Acta physiol. scand. 1974. 92. 21-26 From the Department of Physiology, Gymnastik- och idrottshogskolan, Stockholm, Sweden

Substrate Activation and Product Inhibition of LDH Activity in Human Skeletal Muscle

BY JAN KARLSSON, BODIL HULT~N AND BERTIL SJODIN

Received 15 February 1974

Abstract

KARLSSON, J., B. HULTBN and B. SJODIN. Substrate activation and product inhibi- tion of LDH activity in human skeletal muscle. Acta physiol. scand. 1974. 92. 21-26.

Substrate activation and product inhibition have been studied on LDH activity in both direc- tions in human skeletal muscle. Km for the forward and backward reactions for pyruvate and lactate were found to be 0.8 x M, respectively. For increases in concentra- tions of NAD and NADH the LDH activity was found to increase curvilinearly and no Km value could be obtained. Inhibition of the LDH activity corresponding to 50 % of Vmax was obtained for the forward reaction with a lactate concentration equal to 0.3 x M and for the backward reaction (pyruvate) the corresponding value was 1.0 x 10-4 M. With concentra- tions of lactate and pyruvate approximately corresponding to maximal values observed in skeletal muscle with exercise, the inhibition of LDH activity in both directions was equal to or more than 70 % of Vmax. I t is concluded that in an in uitro system concentrations of lactate and pyruvate similar to what are observed under physiological conditions in human skeletal muscles will induce a significant product inhibition on LDH activity obtained from human skeletal muscles in both reaction directions. I t seems reasonable to assume that the present in nitro findings might reflect functions operating in an in situ system. Thus it is possible that the products of the LDH catalysed reactions (lactate or pyruvate) might have a regulatory effect on the anaerobic glycolysis as well as the oxidation of lactate in human skeletal muscle.

and 0.9 x

Human skeletal muscle possesses great capacities for anaerobic glycolysis. The limit- ing factor for production of lactate is suggested to be either in the onset of the glycolysis (activation of the enzyme phosphorylase) or the phosphorylation on hexosmonophosphate residues (by the enzyme phosphofructokinase, PFK) (for ref. see Karlsson 1971) or both. Later steps in the glycolytic pathway as e.g. reduction of pyruvate to lactate by the enzyme lactate dehydrogenase (LDH) has been neglected as to whether this step might exert a regulatory function in the reduction of pyruvate to lactate. The reason has been that the LDH activity determined as V,,, (optimal rate of reaction) has been found to be 3-5 times the highest rate of lactate formation observed (Karlsson 1971). Although the activity of LDH is far in excess of the maximal rate of lactate formation it does not necessarily mean that

21

22 J A N KARLSSON, BODIL H U L T ~ N AND BERTIL SJODIN

TABLE I. Highest observed muscle tissue lactate concentrations in healthy or diseased muscle of man. ~ ~~~~

Conditions when the samples Muscle lactate concentration Reference were obtained mmol x kg-1 wet muscle

Untrained subjects, short time maximal exercise 16.9 Trained subjects, short time maximal exercise 22.7 Exhaustive isometric contractions 21.8 Cardiogenic shock 34.1 Maximal exercise in patients with Anorexia nervosa 31.5 Maximal exercise in patients with tetralogy of Fallot 30.5

Karlsson 197 1

Karlsson 1971

Karlsson and Ollander 1972 Karlsson et al. 1972

ThorCn, personal comniunication

Eriksson: personal communication

an unlimited amount of lactate will be formed. On the contrary the highest values observed for lactate accumulation in skeletal muscle of man during muscular exer- cise (dynamic or isometric) or in different diseases (Table I) are in close agreement and might indicate a significant product inhibition at a level corresponding to 20- 30 mmol x kg.'.

The aim of the present study was therefore to reinvestigate and to extend the knowledge about the kinetics of the enzyme LDH and to further define the proper- ties of this enzyme in human skeletal muscle.

Methods and material Needle biopsy material (Bergstrom 1962) were obtained from subjects, who from previous studies were known to possess about equal distribution of fast twitch and slow twitch muscle fibres (for further information, see Gollnick et al. 1972) in their examined muscle (vastus lateralis). The biopsy material was stored frozen (below -80' C ) until further analyzed. After thawing and sonification in 200 p l of 0.1 M Tris, pH 7.5 the homogenate was centri- fuged and exempted on fibre debris. The supernatant was then further diluted with 0.1 Tris buffer pH 7.5, to a final volume giving a dilution of 1 : 5000 [wwlv). This dilution was used over a period of 2-3 days for different studies of LDH kinetics. Every day VmaX of the homogenate was reassayed in both directions to ascertain any activity loss of the tissue homo- genate that could endanger the interpretation of the results.

