transaminase activity in extracts of cod muscle studied by the use of a 14c-labelled amino acid...

J . Sci. Fd Agric. 1974,25, 11-20

Transaminase Activity in Extracts of Cod Muscle Studied by the Use of a 14C-Labelled Amino Acid Mixture"

John Murray and James R. Burt

Torry Research Station, Ministry o j Agriculture, Fisheries and Food Aberdeen AB9 8DG, Scotland (Manuscript received 21 May 1973 and accepted 12 September 1973)

Changes in the amino acid fraction of buffered extracts of cod muscle during incubation in the presence of a-ketoglutarate have been studied using a 14C-labelled amino acid mixture. The patterns of radioactive amino acids pre and post incubation suggest that transamination reactions are responsible for marked changes in the levels of aspartic acid, glutamic acid, alanine, valine, leucine and iso-leucine. No changes were noted in the levels of threonine, serine, proline, glycine, lysine and arginine. Slight falls in the concentration of tyrosine and phenylalanine were seen after extended incubation times.

1. Introduction

Carbonyl compounds are known to be present in the volatile flavour fractions of many products including such diverse food-stuffs as cheese' and various fruits2 as well as flesh foods such as meat3 and Their importance to overall flavours has been stressed frequently and present work in this laboratory includes studies designed to identify the volatile flavorous constituents of cod flesh, their precursors and the mechan- isms of their production. In view of the highly unsaturated character of the fatty acids of fish lipids, oxidation of these might be expected to contribute largely to the carbonyls which are produced but it was also considered possible that a variety of a-keto acids could be produced from a general transaminase reaction of the type

R.CO.COOH + R'.CHNH2.COOH + R.CHNH2.COOH + R'.CO.COOH using the spectrum of free amino acids known to be present in cod muscle.6 Some of these keto acids could be volatile and in addition some might be decarboxylated and contribute a variety of aldehydes to the carbonyl pool.

Transaminase activities have been demonstrated previously in the muscle of cod' and in other species of fish8 using methods which relied on the demonstration of net glutamate synthesis by paper chromatographic, by thin-layer chromatographic or by enzymic techniques. Consideration of the ease with which measurement of radioactivity, as opposed to most other analytical methods, can be used to measure concentration suggested that the use of high specific activity amino acids would allow a marked improvement in the sensitivity with which potential transamination reactions could be

11 Crown Copyright reserved.

12 J. Murray and J. R. Burt

assayed. This factor is obviously important when one considers the extremely small concentrations of certain constituents which can exert a marked effect on the senses of taste and smell. Studies using labelled amino acids or aKG" have been previously reported9-" but suffer from the disadvantage that activity towards single substrates only was assayed by such systems.

The study reported here was initiated by the observation that addition of crKG and l4C-label1ed amino acids to an extract of cod muscle resulted in the conversion of approximately 20:4 of the label to volatile material capable of being distilled under reduced pressure.

2. Methods 2.1. Fish Cod were caught by trawl in the North Sea and were held live in an aquarium until required.

2.2. Preparation of extracts Fish were killed by a blow on the head and gutted immediately. After filleting and removal of the skin a 10-g portion of muscle was homogenised with 40 ml of chilled 0.02~-potassium phosphate buffer, pH 7.6. Toluene, 0.5 ml, was added before honiogen- ising in order to inhibit bacterial growth. In certain cases other portions of the same fillet were frozen in liquid nitrogen and held at -30 "C until used. The Iiomogenate was centrifuged at 3000 g for 10 min and the almost clear supernatant solution decanted for use.

2.3. Determination of GPT and GOT activities Measurement of these activities was made by the use of commercial test kits (Boehringer Corporation, Uxbridge Road, London W5) by following the rate of fall of optical density at 340 nm (Bergmeyer and Bernt).12

2.4. Conversion of 14C-labelled amino acid activity to volatile products A solution of amino acids, code CFB 104, uniformly labelled with 14C was obtained from the Radiochemical Centre, Amersham. It had the following composition by activity: L-alanine 10 x, L-arginine monohydrochloride 6.5 x, L-aspartic acid 9.0 %, L-glutamic acid 12.5 %, glycine 5.0 %, L-leucine 12.0 %, L-iso-leucine 5.0 %, L-lysine 5.5 %, L-phenylalanine 7.0 %, mo pro line 6.0 :<, L-serine 5.0 %, L-threonine 6.0 %, L-tyrosine 3.5 %, L-valine 7.0 %. The radioactive concentration was 63 pCi/ml and the specific activity 52 mCi/m atom carbon. The solution was supplied in sterile multidose vials.

