The relative rates of adenosinetriphosphatase and phosphatidic acid synthesis in microsomes from rat brain

211 THE

RELATIVE

RATES

PHOSPHATIDIC

OF ADENOSINETRIPHOSPHATASE ACID FROM

SYNTHESIS RAT

AND

IN MICROSOMES

BRAIN

J. JhiRNEFELT Wenner-Gren

Institute,

University Received

of Stockholm,

July 6,

Stockholm,

Sweden

1961

Two reactions catalyzed by isolated microsomes from nervous tissue have been postulated to reflect a mechanism for the active transport of sodium ions through the cell membrane. The adenosine triphosphatase of microsomes is stimulated by sodium ions [3-6, g-111 in a manner strongly suggesting a close relationship to an active transport mechanism. On the other hand, the existence of a so-called “phosphatidic acid cycle” as a mechanism for active transport has also been postulated [l, 21, and it has been shown that this cyclic mechanism occurs in isolated microsomes from brain [a]. The phosphatidic acid cycle involves the cyclic synthesis and breakdown of phosphatidic acid from diglyceride and ATP, the overall reaction being that of ATP hydrolysis to ADP and inorganic phosphate. It would thus manifest itself as an ATPase, and it is therefore of some interest to compare this reaction with the ordinary ATP-ase. Since the incorporation of phosphate in the phosphatidic acid occurs best in the presence of deoxycholate (DOC) [a], the two reactions were compared in the presence of 0.04 per cent DOC. Parallel experiments on the microsomal ATP-ase have shown, that although this concentration of DOC inhibits the reaction to some extent, the proportional stimulation of the ATP-ase by Na ions is even higher than in the absence of DOC. It was therefore felt that a comparison between the rates of the ATPase and the phosphatidic acid synthesis would be meaningful from the standpoint of assessing their relative importance and possible involvement in active Na+ transport. Table I contains data on two experiments, where the rates of the ATP-ase and the phosphatidic acid synthesis in one and the same assay are compared. The rate of the ATP-ase was determined from the increase of inorganic phosphate in the trichloroacetic acid supernatant according to Lindberg -and Ernster [7]. As the rate of phosphatidic acid synthesis was taken the rate of phosphate incorporation in the chloroform-ethanol extract of the washed trichloroacetic acid precipitate. Quite clearly, the rate of ATP-ase is very much, at least several hundredfold, higher than the rate of phosphatidic acid synthesis. Even taking the data giving the most favourable ratio, namely the difference between the 120 set and 6 set incubations, gives an ATP-ase activity of about one hundredfold the synthesis of phosphatidic acid. In order to exclude the possibility that a rapid breakdown of phosphatidic acid would cause the low incorporation rate, the release of incorporated phosphate from the microsomes was followed. No significant release was observed under several different conditions (see Table II), and it is concluded that the incorporation rates given in Table I depict the true reaction rate. Experimental

Cell Research

25

J. Jiirnefelt

212

TABLE

The rate of the A TP-ase

I.

and the phosphatidic mzcrosomes.

acid synthesis

in rat brain

The assay mixture contained about 2 mg microsomal protein, 20 mAl Tris-chloride, pH 7.4. 2 mM MgCl,, 0.04 :$ (N 1mM) DOC, 0.1 M sucrose and an ATS2P-generating system consisting of 4 mM carbamyl-32P, 0.5 mM ADP and an excess of purified carbamyl kinase [8] in a total volume of 0.5 ml. The samples were incubated for the periods indicated at 3O’C. The reaction was stopped by adding 0.5 ml 20 y0 TC4. The values given rcprcsent means of two closely agreeing duplicates. ATP Esp.

no.

Incubation seconds

1

2

Total j0noles

mp moles/ min

Counts: 10 min

m~moles/ min 2.3 1.09 0.68 0.85 0.80

6

0.35

3500

0.48

240

184 420 1041

0.28 0.40

280 ‘400

943 888

assay

TABLE

32P incorporation

30 120 60 6Oa

a In this

The this

time

hydrolysis

Na+,

0.1 A4 and

II. Release

of

Ii’-,

5 mM,

incorporated

were

present.

