrifuged at 50,000 × g for 120 min in a SW-27 rotor in a Beckman L-2 ultracentrifuge. The subcetlular fractions myelin (PA), synaptosomal (PB), and mitochondrial (Pc) were obtained. P1 and P2 were submitted to hypotomc shock with the same volume of NaC1 solution (0.05 M) in the cold room (4 °C} for 3Omin. The solutions were then separated into soluble and particulate fractions by centrifugation at 17.000 ~ g for 1 h.
Assay of prolidase activity Determination of prolidase activity was according to our method published elsewherelL One unit of prolidase activity was defined as the hydrolysis of I #mole o f Ala-Pro per min under the experimental conditions of our method. Ala-Pro was obtained from Sigma (St. Louis, Mo.). Brain protein was determined by the method of Lowry et al. 17, with calf serum albumin used as the standard. RESULTS
Developmental changes of prolidase activity Prolidase activity was found in the immature rat brain at high levels. The specific activity in the fetal brain (19 days) was 70 % higher than in adult (55 and 33 nmole/mg protein per min respectively). Developmental changes in the specific activity (curve A. Fig. 1) were complex: activity increased in the fetus to a maximum, and decreased
81
8
~0
.
.
.
.
.
.
.
"
sI
I
50
~-
/
B \
~,~2
\
/
. . . . I["
/' 2.q -
150
~, ~r"
........ ............ ,
. - ...o 3c
~
,,,:: .......
/
' c~
,.,"* :...:. ......... ,L
2
_-L~""
..'"
lC '
- "y."
,r,z . . . . . ....P ,,,,"
-
.." . 0 / "£3"O"0. j *
......W;C4N~.-" 0
0
"t"*
I
Birth
I
5
I
I
10
I
15
20
I
28
~)0
50
Age (days) Fig. 1. Developmental change of prolidase activity in rat brain. Each point represents the mean ± S.E.M. of 4 5 experiments. A: specific activity (nmole Ala-Pro split/mg protein per rain); B: total activity, units (t~mole/min) per brain; C: brain weight (g); D: protein content (rag/g) of the brain. TABLE 1 Prolidase activity in rat brain regions Values represent mean ~: S.E.M. (n = 4). Brain regions'
Cerebellum Medulla Hypothalamus Striatum Midbrain Hippocampus Cortex
Specific activity (nmole/min/mg protein)
Per cent total activity*
28-day
90-day
28-day
90-day
41.2 ± 0.8 30.3 ± 2.6 16.0 ± 1.4 23.5 ± 1.7 28.3 ± 2.1 27.0 ~- 2.0 28.8 ± 0.4
22 13 3 4 II 6 43
20 11 1 4 11 5 44
50.8 ± 44.1 ± 37.1 ± 31.4 ± 29.0 ± 35.4 ± 35.7 ±
1.8 1.7 0.6 1.5 0.9 1.7 0.9
* Total activity as percentage of the whole brain in rat brain regions. a r o u n d birth to a m i n i m a l value. Postnatally it increased up to 21 days of age, then declined somewhat to adult values. The total enzyme c o n t e n t per b r a i n (curve B, Fig. 1) changed to a lower degree, showing mostly an increase with development, except in the perinatal period. Regional distribution o f prolidase activity Prolidase activity was f o u n d to be high in all brain regions of y o u n g (28 days) and adult rats (Table I). It was distributed heterogeneously, with the highest levels in the cerebellum in y o u n g and adult. The activity in all brains areas was lower t h a n that
82 TABLE II Distribution of prolidase activity in subcellular fractions o f rat brain
Values represent mean z: S.E.M. of 4 experiments for subcellular fractions and 10 for the homogenate~ Fractions
Homogenate P1 $2 P2 PA Pn Pc
Specific activity f nmole/mg protein/min )
Per cent total activity*
28-day
90.day
28-day
90-day
41.7 :~ 0.8 15.9 4- 2.7 90.6 ~. 1.3 22.9 z: 2.4 3.6 :~ 2.1 6.2-r 0.5 30.9 ~T 2.4
32.6 z 0.7 24.2 z: 3.3 68.5 _~ 4.1 16.7 ~ 1.0 13.1 :~ 1.9 14.1 _x 2.1 12.5 x: 2.5
100 13 56 30 0.6 1.7 6.8
100 22 55 23 2.9 3.7 3.6