To one limb of an H-tube fitted with B24 sockets the following reagents were added: 0.2 ml of 0.25 M-neutrahed xKG solution, pH 7.0, 0.1 nil of PP solution containing 350 pg of PP/ml, 0.5 ml of active amino acid solution prepared by diluting 0.15 ml original to 5.0 ml with water (i.e. approximately I pCi activity) and 2.0 in1 of fish

Abbreviations used are: UKG, cc-ketoglutarate; GPT, glutamate-pyruvate transarninase; GOT, glutarnate-oxalacetate transaminase; NAD, nicotinamide adenine dinucleotide; NADH2, reduced NAD; PP, pyridoxal-5-phosphate; PPO, 2,5-diphenylouazole; POPOP, 1,4 bis-2-(5-phenyl-oxazolyl)- benzene.

Transaminase activity in cod muscle 13

extract. After incubation at 23 "C for various times 0.5 ml of 3.6 N-sulphuric acid was added to terminate the reaction. Similar assays were done in the absence of clKG and PP by replacing these with 0.3 ml of water and blank values were obtained from assays in which the sulphuric acid was added before the fish extract. Volatile products were distilled according to the method of Keay and M~Gi l1 . l~ The volume of the distillate was made to 10 ml with water and aliquot portions, usually 1 ml, taken for liquid scintillation counting with 10 ml of toluene/triton XlOO (2/1, v/v) containing 0.6% PPO and 0.006 % POPOP.14,'s All counting was done with a Nuclear Chicago Unilux 1 model using the channels ratio method for determining efficiency.I6

In order to check potential transaminase activity in spoiling cod muscle, assays identical to those outlined above were made using extracts from gutted cod which had been held for 7 and 14 days in ice.

2.5. Ion-exchange chromatography of incubates The following reagents were added to a series of test tubes: 0.1 ml of 0.5 M - ~ K G , pH 7.0; 0.1 ml of PP solution containing 50 pg of PP; 0.2 ml of water; 0.5 ml of diluted (1.45 pCi/ml) active amino acid solution and 2.0 ml of buffered cod muscle extract. A further series of tubes was prepared in which the water was replaced by 0.2 ml of an inactive amino acid solution. This solution consisted of a mixture of all 14 ainino acids present in the active solution at a concentration of 0.5 pmol/ml, except for threonine and isoleucine which were at 0.25 pmol/ml and tyrosine at 0.125 ,umol/ml.

After incubation at 23 "C the reaction was stopped by addition of 0.5 ml 3 N-

perchloric acid. The inactive amino acid solution (0.5 ml) was then added to the carrier- free incubates and the mixture filtered after centrifuging at 3000 g in order to remove precipitated protein. To those tubes which already contained 0.2 ml of inactive amino acid solution before incubation a further 0.3 ml only was added followed by 0.2 ml of water before addition of acid. Blanks were prepared by adding perchloric acid before fish extract and carrier solution. A portion (2.5 ml) of each filtrate was adjusted to pH 7.6 by the addition of 0.5 ml of 2.2 N-potassium hydroxide and the precipitated potassium perchlorate was spun off at I "C.

A preliminary indication of the extent of conversion of radioactivity to non-cationic compounds, both volatile and non-volatile, was obtained by running 2.0-ml portions of the deproteinised, neutralised incubates through Zeocarb 225H' columns (1 8 cm x 1.4 cm). After eluting the column with 50 ml of water, the radioactivity in the pooled effluent was determined.

In order to determine which of the individual amino acids of the mixture had been converted, a similar series of incubates was prepared as outlined above and, in this instance, the 2-ml portions of the deproteinised, neutralised solutions were frozen and lyophilised over P,O,. Citrate buffer (1 .O nil, pH 2.2) was added to the dry residue and a 0.3-ml aliquot used for the separation of individual amino acids using an automatic amino acid analyser (The Locarte Company, 24 Emperor's Gate, London SW7).