LHD activity was assayed in both directions, i.e. reduction of pyruvate (forward reaction) and oxidation of lactate (backward reaction) with a methodological error of less than 2 %. The basic medias were 0.02 M Imidazol buffer pH 7.5, 1 x 10.3 M pyruvate, and 1 x 10.0 M NADH for the forward reaction and 0.1 M Tris buffer pH 8.2, 2 x 10.2 M lactate and 1 x 10.3 M NAD for the backward reaction. The reactions were run on Farrand photo- electric fluorometer as earlier described (Karlsson, Diamant, and Saltin 1968). Different pH levels were obtained with addition of either HCI or NaOH. Varying concentrations of sub- strates as well as reaction products of the test medias were obtained by addition of the above mentioned chemicals. They were taken from the same batches from which the basic test medias were made up. The chemicals used were of highest purity and obtained either from Sigma Chemical Go., or Boehringer, Mannheim GmbH. The pH of the basic test medium for either reaction direction as well as concentrations of substrates were chosen to give a high rate of reaction (Vmax). The similarly obtained LDH activity was set to 100 70. Results obtained

LDH ACTIVITY IN SKELETAL MUSCLE 23

m

I/

PYRUVATE + LACTATE LACTATE -+ PYRUVATE

v6 a - 4 0.3 0

6 PYRUVATE. M LACTATE, M

LACTATE, M PVRUVAlE. M

Fig. 1. Effect of different concentrations of pyruvate and lactate as substrates or products on the LDH reaction in the forward (pyruvate reduced to lactate) or the backward reaction (lactate oxidized to pyruvate) . The shaded areas indicate the normal concentration ranges for these metabolites in human skeletal muscle (Karlsson 1971). Each point represents the mean of not less than 3 separate analysis.

with test medias made up with different pH, concentrations of substrates etc. will be expressed in percent of V,,,. Incubation and fluorometer readings were performed either at room temperature (21" C) or at 37" C, and interpretations of nonlinear relationships were made as previously described (Karlsson et al. 1968).

Results The V,,, data obtained for LDH activity, ranged 0.6-1.4~ and 0.3-1.1 x

mol x g-* x rnin-' in the forward and backward reactions, respectively, and were consequently in agreement with previous data (Karlsson et al. 1968). In respect to

NAD, M NADH,M

a b

Fig. 2. The effects of different concentrations of NADH and NAD on the activity of the forward and backward reactions (see Fig. 1). Each curve is obtained from 6 or more means (see Fig. 1 ) .

24 JAN KARLSSON, BODIL HULT~N AND BERTIL SJODIN

PYRUVATE - LACTATE LACTATE II) PY RUVATE

- . 1.0 mM Py .____-.. 0.1 -*I-

............ Qoo( - II -

--- .................... ................ ......... ......................

-. 20 mM L. .--- -. 1.0 - "- ............. 0.1 - * I -

10

26 .......... ' ........ ......

0 - .......

t r M 70 M M M 7.0 M I

i " * Y 0 -.o mMLa c 3 w 0

_-----. 1.0 - I*- --. o mM Py -20 - ' I - ____- . a1 - ,. -

* ,.o - * I - ...........

........... ; 75

so

25

'..."I..........

Po 8n 10 M M M 70 M

Fig. 3. The effect of pH variations on the forward and backward reactions (see Fig. 1 ) with different levels of substrates and products. For further information see Fig. 2.

pyruvate and lactate, K, for the forward and backward reactions were 0 . 8 ~ lo-* M and 0.9 x M (Fig. 1) , respectively. The corresponding values for NADH and NAD could not be given as the activity increased curvilinearly with increases in NAD and NADH concentrations (Fig. 2 a and b) .