* Total activity as percentage of the whole brain in rat brain subcellular fractions. found previously in the kidney and sciatic nerve, but it was equal t o o r higher than that in liver and spinal cordlL The specific activity of prolidase in the brain regions of the 28-day-old rats was higher than in the adult ones, except in the midbrain, where the activity was similar. There was some difference in the regional distribution ofenzyme activity between the two ages; at 28 days of age the midbrain, and at 90 days the hypothalamus, showed the lowest specific activity. The largest part of total activity was in the cortex, with only small amounts in the hypothalamus (Table I). Subcellular distribution o]'prolidase activity in rat brain The major portion of the enzyme was not particulate-bound. In the rats, half of the total prolidase activity was located in the $2 fractions, The P1 fraction and Pz fraction had similar but lower prolidase activity (Table 11). Specific activity was highest in the $2 fraction at both ages; it was twice as high in the supernatant as in the whole brain homogenate, and several-fold higher than in any other fraction. There were some developmental differences in the particulate distribution of the enzyme, the mitochondrial fraction (Pc) in the young had a higher and the myelin (PA) fraction a lower activity as compared to that in the mature brain. There was no indication that the enzyme was membrane-bound, since hypotonic shock released m o r e t h a n 90 ~ of the enzyme from fractions P1 and Pz into the soluble supernatant (Table III). The subcellular and regional distribution of the enzyme was measured in brains from young and adult male rats. We found a small but significant difference in cerebral prolidase activity between male and female rats; the specific activity in I0 samples from males was 32.6 :~ 0.7 and in 10 samples from females was 30.7 ± 0.2 nmole/min per mg protein in brain, with a P < 0.05. Total enzyme activity per brain was 7.5 units in brain of male and 5.8 units in brain of female rat. For these experiments homogenates of adult brain corresponding to 0.5 mg fresh tissue were incubated with 50/~1 of 3.6 m M Ala-Pro solution and 100/~1 of 0.1 M phosphate buffer pH 6.8 for 30
min.
83 TABLE III Prolidase activity in soluble and particulate fractions qf P1 and P2 *
PI and P2 were submitted to hypotonic shock with same volume of NaCI solution (0.05 M) in a cold room (4 c~C)for 30 rain. The solutions were then separated into soluble and particulate fractions by centrifugation at 17,000 × g for 1 h. Fractions
P1 Soluble Particulate P2 Soluble Particulate
Specific activity (nmole/min/mg protein)
Total activity (units)
77.4 ± 7.0 0.5 ± 0.4
0.60 0.05
116 ± 7.4 0.9 ± 0.7
0.96 0.07
DISCUSSION Knowledge concerning mechanisms and function of protein breakdown, which is obviously an integral part of the regulatory mechanism affecting protein level and composition in the brain, is rudimentary compared to knowledge of biosynthesis. Recent data show that a number of biologically active peptides or hormone releasing factors are formed from peptides or proteinsl,~, 18,2a. This indicates a role for peptidases in the formation of physiologically active substances. It is interesting that many biologically active peptides contain a proline residue (substance P, melanocyteinhibitory factor, thyrotropin-releasing factor, fi-lipotropin, fl-melanocyte stimulating hormone, oxytocin, vasopressin, angiotensin, and LH- and FSH-releasing hormone). Prolidase therefore would be an essential, at times rate-limiting, enzyme in the metabolism of these compounds. Prolidase, in addition to regulating the level of proline peptides, may influence the level of free proline, which, like some of its peptides, has been regarded as a potential neurotransmitter 5. The greatest developmental change in prolidase occurs in the fetal brain and in the per±natal period, where first the maximal value is reached before birth, then the minimal value just after birth (Fig. 1). The rapid drop of prolidase activity parallels per±natal changes in amino acid content 16. Some of the changes in protein metabolism defend on the date of birth rather than on the gestation age 20. We found that the in vivo rates of protein breakdown were higher in the immature as compared to adult brain 3. This may be due to the need to alter the protein composition during development of the nervous system. Prolidase may participate in this phenomenon. Prolidase activity in males was greater than in females, indicating differences of cerebral metabolism. This sexual difference is similar to that found for the LH-RH peptidases and T R H peptidases in the brainS, 9, and to that for hypothalamic peptidases inactivating oxytocin, which can be reversed by neonatal treatment v. There are few data available on regional protein and peptide catabolism in brain. Hydrolysis of T R H and LH-RH, like prolidase activity reported here, was higher in