Simultaneous measurement of radioactivity and amino acid concentration of eluted peaks was obtained by inserting an anthracene-packed flow cell (NE 806; Nuclear Enterprises, Sighthill, Edinburgh) into the flow stream at the end of the ion exchange column, the flow cell being coupled to the face of a photomultiplier tube (EM1 6097 C)


held within a lead castle. The effluent from the cell was passed back to the analyser to be processed with ninhydrin. High voltage to the photomultiplier was provided by a GSA 2 module and the output from this was passed to a ratemeter (RTM 5) (Panax Equipment Ltd, Redhill, Surrey). The output from the ratemeter was taken to a 10-ohm wire-wound resistor and the voltage tapped from this source was fed to the channel of the recorder which normally accepts the output from the 570 nm x 3 photocell.

The analyser had already been modified for routine amino acid analysis so that parallel outputs from all three recorder terminals were passed to a digital voltmeter (Solartron Electronic Group Ltd, Farnborough, Hampshire) and then to a data logging tape punch (type ADD0 X, supplied by the same group). Thus it was possible to record, in digital terms on paper tape at 10-s intervals, a voltage proportional to the activity present in the flow cell and also to have a graphic record of activity displayed on the recorder trace.

To estimate the extent to which individual amino acids had changed the punched paper tape recording the output from the digital voltmeter from each run was processed by an IBM 1 130 computer to give a print-out of 2000 consecutive readings. A comparison of this print-out with the graphic recordings of radioactivity and of ninhydrin reactivity was made in order to determine the start and finish point of each peak of activity and to assign this activity peak to a definite amino acid or to the forerunnings, represented by the activity emerging before aspartic acid. A computer program was written which allowed the average background value to be computed from the period before the emergence of any radioactive peak, the activity values relevant to each peak to be summated and the appropriate blank value to be subtracted. Thus a value which represented the activity within each peak, expressed as a percentage of the total activity, was obtained. Activity peaks of the forerunnings were occasionally outwith the scale of the recorder (see Figure 2), but since the voltage output of the ratemeter was also passed to the digital voltmeter these readings, greater than 5 mV, were faithfully recorded on the tape punch.

In order to reduce background count rates to levels of approximately 30 cts/min the GSA 2 module was operated at a discriminator bias of 10 mV with 800 V supplied to the photomultiplier. Although these settings do not result in maximal E Z / B ratios it was considered preferable to work at low background levels even though this resulted in some loss of detection efficiency. The calculated efficiency of the flow cell at the settings used was 12 "/, with ['4C]carbonate. PiezI7 has shown the efficiency of detection of activity is independent of the eluting buffer used in systems similar to that outlined above.

The sodium citrate buffer systems adopted for elution of amino acids from the column were as follows: pH 3.25, 0.2 M for 45 min, followed by pH 4.25, 0.2 M for 75 min and finally pH 6.65, 1.0 M for 150 min. If the analyser was used in the automatic mode sodium hydroxide (0.2 N) was passed for 30 min after the pH 6.65 buffer and this was followed by the pH 3.25 buffer for 80 min.

3. Results

Since the products of transaminase action on the substrates alanine and aspartic acid, pyruvic and oxalacetic acids, respectively, might be expected to be volatile under the


conditions used here for trapping volatile materials it was obviously of interest to determine the GPT and GOT activities in cod extracts. The activities found in the present study were 2.4 and 21.8 units/g muscle for GPT and GOT, respectively, one unit of activity being defined as that which will convert 1 pmol of substrate per min at 25 "C. These figures are substantially higher than those found in extracts of frozen cod muscle7 but this is to be expected since under the conditions of the present assay the products of transamination were removed when formed by the linked enzyme systems and since NADH, was present in excess.

TABLE 1. Percentage of added 14C-labelled amino acid activity converted to volatile products