32P from brain

microsomes.

microsomes were incubated for 3 min in a mixture similar to that described in Table I. After period the mixture was diluted with 3 ml of a solution containing (A) 20 mM Tris-chloride, pH 7.4, 2 mM MgCl,, 0.1 iVf SaCl, 5 m&Z KC1 and (B) the same mL0.04 “: DOC. Counts/IO Incorporation A, 6 see A, 60 see B, 6 set B, 60 see

TABLE

III.

during

Stimulation

of

3 nun

A TP-ase

pre-incubation

min

1021 1180 1178 1120 1091

by Na ions in presence

of

deoxycholate.

The assay mixture contained 20 mM Tris-chloride buffer, pH 7.4, 4 mM MgCl,, 5 mM Tris-ATP, 0.04 76 DOC (tris-salt) 50 mM sucrose and microsomes (about 0.1 mg protein). Other additions as indicated. The incubation time was 10 min, at 3O’C. Phosphate was determined according to Lindberg and Ernster [7]. pmoles

Additions KCl, 5 mAl KaCl, 0.1 iU NaCl, 0.1 A1, KCl, Experimental

Cell Reseurch

25

5 m.All

Pi liberated 0.28 0.25 0.30 0.55

ATPase

and phosphatidic

213

acid cycle

Under the conditions used, the phosphatidic acid cycle can thus not account for a major portion of the microsomal ATP-ase. That the ATP-ase under these conditions still fully exhibits the property of being stimulated by Na+ in presence of K+ is partly shown in exp. 2 in Table I and also in Table III. Thus we have, in the same experiment, two systems in operation, both of which have been claimed to be connected with the active transport of sodium ions, and which under the particular conditions used exhibit the properties which have been used as evidence in favor of their involvement in sodium transport. The ATP-ase is at least one hundredfold as active as the phosphatidic acid synthesis. At present, when no direct evidence exists on the mechanism of active sodium transport, it would seem reasonable to give the phenomenon manifested in the ATP-ase a preference over the phosphatidic acid cycle. Such a conclusion must, of course, be tested with experiments that could directly measure the rates of the two reactions in relation to actual sodium transport. Summary.-It is shown that in isolated rat brain microsomes the rate of the ATPase is at least one hundred times the rate of phosphatidic acid synthesis. The relative importance of the two reactions as possible manifestations of active sodium transport is discussed. The experiments reported here will be discussed in more detail elsewhere. REFERENCES 1. HOKIS, L. E. and

HOKIS.

AI.

R., .I. (&II. I’hysiol. 44, 61 (1960).

2. -J. Bid. Chem. 234, 1381 (1959). 3. JXRSEFELT, .J., Expfl. Cell Research 21, 4. -Biochim. et Biophys. Acfn 48, 104 5. ibid. 48, 111 (1961).

214 (1960). (1961).

6. ~ Suture 190, 694 (1961). 7. lA~~~~~~~;, 0. and ERSSTER, L., in D. GLICK, Etl., Methods of Biochemical Analysis, Vol. 3, p. 1. Interscience Publ., skew York, 1956. 8. MOKRASCH, L. C., CAR.~V~CA, .J. and GRISOLIA, S., Biochim. et Biophys. Acfa 37, 442 (1960). 9. POST, R. L., MERRITT, C. R.. KISSOLVI~G, C. R. and ALBRIGHT, C. D., -7. Bid. Chem. 235, 1796 (1960). 10. SKOU, .J. C., Biochim. ef Hiophys. .4cfa 23, 394 (19.57). 11. ~-

ibid.

THE

42, 6 (1960).

OCCURRENCE

FUNCTION

IN THE

OF DENSE GRANULES NUCLEOLI

OF UNKNOWN

OF CERTAIN

PLANT

CELLS

C. N. SUN Washington

University,

St. Louis,

Missouri,

U.S.A.

Received July 19, 1961

D

ENSE spherical or irregularly shaped granules have been demonstrated in the nucleoli of the protozoa Amoeba proteus [2] and in these root meristematic cells of AIlium cepa and Vicia fuba [3, 41. But such granules are not generally present in other species and the significance of these exceptionally high electron density particles

Experimenfnl

Cell

Research

25