84 the cerebellumS,L It is not k n o w n whether this activity represents specific peptidases for these substrates. The higher activity of prolidasein the cerebellum may have some relationship to the high rate o f T R H and L H - R H breakdown in this area: in further support, our findings o f low prolidase activity in the hypothalamus are consistent with those of Griffiths et al. on peptidases splitting T R H and LH-RHS, 9. This indicates that in the hypothalamus, the region in which the peptide hormones are synthesized and released, most of the peptides are fairly stable. Prolidase activity in the pituitary is two times the activity in hypothalamus (27 vs. 16 nmole/min per m g protein) 4. Plasma, the carrier of peptide hormones, like hypothalamus, has low prolidase activity 114 nmole/min per mg protein) 12. It has to be emphasized that hormone-releasing factors have functions outside the h y p o t h a l a m u s - p i t u i t a r y - t h y r o i d axisg, 10 Prolidase is a soluble enzyme; the major portion was found in the $2 fraction, and most (90 700)o f the prolidase activity was in the soluble fractions o f P1 and P2 fractions. Brain peptidases hydrolyzing T R H and L H - R H are located mostly in the soluble fractions o f all brain regionsS, 9. The function of prolidase in the nuclei is u n k n o w n : it m a y play a role in the metabolism o f the histones. The changes in regional and subcellular distribution during development may help us to understand the function of prolidase in the nervous system. ACKNOWLEDGEMENT This investigation was supported in part by National Institute o f Health Grant N B 03226
REFERENCES 1 Austin, B. M. and Smyth, D. G., Specific cleavage of lipotropin C-fra~nent by endopeptidases: evidence for a preferred conformation, Bioehem. biophys. Res. Commun., 77 (1977) 86-94. 2 Chretien, M., Benjannet, S., Dragon, N., Seidah, N. G. and Lis, M., Isolation of peptidases with opiate activity from sheep and human pituitaries: relationship to beta-lipotropin, Biochem~ biophys. Res. Comraun., 72 (1976) 472-478. 3 Dunlop, D., Lajtha, A. and Toth, J., Measuring brain protein metabolism in young and adult rats. In S. Roberts, A. Lajtha and W. H. Gispen (Eds.), Mechanisms, Regulation and Special Functions of Protein Synthesis in the Brain, Elsevier, Amsterdam, 1977, pp. 79-96. 4 Dunlop, S., van Elden, W. and Lajtha, A., Developmental effects on protein synthesis rates in regions of the CNS in vivo and in vitro, J. Neurochem., 29 (1977) 934--945. 5 Felix, D. and Kunzle, H., The role of proline in nervous transmissions, Advanc. Biochem. Psychopharmacol., 15 (1976) 165-174. 6 Glowinski, J. and Iversen, L. L., Regional studies of catecholamines in the rat brain, i. The deposition of [a H]n orepinephrine, [aH]dopamine and [SH]DOPA in various regions of the brain. J. Neurochem., 13 (1966) 655-669. 7 Grifliths, E. C. and Hooper, K. C., The effect ofneonatal androgen on the activityofcertain enzymes in the rat hypothalamus, Acta Endocr. (Kbh.), 70 (1972) 767-776. 8 Grifliths, E. C., Hooper, K. C., Jeffcoate, S. L. and Holland, D. T., Peptidases in different areas of the rat brain inactivating luteinizing hormone-rdeasing hormone (LH-RH), Brain Research, 85 (1975) 161-164. 9 Grifliths, E. C., Hooper, K. C., Jeffcoate, S. L. and White, N., Inactivation of thyrotrophin releasing hormone (TRH) by peptidases in different areas of the rabbit brain, Brain Research, 105 (1976) 376-380.