Percentage conversion Time in ice < ,

(days) Added aKG 4-h incubation 23-h incubation

7 0 0 + 21 24

1.6 7 7 + 19 23

14 - 0.2 14 + 16 19

-

-

Having confirmed that a potential existed for the transamination of aspartic acid and alanine in what might be regarded as model systems, attempts were then made to determine whether and to what extent these and other amino acids might be converted to volatile products in muscle extracts containing added aKG and PP only from cod which had been held gutted for various times in ice. Table 1 gives the results of these experiments for assays in the presence and absence of added aKG. The figures given in the Table are corrected for the extent of the added radioactivity found to be volatile after incubation in the presence of denatured fish muscle extract. This blank which was approximately 1 % was almost certainly due to radiolytic decomposition of the active amino acid mixture prior to assay. Although the extent of conversion in the absence of added aKG falls from 7 to 0.2% on holding the fish in ice from 0 to 14 days enzyme activity is apparently unimpaired since the conversion figures in the presence of added acceptor acid fall only slightly during this period. It is thus likely that the decline in the conversion rate is due to a fall in concentration of aKG in the fish muscle extracts. Incidentally, similar experiments done with extracts of fresh fish at still higher added aKG concentrations exhibited substrate inhibition ; the conversion rate fell to approximately half when the aKG level was increased fourfold.

Since the complete conversion of alanine and aspartic acid would account for only 19 % of the total activity added, and since volatile components frequently accounted for up to 24 %, it was obvious that substrates other than these two were undergoing change. In order to widen the scope of this investigation and determine the total extent of conversion of radioactivity deproteinised incubates from fresh cod were subjected after neutralisation to cation exchange treatment. After allowance for blanks it was


calculated that 52 % of the original activity was unretained by the column. Thus more than halr the original activity added as amino acids had been converted to non-cationic material after an incubation period of 6 h. Attempts to identify the products of reaction using thin-layer chromatography followed by measurement of activity in the developed plates were not successful but it is planned to repeat this work using radio-gas chromatographic techniques in the near future. It was reasoned however that if the individual amino acids could be separated and their associated radioactivity measured during the separation the extent to which these amino acids had entered into transamination reactions should be immediately obvious. Typical separations obtained using the amino acid analyser from mixtures containing no carrier amino acid, pre and post incubation, are given in Figures 1 and 2, respectively. A fixed time delay of 15 min is apparent between any activity peak and the corresponding ninhydrin peak due to the hold up in the reaction coil of the 100 ‘C water bath. Table 2 lists the values obtained

TABLE 2. Percentage total activity associated with individual amino acids after ion exchange separation

Compound Run1 Run2 R u n 3 Run4

Forerunnings Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Iso-leucine Leucine Tyrosine Phenylalanine Lysine Arginine

1.8 49.1 55.0 51.7 9.2 0.6 1.8 0.5 5.0 5.4 5.6 5.2 4.8 5.2 5.2 4.8

12.3 0.9 0.8 0.8 5.7 6.0 5.9 6.0 4.9 5.0 5.3 4.9

10.1 0.3 0.2 0.3 7.3 3.3 0.4 3.3 4.8 0.4 0.0 0.3

12.0 I .2 0.2 1.5 3.4 3.6 2.8 3.3 7.4 7.3 6.0 6.8 5.5 5.3 5.3 5.3 5.8 5.9 5.7 5.4

from the print-out of the computer after feedingin the appropriate data and programme tapes for each run. Runs 2 and 3 were obtained from 6-h and 23-h incubates, respectively, in the absence of carrier amino acid during incubation and run 4 was obtained from a 6-h incubate when carrier amino acid had been present during incubation. Run 1 represents the blank where enzyme activity was destroyed by the addition of perchloric acid to the assay system before the fish muscle extract. The percentage activities found in run 1 agree closely with those published by the Radiochemical Centre for the amino acid mixture supplied by them and gives an indication of the accuracy of the total process. It is immediately obvious from Table 2 that aspartic acid, glutamic acid, alanine, iso-leucine and leucine have undergone rapid and almost complete conversion, with valine being converted to a slightly lesser extent. Indeed, the only significant feature during the next 17 h of incubation is the further loss of valine. The small falls in the

5-

10

y-

.”

v) t,

20

25

Tim

e (

min

)

Figu

re 1

. T

raci

ng o

f ch

rom

atog

ram

obt

aine

d fr

om th

e am

ino

acid

ana

lyse

r of

an ex

trac

t of

cod

mus

cle

in th

e pr

esen

ce o

f ct

KG

, PP

and

14C

-labe

lled

amin

o ac

id m

ixtu

re a

t zer

o tim

e (r

un 1

see

text

). C

arri

er a

min

o ac

id w

as a

dded

bef

ore

sepa

ratio

n. O

ne p

en o

f th

e re

cord

er w

as u

sed

to d

ispl

ay a

ctiv

ity

(sol

id li

ne).

