Incorporation of 33P in soybean phosphatides

SHORT

nn*

COMMUNICATIONS

53253

Incorporation Recent

of 33P in soybean studies

phosphatides

in this laboratoryl+

showed that the lipids of immature

beans are a heterogenous mixture of triglycerides, the early stages of development, the lipid consists

soy-

glycolipids and phospholipids. In of larger proportions of the latter

two components; as the beans mature, the triglycerides become the major component. A number of new cerebrosides and phospholipids were isolated from immature soybeans in these studies3. Identified among these compounds by chromatographic properties was N-acyl phosphatidyl ethanolamine4$5 which has recently been reported in soybean

lecithin6

and germinating

peas7. As a prelude to further

studies

on the

structures and metabolism of these compounds, several experiments were carried out on the incorporation of phosphorous (““P) and acetate (14C) into the polar lipids of immature soybeans. The isotopically labeled 33P as Na,H33P0, or sodium [I-%]acetate (obtained from New England Nuclear Corp.) were injected directly into the immature seed of Chippewa 64 soybeans (approx. 30 days after flowering). The plants were brought from the field about z h previous to injection. The beans were then harvested at selected (2

: I,

v/v,

washing Packard

intervals (up to 24 h). The lipid was extracted with chloroform-methanol twice and then I : 2, v/v, once). The non-lipid impurities were removed by the extract with 0.76 ‘$6 aq. NaCl. Aliquots of extract were counted in a Tri-Carb liquid scintillation spectrometer using a toluene scintillation solu-

tions. After separation of the lipids by two-dimensional thin-layer chromatography, the spots were made visible by exposing the plates to I, vapor. The compounds were marked and after evaporation

of I, under a stream of nitrogen,

scraped and the radio-

Fig, I, Two-dimensional thin-layer chromatography of polar lipids of immature soybean. a. Visualized by charring after spraying with K,Cr,O,-H,SO, b. Autoradiogram of 14C-labeled lipids. c. Autoradiogram of 33P-labeled lipids. Thin-layer chromatography was done on layers of silica gel H, 250 p thick and activated at IZOO for z h. The chromatograms were developed in the vertical ‘$A (w/w) aq. ammonia (65:x5:5, by vol.) followed by direction with chloroform-methanol-28 drying for IO min under nitrogen and development in the second dimension (right to left) with chloroform-acetoneemethanol-acetic acid-water (IOO:40:zo :zo :IO, by vol.), i\bbreviations : ; DGDG, digalactosyl diglyceride ; SL, sulfolipid ; PSG, phytosterol glycoside ; Ce, cerebrosides; NL, neutral lipids; PA, phosphatidic acid; PI, phosphatidyl inositol; PS, phosphatidyl serine; PC, phosphatidyl choline ; PE, phosphatidyl ethanolamine; PG, phosphatidyl glycerol ; APE, Nacyl phosphatidyl ethanolamine; X, uncharacterized compound : 0, origin. Biochim.

Biophys.

Acta,

202 (1970) zoo-z02

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SHORT COMMUNICATIONS

activity counted in dioxane solution *. Some plates were exposed to X-ray film for autoradiography of labeled lipidss. The detection of lipids was also carried out by charring (K~Cr~O~-H~SO~)@, phospho~pids by spraying with phosphomoIybdate spray (ref. IO), and compounds with free amino groups by spraying with ethanolic ninhydrin solution. Identifications were made by comparison of chromatographic properties with authentic samples. Two-dimensional thin-layer chromatograms of immature soybean lipids after charring and autoradiography are shown in Fig. I. The distribution of 33P activity among various phosphatides and the changes that take place with time are given in Table I. TABLE

I

DISTRIBUTION

OF

33P

ACTIVITY

AMONG

LIPID

COMPONENTS

OF IMMATURE

SOYBEAK

AFTER

INJECTING

Na,H=PO 4 Radioactivity

remaining at the origin and minor compounds

Lipid comporaent

Xl Phosphatidylinositol Phosphatidylse~ne Phosphatidic acid Phosphatidylcholine Phosphatidylethanolamine Phosphatidyl glycerol N-Acyl phosphatidylethanolamine ---___-.-

(
_I-.

3SP activity (“ib of total cou~ts~~~n)

--____ Time of harv~s&ng the seeds aftftevthe administration of the isotofie: ___.-._. ~_ 2h “4 h 15 min 30 min I h 12 h qh -~8.2 2.4 1.0 I.2 I.2 8.9 2.4 21.0 26.2 12.3 18.4 26.5 22.7 23.3 0.6 0.9 2.3 0.5 2.7 I.4 0.3 58.2 8.1 IO.9 5.9 33.4 5.3 45.5 12.6 13.6 14.6 9.8 5.8 3.0 3.4 12.9 18.6 12.1 10.3 4.7 5.7 7.4 6.1 6.5 5.0 6.1 3.1 5.2 3.9 22.0 26.2 7.0 IO.5 30.3 35.8 42.4 -__.____