85 10 Griffiths, E. C., Jeffcoate, S. L. and Holland, D. T., Local degradation of growth hormone-releasing inhibiting hormone (somatostatin) in the central nervous system, Neurosci. Lett., 4 (1977) 33-37. 11 Hui, K.-S. and Lajtha, A., Prolidase activity in the nervous system, Trans. Amer. Soc. Neurochem., 8 (1976) 139. 12 Hui, K.-S. andLajtha, A.,Prolidaseactivityinbrain:comparisonwithotherorgans, J. Neurochem., 30 (1978) 321-327. 13 Lajtha, A. and Dunlop, D., Alterations of protein metabolism during development of the brain. In A. Vernadakis and N. Weiner (Eds.), Drugs andthe Developing Brain, Plenum Publ., New York, 1974, pp. 215-229. 14 Lajtha, A., Latzkovits, L. and Toth, J., Comparison of turnover rates of proteins of the brain, liver and kidney in mouse in vivo following long-term labeling, Biochim. biophys. ,4cta (Amst.), 425 (1976) 511-520. 15 Lajtha, A. and Toth, J., Instability of cerebral proteins, Biochem. biophys. Res. Commun., 23 (1966) 294-298. 16 Lajtha, A. and Toth, J., Perinatal changes in the free amino acid pool of the brain in mice, Brain Research, 55 (1973) 238 241. 17 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265 275. 18 Marks, N., Conversion and inactivation of neuropeptides. In H. Gainer (Ed.), Peptides in Neurobiology, Plenum Publ., New York, 1977, pp. 221 258. 19 Marks, N., Stern, F. and Lajtha, A., Changes in proteolytic enzymes and proteins during maturation of the brain, Brain Research, 86 (1977) 307-322. 20 Schain, R. J., Carver, M. J., Copenhaver, J. H. and Underdahl, N. R., Protein metabolism in the developing brain: Influence of birth and gestational age, Science, 156 (1967) 984-980. 21 SjOstr6m, H., Norin, O. and Josefsson, L., Purification and specificity of pig intestinal prolidase, Biochem. biophys. Acta (Amst.), 327 (1973) 457-470. 22 Whittaker, V. P. and Barker, L. A., The subcellular fractionation of brain tissue with special reference to the preparation of synaptosomes and their component organelles. In R. Fried (Ed.), Methods of Neurochemistry, Marcel Dekker, New York, 1972. 23 de Wied, D., Peptides and behavior, Life Sci., 20 (1977) 195 204.
79
PROLIDASE ACTIVITY IN RAT BRAIN; DEVELOPMENTAL, R E G I O N A L AND SUBCELLULAR DISTRIBUTION
KOON-SEA HUI and ABEL LAJTHA hlstitute of Neurochemistry, Rockland Research Institute, Ward's Island, New York, N. Y. 10035 ( U.S.A.)
(Accepted January 4th, 1978)
SUMMARY We determined the regional and subcellular distribution of prolidase in rat brain and its changes with development. The most rapid changes in enzyme activity occurred perinatally, with a maximum level of activity 2 days before birth and a minimum 1 day after birth. Of the 7 regions examined, cerebellum had the highest enzyme level, followed by medulla. The lowest levels were found in the hypothalamus in the adult and in the midbrain in the young. Prolidase was mainly soluble; over 55% was recovered in the $2 fraction, and the rest was released from the particulate fractions by hypotonic shock. Brains of male rats contained a slightly higher level of the enzyme.
INTRODUCTION Protein turnover is highly active in the brain, and most brain proteins are in a dynamic state14,1.~.. Rates of turnover, synthesis and breakdown change during development 4,13. Proteinase levels also change during development 19, but the relationship of enzyme levels to turnover and to the mechanism and control of development needs further clarification. The role of peptidases in development is not known; however, it is likely that they regulate the rate of a number of the metabolic reactions. We report here our studies on prolidase (imidodipeptidase, EC 3.4.3.7), the only enzyme that splits the peptide bonds of X-Pro peptides el. This enzyme is greatly in excess (100-1000-fold) of that needed to account for the turnover of cerebral proteins 11,r'. Since a high level of the enzyme would result in low levels of proline peptides, it is likely that this enzyme regulates the level of a number of hormone-releasing factors and neurotransmitters containing proline peptide bonds. To throw some light on the function of prolidase in the brain, we studied the regional and subcellular distribution of cerebral prolidase activity and its pre- and postnatal changes. Regional and subcellular distribution of prolidase activity in young (28 days old) and adult rats was also compared.