Rat

emet

er ra

nge

was

30

cts/

s ful

l sca

le d

efle

ctio

n and

a ti

me

cons

tant

of

10 s

was

em

ploy

ed. A

bsor

ptio

n at

570

nm

is s

how

n (d

otte

d lin

e) a

nd

the

abso

rptio

n at

440

nm

is a

lso

give

n fo

r pr

olin

e on

ly (d

ashe

d lin

e).

10

o

e

06

-A

g

o4

15-k

20

2

- 0

1

-

..

....

._

^.I

... ..

..... .

,,.

. ..

. .

..

Tim

e (

min

)

Figu

re 2

. T

raci

ng o

f chr

omat

ogra

m o

btai

ned

from

an

extr

act o

f co

d m

uscl

e in

the

pres

ence

of

GIK

G, PP

and

“G

labe

lled

amin

o ac

id m

ixtu

re a

fter

in

cuba

tion

for 6

h a

t 23

“C (r

un 2

, see

text

). O

ther

det

ails

as

for F

igur

e 1.


percentage activities associated with tyrosine and phenylalanine between 6 and 23 h incubation may indicate a slight degree of transaminase activity towards these particular substrates.

Since the 0.75 pCi of radioactivity added to these incubates represents less than 0.2 pg total carbon it was possible that at these extremely small mass levels atypical reactions could take place leading to false impressions of the extent of transaminase activity. For this reason additional inactive amino acids were added before incubation, total carbon added in this form being 72 pg, nearly 400 times that of the added active carbon. The results, run 4, show that the extent of conversion of individual amino acids is essentially identical to that observed in the absence of added carrier. Subsequent qualitative amino acid analysis of the fish muscle extract alone at the level at which it was added to incubates showed the presence of all 14 amino acids with the possible exception of aspartic acid and lysine the latter being improperly resolved from 1-methyl histidine and anserine in the elution system employed. The amino acid profile found is in agreement with the results of previously published work.6

4. Discussion

Hodgkiss and Jones" and Shewan and JonesIg reported on the changes taking place in the individual constituents of the free amino acid pool in sterile blocks of cod muscle held at 0 "C. Their findings that the levels of alanine and leucine fell continuously over a 25-day period of storage whereas glutamic acid levels increased by over 300% in the same time are entirely explicable in the light of the present results. The fall in lysine levels also noticed by them probably does not result from transaminase activity, since lysine appears to be particularly stable in this respect (Table 2). The results of Siebert, Schmitt and Bottke7 who studied, using extracts of frozen cod muscle, the transamination potential of a wide range of amino acid/keto acid pairs appear to be in general agreement with the present findings except that the extent of conversion of tyrosine and phenylalanine with aKG noted by them seems to be greater than that found here. Whether this difference is due either to the possible inhibition of transaminase action on these specific substrates by other amino acids or to the presence of toluene in the homogenates used here must remain open to question. Patrick20 has shown that rat liver transaminase activities toward specific substrates can be inhibited by the presence of other amino acids. Published findings for transaminase activities in the white muscle of carp" agree with current findings for cod except that activities towards lysine and tyrosine appear to be higher for the former species.

Comparison of the ninhydrin traces in Figures 1 and 2 show a marked drop in the level of alanine and smaller falls in the level of valine, iso-leucine and leucine. These changes tend to be obscured due to the fact that carrier amino acid was added to the reaction mixture after completion of the incubation period and thus, unless an amino acid was present at an appreciable level before incubation, changes in that level will not be sensed.

An increase in concentration of the ninhydrin peak immediately following phenylalanine is observed. This peak is almost certainly p-alanine, whose concentration


increases as a result of the anserinase activity which exists in cod m u ~ c l e . ~ ~ ~ ~ ~ Anserine, lysine and 1-methyl histidine are unresolved by the elution system used but examination of the figures suggest that the first peak of this combination is anserine since its level falls on incubation.