With increasing concentrations of products in the forward as well as the back- ward reactions a significant inhibition of LDH activity as compared to V,,,:,, was obtained at lower concentrations of lactate and pyruvate than observed under physiological conditions (Karlsson 1971 ) (Fig. 1) . An inhibition corresponding to 50 % of V,,,,, were obtained at lactate and pyruvate concentrations equal to 3~

M and 1 x loo4 M, respectively. With lactate and pyruvate concentrations approximately equal to maximal concentrations observed in human skeletal muscle the inhibitions were equal to or more than 70 % of VmtLx.

Different pH levels of the test medias gave different results if the reaction was performed in the forward or in the backward direction. Moreover, for the forward reaction a peak activity was obtained within the pH limits studied (pH 9.0-6.0) and this peak was shifted to a higher pH with increasing concentrations of pyruvate (Fig. 3 ) . For a pyruvate concentration corresponding to 1 x M the peak was

L D H ACTIVITY IN SKELETAL MUSCLE 25

found at a physiological pH level. Increased lactate concentration in the medium (i.e. product inhibition) shifted the peak further to the basic side. Moreover, at a pH level corresponding to 7.0 and 20 mM of lactate the product inhibition present- ed above was further augmented. For the backward reaction the activity seemed to increase the higher the pH. Different levels of substrates (lactate), or products (pyruvate) did not change this general pattern although the curves were on a lower level (Fig. 3 ) .

The above presented results were obtained a t room temperature. To ascertain that no changes were related to a difference in temperature between the in vitro experiments (21O C) and the expected temperature in situ in the muscle additional readings were made for product inhibition at 37O C. Practically identical results were obtained for the inhibitory effect of the products in the forward as well as the backward direction indicating that the temperature difference had no other effects than those expected from the increase in temperature per se.

Discussion The major findings from the present study are that in an in vitro system, both the reduction of pyruvate (the forward reaction) and the oxidation of lactate (the backward reaction) are operating at the optimal part of the affinity curve as inter- preted from the K,, values obtained. Moreover, at low concentrations of lactate as well as of pyruvate a significant product inhibition were present. Although these results are derived from in vitro experiments and crude muscle biopsy homogenates it seems reasonable to assume that the results might be extrapolated to the in situ system and the living cell.

I t is well documented that human skeletal muscle contains all the 5 LDH isozymes in a pattern which is proportional to the percent slow twitch fibres of the muscle (Karlsson et al. 1974). Thus in a muscle with a high percent slow twitch fibres the major portion of the total LDH activity is attributable to the more heart specific LDH isozymes, whereas in a muscle with a low percent of slow twitch fibres it is vise versa. Moreover, with an increasing number of slow twitch fibres the total LDH activity in both directions is decreased.

As these different LDH isozymes possess different affinities for substrates (or dem- onstrate different substrate inhibition) (for ref. see Sund 1968), the present re- sults have to be interpreted with the above mentioned knowledge as a background. I t might be argued that the present data are obscured by these facts. Although this to a certain extent is true, it cannot be denied that the present data will exhibit a general physiological pattern which might be modified for the individual muscle in respect to the actual muscle fibre composition.

References BERGSTROM, J., Muscle electrolytes in man. Scand. I. clin. Lab. Invest . 1962. Suppl. 68. KARLSSON, J., B. DIAMANT, and B. SALTIN, Lactate dehydrogenase activity in muscle after

prolonged severe exercise in man. /. appl. Physiol. 1968. 25. 88-91.

26 JAN KARLSSON, BODIL HULTBN AND BERTIL SJODIN

KARLSSON. I.. Lactate and ohomhaen concentrations in working muscle of man. Acta bhvsio2. . . - I . . stand. isii. Suppl. 358.

KARLSSON. I.. and B. OLLANDER. Muscle metabolites with exhaustive static exercise of differ- ent duraGon. Acta physiol. scand. 1972. 86. 309-314.

KARLSSON, J., J. T. WILLERSON, S. J. LESHIN, C. B. M U L L I N ~ S and J. H. MITCHELL, Skeletal muscle metabolites in patients in cardiogenic shock or congestive heart failure. Circulation 1972. 46. Abstr. 420.

KARLSSON, J., K. FRITH, B. SJBDIN, P.D. GOLLNICK, and B. SALTIN. Distribution of LDH isozymes in human skeletal muscle. Scand. J . clin. Lab. Inuest. 1974. In press.

SUND, H., The pyridine nucleotide coenzymes. In Biological Oxidations. 1968. Interscience Publ. New York. 603-639.