80 MATERIALS AND METHODS Fetal, neonatal and adult Wistar rats (250-300 g) bred in our colony were used in this investigation. To determine fetal age, we kept one male and 6 female adult rats in a cage overnight and then removed the male. This was taken as the beginning of the first day of pregnancy. The animals were killed 15 or 19 days later, and the fetuses were removed. Newborn rats (3 and 24 h after birth) were compared with the same litter. Other rats were male, unless otherwise stated. The tissues were homogenized with 9 parts of ice-cold phosphate buffer, 0.1 M, pH 6.8, with a glass pestle homogemzer (Dounce homogenizer; Kontes Products, Vineland, New Jersey) in a cold room (4 °C).
Isolation of brain regions and subcellularfractions Brains were removed as quickly as possible after killing the rats by decapitation. The brains were dissected on a cold glass plate (0 °C) according to the procedure outlined by Glowinski and Iversen 6 into regions listed in legends to Table I. The subcellular fractions were prepared by the procedure of Whittaker and Barker~L The rat brains were chilled, and homogenized in 0.32 M sucrose (1:9 w/v). The homogenate was centrifuged at 800 × g for 10 min to obtain the crude nuclear fraction (P1) and then at 12,000 × g for 20 min to separate the crude mitochondrial fractions (P~). The supernatant was the Sz fraction. For preparation of subfractions, the P2 fraction was resuspended in 0.32 M sucrose, and this suspension was layered over a discontinuous density gradient consisting of 10 ml each of 0.8 M and 1.2 M sucrose. The gradient was centrifuged at 50,000 × g for 120 min in a SW-27 rotor in a Beckman L-2 ultracentrifuge. The subcetlular fractions myelin (PA), synaptosomal (PB), and mitochondrial (Pc) were obtained. P1 and P2 were submitted to hypotomc shock with the same volume of NaC1 solution (0.05 M) in the cold room (4 °C} for 3Omin. The solutions were then separated into soluble and particulate fractions by centrifugation at 17.000 ~ g for 1 h.
Assay of prolidase activity Determination of prolidase activity was according to our method published elsewherelL One unit of prolidase activity was defined as the hydrolysis of I #mole o f Ala-Pro per min under the experimental conditions of our method. Ala-Pro was obtained from Sigma (St. Louis, Mo.). Brain protein was determined by the method of Lowry et al. 17, with calf serum albumin used as the standard. RESULTS
Developmental changes of prolidase activity Prolidase activity was found in the immature rat brain at high levels. The specific activity in the fetal brain (19 days) was 70 % higher than in adult (55 and 33 nmole/mg protein per min respectively). Developmental changes in the specific activity (curve A. Fig. 1) were complex: activity increased in the fetus to a maximum, and decreased
81
8
~0
.
.
.
.
.
.
.
"
sI
I
50
~-
/
B \
~,~2
\
/
. . . . I["
/' 2.q -
150
~, ~r"
........ ............ ,
. - ...o 3c
~
,,,:: .......
/
' c~
,.,"* :...:. ......... ,L
2
_-L~""
..'"
lC '
- "y."