The results in Table I show that phosphatidic acid is labeled rapidly. The 33P activity in phosphatidic acid in the first 15 min accounted for 58.2% of the total 33P incorporated in lipid phosphorus and this decreased progressively with time to a value of about 5%. At the same time the proportion of 8sP in N-acyl phosphatidyl ethanolamine increased rapidly over the first 4 h, then gradually, reaching a value of about 42% at 24 h. The proportion of 33P in phosphatidyl inositol and phosphatidyl ethanolamine also increased over the first 4 h reaching a maximum and then decreasing to approx. 23% and 12%, respectively. In contrast, the proportion of s3P in phosphatidy1 choline increased slowly and reached a plateau at approx. 2 h. The amount of phosphatidyl glycerol in total lipids is very low but it has a relatively high specific activity as evident by comparison of Figs. I a and I c. 33P (2%) is also incorporated into other minor uncharacterized phosphatides. Acetate (W) is incorporated into all the major phospholipids. PhosphatidyI glycerol had particularly high specific activity as is evident from comparison of Figs. ~a and I b. On the other hand, the major glycolipids, e.g. digalactosyl diglyceride, phytosterol glycoside etc., did not incorporate the label from acetate during the 24 h of the experiment (Fig, I b). These results indicated that phospholipids turn over much more rapidly than the glycolipids and that the pathways for biosynthesis of these compounds are independent of each other. Recent reports on the wide distribution of N-acyl phosphatidyl ethanolamine in seed lipids and its rapid metabolism in germinating peas7 suggest it might be involved in the metabolism of seed lipids. This compound is highly labeled with both Biochim. Biophys.

Acta,

202

(xg?a)

200-202

202

SHORT COMMUNICATIONS

33P and 14Cin the above experiments with immature soybeans. This, together with the fact that it is present in relatively large amounts in the lipid of immature bean and decreases in concentration as the bean matures3 indicate that it is involved in lipid metabolism associated with maturation. This investigation was supported in part by U.S. Public Health Service Research Grant HE 05735 and U.S. Public Health Service Program Project Grant HE 08214. The excellent technical assistance of Mark Stadtherr is appreciated.

I 0.

S. PRIVETT,

for publication.

‘2 3

J. N. RoEah% H. SINGES AXD

R. A. GROSS,K. BEUTELAND H. SINGH,/. Apz. Oil Chrmnisls’ Sec., submitted PRIVETT,Lzpids, in the press. 0. S. PRI~ETT,paper presented at jg~d Annual Meeting Am. Oil

AND 0. S.

Chemists’ Sot., ~~/~~.,~z~e~~~~~s, Minn., ~969, Abstr. No. 143, Lipids, submitted for publication. 4 R. ROMSTEIN, Biochem. Bzophys. Res. Commztn., 21 (1965) 49. 5 VOX I-l. DEUUCHAXDG. WENDT,2. Physiol. Chum., 348 (1967) 471. 6 K. ANEJA, J. S. C~antr.4AND J. -4. KNAGGS,Biochem. Biophys. Res. Cornmu?%, 36 (1969) 401. 7 R. M. C. DAWSON, N. CLARKE AND R. H. QUARLES, Biochem. J., 111 (1969) 1860. 8 I;. SNYDER. AnaE. Biochrm., 9 (1964) IS~. 9 M. L. BLANK, J. A. SCKMIDT AND 0. S. IJRIVETT, J. dm. Oil Ck~rni~i~ts’ Sot., .ir (1964) 371. IO V. E. YASKO~SKY ASI) E. Y. KOSTETSKY, J. Lipid Res., 9 (rg68) 396.

Received

October z7th, 1969

Biochim. Biophys. .4&a, 202 (1970) 200-202

BBA 53255

Incorporation

of phosphatide precursors from serum into erythrocytes

Previous studies by HAHN AND HEVESAY~ and others as reviewed and extended by REEDS have shown that some renewal of erythrocyte membrane phosphatides occurs by means of passive equilibration of membrane phosphatides with preformed serum phosphatides. Further studies by OLIVEIRA AND VAUGHAN~ and later TARLOVZ have shown that another pathway for phosphatide renewal occurs via the acylation of lysophosphatides with free fatty acids. Since both lysophosphatides and free fatty acids are normally carried on serum albumin, it is possible for the erythrocyte to assemble phosphatides from these two building blocks found in normal serum. TARLOV”, and SHOHET et al.= showed by independent approaches that phosphatide renewal by these two routes was approximately equal. Studies by MULDER et al.@ showed that the transfer of fatty acid from one lysophosphatide molecule to another to produce I molecule of phosphatide and I molecule of glycerophosphatidyl choline’ could alsooccurinred blood cell homogenates. However, at normal pH, this reaction was of colnparatively little importance when compared with the direct acylation pathway. In a search for any other potential sources of phosphatide renewal in the intact erythrocyte and in an effort to establish the relative incorporation rates of lipid precursors we have incubated erythrocytes with several of these precursors and with preformed phosphatides. These compounds were radioactivity labeled as indicated Biociaim. Biophys.

Acta, 202 (Ig7o)

ZOZ-205