The marked rise in the level of glutamic acid, which is present at only extremely low concentrations in fresh extracts, is to be expected, since the net result of transaminase activity in the presence of aKG must be the production of this amino acid. What is perhaps surprising is the sharp fall in the activity level of this acid simultaneous with the production of relatively large amounts of the inactive acid. The latter effect might be expected to lead at the end of incubation to amounts of labelled glutamic acid greater than those actually found. Glutamic acid has been shown to transaminate with aKGZ4 and glutamic dehydrogenase is reported to be present in cod muscle;7 both these activities might be expected to lead to an early disappearance of the label from active glutamic acid. A comprehensive review of the methods of measurement of transaminase activity has been published by Aspen and MeisterZ5 and in most cases activities have been assayed by addition of the individual amino acid to the reaction system followed by estimation of glutamic acid using a variety of methods. The inhibition of activity towards substrates by other amino acidsZo suggests that it would be advantageous to adopt some system of measurement whereby the system being studied is modified to the least possible degree. This can be achieved by the addition of high specific activity amino acids as a mixture to reaction systems. Subsequent estimation, by measurement of activity, of the extent of transamination of the separated amino acids offers a number of advantages, including that of sensitivity, over previously described methods of assay. The analytical system described here, which is an extension of that originally proposed by Schram and Lombaert,26 is inferior in some respects to that used by Piez17 for the measurement of I4C and 3H in synthetic mixtures of amino acids. However, our method has the advantage that aliquid scintillation spectrometer is not required. Further savings in cost, at small extra effort, can be obtained by manual calculation of the activity peak areas.

Acknowledgments Mr P. F. Howgate wrote the computer programs and also modified the amino acid analyser to give a print out of the voltages present at the recorder terminals.

References 1. Day, E. A.; Libbey, L. M. J. Fd Sci. 1964,29, 583. 2. Nursten, H. E.; Williams, A. A. Chemy Znd. 1967,12,486. 3. Hornstein, I. In Chemistry andf'hyisology of Flavors (Schultz, H. W.; Day, E. A.; Libbey, L. M.,

eds.) Avi Publishing Company Inc., Westport Connecticut. 1967, p. 228. 4. Jones, N. R. In Chemistry and Physiology of Flavors (Schutlz, H. W.; Day, E. A.; Libbey, L. M.,

eds.) Avi Publishing Company Inc., Westport Connecticut. 1967, p. 267. 5. Wong, N. P.; Damico, J. N.; Salwin, H. J. Ass. Off. Analyt. Chem. 1967, 50, 8 . 6. Jones, N. R. Biochem. J . 1954, 58, XLVII. 7. Siebert, G.; Schmitt, A.; Bottke, I. Arch. Fisch Wiss. 1965, 15, 233. 8. Bell, G. R. J. Fish. Res. Bd Can. 1968,25,1247. 9. Gabay, S.; George, H. Analyt. Biochem. 1967, 21, 111.

10. Weinstein, A.; Medes, G.; Litwack, G. Analyt. Biochem. 1967,21, 86.


Wilson, D. G . ; King, K. W. ; Burris, R. H. J. biol. Chem. 1954,208, 863. Bergmeyer, H. U.; Bernt, E. In Methods ofEnzymatic Anuly~is (Bergmeyer, H. U., ed.) Academic Press, New York. 1963, p. 837. Keay, J. N.; McGill, A. S. J. Fd Technol. 1968, 3, 345. Patterson, M. S.; Greene, R. C. Analyf. Chem. 1965, 37, 854. Turner, J. C. Int. J . appl. Radiat. Isotopes 1968, 19, 557. Baillie, L. A. Int. J. uppl. Radiat. Isotope3 1960, 8, 1. Piez, K. A. Analyt. Biochem. 1962,4,444. Hodgkiss, W.; Jones, N. R. Biochem. J . 1955, 61, IV. Shewan, J. M.; Jones, N. R. J. Sci. Fd Agric. 1957, 8, 491. Patrick S. J. Can. J . Biochem. Physiol. 1963,41, 1163. Creach, Y. Archs Sci.physio1. 1967, 21, 443. Jones, N. R. Biochem. J. 1955,60, 81. Trommsdorff, H. 2. Lebensmittelunters. u.-Forsch 1971,147, 133. Nisonoff, A. ; Barnes, F. W. ; Enns, T. ; Von Schuching, S. Bull. Johns Hopkins Hosp. 1954,9411 7. Aspen, A. J.; Meister, A. In Methods of Biochemical Analysis Vol. 6 (Glick, D., ed.) Interscience Publishers Inc. New York. 1958, p. 131. Schram, E.; Lombaert, R. Analyt. Biochem. 1962,3,68.

11. 12.

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

26.

transaminase activity in extracts of cod muscle studied by the use of a 14c-labelled amino acid...

Documents