,r,z . . . . . ....P ,,,,"
-
.." . 0 / "£3"O"0. j *
......W;C4N~.-" 0
0
"t"*
I
Birth
I
5
I
I
10
I
15
20
I
28
~)0
50
Age (days) Fig. 1. Developmental change of prolidase activity in rat brain. Each point represents the mean ± S.E.M. of 4 5 experiments. A: specific activity (nmole Ala-Pro split/mg protein per rain); B: total activity, units (t~mole/min) per brain; C: brain weight (g); D: protein content (rag/g) of the brain. TABLE 1 Prolidase activity in rat brain regions Values represent mean ~: S.E.M. (n = 4). Brain regions'
Cerebellum Medulla Hypothalamus Striatum Midbrain Hippocampus Cortex
Specific activity (nmole/min/mg protein)
Per cent total activity*
28-day
90-day
28-day
90-day
41.2 ± 0.8 30.3 ± 2.6 16.0 ± 1.4 23.5 ± 1.7 28.3 ± 2.1 27.0 ~- 2.0 28.8 ± 0.4
22 13 3 4 II 6 43
20 11 1 4 11 5 44
50.8 ± 44.1 ± 37.1 ± 31.4 ± 29.0 ± 35.4 ± 35.7 ±
1.8 1.7 0.6 1.5 0.9 1.7 0.9
* Total activity as percentage of the whole brain in rat brain regions. a r o u n d birth to a m i n i m a l value. Postnatally it increased up to 21 days of age, then declined somewhat to adult values. The total enzyme c o n t e n t per b r a i n (curve B, Fig. 1) changed to a lower degree, showing mostly an increase with development, except in the perinatal period. Regional distribution o f prolidase activity Prolidase activity was f o u n d to be high in all brain regions of y o u n g (28 days) and adult rats (Table I). It was distributed heterogeneously, with the highest levels in the cerebellum in y o u n g and adult. The activity in all brains areas was lower t h a n that
82 TABLE II Distribution of prolidase activity in subcellular fractions o f rat brain
Values represent mean z: S.E.M. of 4 experiments for subcellular fractions and 10 for the homogenate~ Fractions
Homogenate P1 $2 P2 PA Pn Pc
Specific activity f nmole/mg protein/min )
Per cent total activity*
28-day
90.day
28-day
90-day
41.7 :~ 0.8 15.9 4- 2.7 90.6 ~. 1.3 22.9 z: 2.4 3.6 :~ 2.1 6.2-r 0.5 30.9 ~T 2.4
32.6 z 0.7 24.2 z: 3.3 68.5 _~ 4.1 16.7 ~ 1.0 13.1 :~ 1.9 14.1 _x 2.1 12.5 x: 2.5
100 13 56 30 0.6 1.7 6.8
100 22 55 23 2.9 3.7 3.6
* Total activity as percentage of the whole brain in rat brain subcellular fractions. found previously in the kidney and sciatic nerve, but it was equal t o o r higher than that in liver and spinal cordlL The specific activity of prolidase in the brain regions of the 28-day-old rats was higher than in the adult ones, except in the midbrain, where the activity was similar. There was some difference in the regional distribution ofenzyme activity between the two ages; at 28 days of age the midbrain, and at 90 days the hypothalamus, showed the lowest specific activity. The largest part of total activity was in the cortex, with only small amounts in the hypothalamus (Table I). Subcellular distribution o]'prolidase activity in rat brain The major portion of the enzyme was not particulate-bound. In the rats, half of the total prolidase activity was located in the $2 fractions, The P1 fraction and Pz fraction had similar but lower prolidase activity (Table 11). Specific activity was highest in the $2 fraction at both ages; it was twice as high in the supernatant as in the whole brain homogenate, and several-fold higher than in any other fraction. There were some developmental differences in the particulate distribution of the enzyme, the mitochondrial fraction (Pc) in the young had a higher and the myelin (PA) fraction a lower activity as compared to that in the mature brain. There was no indication that the enzyme was membrane-bound, since hypotonic shock released m o r e t h a n 90 ~ of the enzyme from fractions P1 and Pz into the soluble supernatant (Table III). The subcellular and regional distribution of the enzyme was measured in brains from young and adult male rats. We found a small but significant difference in cerebral prolidase activity between male and female rats; the specific activity in I0 samples from males was 32.6 :~ 0.7 and in 10 samples from females was 30.7 ± 0.2 nmole/min per mg protein in brain, with a P < 0.05. Total enzyme activity per brain was 7.5 units in brain of male and 5.8 units in brain of female rat. For these experiments homogenates of adult brain corresponding to 0.5 mg fresh tissue were incubated with 50/~1 of 3.6 m M Ala-Pro solution and 100/~1 of 0.1 M phosphate buffer pH 6.8 for 30
min.
