European Journal of

Europ. J. appl. Physiol. 36, 267--273 (1977)

Applied

Physiology

and Occupational Physiology c by Springer Veriag 1977

Muscle Amino Acid Arylpeptidases and Their Serum Changes with Exercise 1 A. Berg and J. Keul Lehrstuhl f'fir Leistungs- und Sportmedizin (Direktor: Prof. Dr. J. Keul), Medizinische Universitgtsklinik, D-7800 Freiburg i. Br., Federal Republic of Germany

Summary. Activities of serum amino acid arylpeptidases were determined in five groups of healthy male adults at rest and after various exercise types using the following substrates: Ala-, Leu-, Phe-, Tyr-, Gly- and Pro-4-Nitroanilide. The exercise-induced changes were compared to the activities of the respective enzymes in resting leg muscles. A regression function was found, showing a close correlation between the mentioned parameters in all groups. The influence of haemoconcentration and intravascular hemolysis on the postexercise measured activities was of no consequence. Key words: Aminopepfidases - Muscle -- Serum - Exercise.

In a previous paper from this laboratory, activities of amino acid arylamidases in skeletal muscle and serum of healthy male subjects have been investigated [5]. A relationship has been found between the increase of the serum activities with various substrates (Ala-, Leu-, Phe-4-nitroanilides) after physical exercise and their respective activities in the resting leg muscle. It was the purpose of this study to gain more information on the hypothesis that AAP are increasingly released from working muscle. To verify this, investigations were performed: a) on AAP-activities in human muscle with the substrates already described, as well as with other amino acid paranitroanilides (Tyr-, Gly- and Pro-4-nitroanilide) as substrates; b) on changes of serum activities after physical strain using the same substrates. The possibility of intravascular (or in vitro) hemolysis influencing the results was investigated by measuring serum hemoglobin before and after exertion. Eventually the changes of total serum protein were determined, purporting to seek information as to the amount of haemoconeentration. 1

The authors express their thanks to G. Haralambie for his encouragement during this work

268

A. Berg and J. Keul

Subjects and Methods Muscle samples were obtained surgically from male subjects, aged 18--36 years, under conditions described in [5]. In no case there were any symptoms of muscular disease. After removing fat and connective tissue, the samples weight was between 80 and 360 mg. The preparation of a muscle extract in triethanolamine buffer (0.3 M, pH 7.6, containing 150 mM KC1) is detailed in [5]. After centrifugation at 30,000 g for 20 min, the clear supernatant (final dilution 1 : 2 1 w/v) was analyzed immediately. Determinations of the AAP-activities were performed as indicated by Nagel et al. [13]. Activities in muscle extracts were measured at 37~, serum activities at 25 ~. For comparison with the activities in muscle, serum activities were multiplied by the coefficient 2.4 (see ref. [5]). The following substrates were used: L-alanine-4-nitroanilide hydrochloride (Merck, Darmstadt), L-leucine-4-nitroanilide (Boehringer, Mannheim), L-Phenylalanine-4-nitroanilide (Schuchardt, Mtinchen), L-Tyrosine-4-nitroanilide (Merck, Darmstadt), L-glycine-4-nitroanilide (Serva, Heidelberg), L-proline-4-nitroanilide (Sehuchardt, Mfinchen). The final concentration of all substrates used was 0.8 mM with the exception of L-proline-4nitroanilide. Serum analyses with this latter substrate were performed with a final concentration of 15.6 mM. Measurements in muscle extract showed, that there was no difference in the extent of splitting of L-proline-4-nitroanilide, when concentration was raised above 0.8 mM. Serum hemoglobin was determined spectrophotometrically according to Harboe [7]. Serum total protein was measured with the biuret reaction (reaction kit of the Fa. Merck, Darmstadt, Art. No. 3327). Five groups of male subjects, all trained students (groups B, C, E), or trained athletes (groups A, D) aged 16-41 years were examined before and after their respective races (Table 1). All subjects were clinically healthy, i.e. there was no muscular, connective tissue or systemic disease. Blood was drawn from the cubital vein before and 5 - 8 min after the end of the exercise, avoiding stasis and without anticoagulant. After clotting serum was centrifugated twice and analyzed immediately. Mean values and S.D. were calculated and the significance of difference was tested with the Student's t-test for paired data. Correlations were computed as linear regression functions.

Table 1. Data on subjects and type of exercise Group

n

Mean age of subject (years)

Type of exercise

Mean duration (min)

A B C D E

8 13 18 10 10

17.5 29.6 28.5 29.0 32.0

10 20 42 50 60

25.1 80.0 186.0 135.0 340.0

km km km km km

skiing cross running marathon race skiing skiing

Results V a l u e s o f s e r u m - A A P - a c t i v i t i e s at r e s t a n d after t h e different exercise t y p e s are g i v e n in T a b l e 2. T a b l e 3 p r e s e n t s t h e A A P - a c t i v i t i e s w i t h t h e d e s c r i b e d s u b s t r a t e s a t 37 ~ in v a r ious leg- a n d a r m - m u s c l e s . W h e n also t h e v a l u e s o f t h e p r e c e e d i n g d e t e r m i n a t i o n [5]

* See Subjects and Methods

P

Muscle amino acid arylpeptidases and their serum changes with exercise.

European Journal of Europ. J. appl. Physiol. 36, 267--273 (1977) Applied Physiology and Occupational Physiology c by Springer Veriag 1977 Muscle...
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