83 TABLE III Prolidase activity in soluble and particulate fractions qf P1 and P2 *
PI and P2 were submitted to hypotonic shock with same volume of NaCI solution (0.05 M) in a cold room (4 c~C)for 30 rain. The solutions were then separated into soluble and particulate fractions by centrifugation at 17,000 × g for 1 h. Fractions
P1 Soluble Particulate P2 Soluble Particulate
Specific activity (nmole/min/mg protein)
Total activity (units)
77.4 ± 7.0 0.5 ± 0.4
0.60 0.05
116 ± 7.4 0.9 ± 0.7
0.96 0.07
DISCUSSION Knowledge concerning mechanisms and function of protein breakdown, which is obviously an integral part of the regulatory mechanism affecting protein level and composition in the brain, is rudimentary compared to knowledge of biosynthesis. Recent data show that a number of biologically active peptides or hormone releasing factors are formed from peptides or proteinsl,~, 18,2a. This indicates a role for peptidases in the formation of physiologically active substances. It is interesting that many biologically active peptides contain a proline residue (substance P, melanocyteinhibitory factor, thyrotropin-releasing factor, fi-lipotropin, fl-melanocyte stimulating hormone, oxytocin, vasopressin, angiotensin, and LH- and FSH-releasing hormone). Prolidase therefore would be an essential, at times rate-limiting, enzyme in the metabolism of these compounds. Prolidase, in addition to regulating the level of proline peptides, may influence the level of free proline, which, like some of its peptides, has been regarded as a potential neurotransmitter 5. The greatest developmental change in prolidase occurs in the fetal brain and in the per±natal period, where first the maximal value is reached before birth, then the minimal value just after birth (Fig. 1). The rapid drop of prolidase activity parallels per±natal changes in amino acid content 16. Some of the changes in protein metabolism defend on the date of birth rather than on the gestation age 20. We found that the in vivo rates of protein breakdown were higher in the immature as compared to adult brain 3. This may be due to the need to alter the protein composition during development of the nervous system. Prolidase may participate in this phenomenon. Prolidase activity in males was greater than in females, indicating differences of cerebral metabolism. This sexual difference is similar to that found for the LH-RH peptidases and T R H peptidases in the brainS, 9, and to that for hypothalamic peptidases inactivating oxytocin, which can be reversed by neonatal treatment v. There are few data available on regional protein and peptide catabolism in brain. Hydrolysis of T R H and LH-RH, like prolidase activity reported here, was higher in
84 the cerebellumS,L It is not k n o w n whether this activity represents specific peptidases for these substrates. The higher activity of prolidasein the cerebellum may have some relationship to the high rate o f T R H and L H - R H breakdown in this area: in further support, our findings o f low prolidase activity in the hypothalamus are consistent with those of Griffiths et al. on peptidases splitting T R H and LH-RHS, 9. This indicates that in the hypothalamus, the region in which the peptide hormones are synthesized and released, most of the peptides are fairly stable. Prolidase activity in the pituitary is two times the activity in hypothalamus (27 vs. 16 nmole/min per m g protein) 4. Plasma, the carrier of peptide hormones, like hypothalamus, has low prolidase activity 114 nmole/min per mg protein) 12. It has to be emphasized that hormone-releasing factors have functions outside the h y p o t h a l a m u s - p i t u i t a r y - t h y r o i d axisg, 10 Prolidase is a soluble enzyme; the major portion was found in the $2 fraction, and most (90 700)o f the prolidase activity was in the soluble fractions o f P1 and P2 fractions. Brain peptidases hydrolyzing T R H and L H - R H are located mostly in the soluble fractions o f all brain regionsS, 9. The function of prolidase in the nuclei is u n k n o w n : it m a y play a role in the metabolism o f the histones. The changes in regional and subcellular distribution during development may help us to understand the function of prolidase in the nervous system. ACKNOWLEDGEMENT This investigation was supported in part by National Institute o f Health Grant N B 03226
REFERENCES 1 Austin, B. M. and Smyth, D. G., Specific cleavage of lipotropin C-fra~nent by endopeptidases: evidence for a preferred conformation, Bioehem. biophys. Res. Commun., 77 (1977) 86-94. 2 Chretien, M., Benjannet, S., Dragon, N., Seidah, N. G. and Lis, M., Isolation of peptidases with opiate activity from sheep and human pituitaries: relationship to beta-lipotropin, Biochem~ biophys. Res. Comraun., 72 (1976) 472-478. 3 Dunlop, D., Lajtha, A. and Toth, J., Measuring brain protein metabolism in young and adult rats. In S. Roberts, A. Lajtha and W. H. Gispen (Eds.), Mechanisms, Regulation and Special Functions of Protein Synthesis in the Brain, Elsevier, Amsterdam, 1977, pp. 79-96. 4 Dunlop, S., van Elden, W. and Lajtha, A., Developmental effects on protein synthesis rates in regions of the CNS in vivo and in vitro, J. Neurochem., 29 (1977) 934--945. 5 Felix, D. and Kunzle, H., The role of proline in nervous transmissions, Advanc. Biochem. Psychopharmacol., 15 (1976) 165-174. 6 Glowinski, J. and Iversen, L. L., Regional studies of catecholamines in the rat brain, i. The deposition of [a H]n orepinephrine, [aH]dopamine and [SH]DOPA in various regions of the brain. J. Neurochem., 13 (1966) 655-669. 7 Grifliths, E. C. and Hooper, K. C., The effect ofneonatal androgen on the activityofcertain enzymes in the rat hypothalamus, Acta Endocr. (Kbh.), 70 (1972) 767-776. 8 Grifliths, E. C., Hooper, K. C., Jeffcoate, S. L. and Holland, D. T., Peptidases in different areas of the rat brain inactivating luteinizing hormone-rdeasing hormone (LH-RH), Brain Research, 85 (1975) 161-164. 9 Grifliths, E. C., Hooper, K. C., Jeffcoate, S. L. and White, N., Inactivation of thyrotrophin releasing hormone (TRH) by peptidases in different areas of the rabbit brain, Brain Research, 105 (1976) 376-380.
85 10 Griffiths, E. C., Jeffcoate, S. L. and Holland, D. T., Local degradation of growth hormone-releasing inhibiting hormone (somatostatin) in the central nervous system, Neurosci. Lett., 4 (1977) 33-37. 11 Hui, K.-S. and Lajtha, A., Prolidase activity in the nervous system, Trans. Amer. Soc. Neurochem., 8 (1976) 139. 12 Hui, K.-S. andLajtha, A.,Prolidaseactivityinbrain:comparisonwithotherorgans, J. Neurochem., 30 (1978) 321-327. 13 Lajtha, A. and Dunlop, D., Alterations of protein metabolism during development of the brain. In A. Vernadakis and N. Weiner (Eds.), Drugs andthe Developing Brain, Plenum Publ., New York, 1974, pp. 215-229. 14 Lajtha, A., Latzkovits, L. and Toth, J., Comparison of turnover rates of proteins of the brain, liver and kidney in mouse in vivo following long-term labeling, Biochim. biophys. ,4cta (Amst.), 425 (1976) 511-520. 15 Lajtha, A. and Toth, J., Instability of cerebral proteins, Biochem. biophys. Res. Commun., 23 (1966) 294-298. 16 Lajtha, A. and Toth, J., Perinatal changes in the free amino acid pool of the brain in mice, Brain Research, 55 (1973) 238 241. 17 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265 275. 18 Marks, N., Conversion and inactivation of neuropeptides. In H. Gainer (Ed.), Peptides in Neurobiology, Plenum Publ., New York, 1977, pp. 221 258. 19 Marks, N., Stern, F. and Lajtha, A., Changes in proteolytic enzymes and proteins during maturation of the brain, Brain Research, 86 (1977) 307-322. 20 Schain, R. J., Carver, M. J., Copenhaver, J. H. and Underdahl, N. R., Protein metabolism in the developing brain: Influence of birth and gestational age, Science, 156 (1967) 984-980. 21 SjOstr6m, H., Norin, O. and Josefsson, L., Purification and specificity of pig intestinal prolidase, Biochem. biophys. Acta (Amst.), 327 (1973) 457-470. 22 Whittaker, V. P. and Barker, L. A., The subcellular fractionation of brain tissue with special reference to the preparation of synaptosomes and their component organelles. In R. Fried (Ed.), Methods of Neurochemistry, Marcel Dekker, New York, 1972. 23 de Wied, D., Peptides and behavior, Life Sci., 20 (1977) 195 204.
