The Incorporation of Leucine-C 4 into Microsomal Particles

Incorporation of leucine-C ~ into subcellular fractions of the apical section of pea seedlings has been studied as a function of the length of incubation. The specific activity of the microsomes was higher than that of the supernatant for short but not for long incubations, in agreement with observations on other systems. In this developing tissue the nuclei and especially the mitochondria appear to incorporate amino acid very rapidly. An insoluble fraction of the microsome pellet, which is presumably a liponucleoprotein complex, was found to possess, after 1 hour of incubation, a specific activity much greater than that of the purified microsomal particles or the supernatant fraction. Ninety-eight per cent of the leucine-C ~4 in the purified microsomal particles has been shown to possess bound amino groups, presumably in peptide linkages, by the DNP-end group method. These particles liberate but little peptide or protein of very high specific activity when they are destroyed by removal of Mg or by hydrolysis of RNA. Microsomal particles were fractionated into an RNA fraction and five protein fractions by means of density gradient centrifugation. By this method 95 per cent of the RNA can be separated from 90 per cent of the protein of the particle. Furthermore, the RNA fraction has been shown to contain very little protein of high specific activity. A particular protein fraction which contains the remaining 5 per cent of the RNA, possessed after 1 hour of incubation a specific activity 2 to 9 times higher than the protein of the other fractions. Students of protein synthesis have recently focused their at tention on the incorporation of amino acids into microsomal particles (1-3). We have earlier reported on the isolation and characterization of microsomal particles from pea epicotyls (4), and have developed a fractionation procedure for the subcellular components of the apical portion of the growing seedling (5). This procedure emphasizes quanti tat ive separation of microsomal particles from supernatant and mitochondrial fractions as well as the separation of nuclei from the cytoplasmic components. Using this procedure we have now examined the incorporation of leucine into the various subcellular components of this growing plant tissue. * Report of work supported in part by grants No, RG-3977 and No. RG-5143 from the National Institutes of Health, United States Public Health Service. J . BIoPHgsIc. AND ]]IOCHEM. CYTOL., 1959, Vol. 5, No. 1 Kinetic data on the labelling of liver microsomes (1, 6) suggest that about 1 per cent of the microsomal protein is rapidly turning over while the remainder is relatively inert. We have, therefore, looked for a microsomal protein fragment of high specific activity, using the recent information (7, 8) concerning the structure and the subunits of the particle. A procedure has been developed for the fractionation of microsomal material which is based on the density of the various fractions. Materials and Methods Chemicals.--Pancreatic ribonuelease, three times crystallized and salt-free, was obtained from Worthington. Leucine-C 14, uniformly labelled, was obtained from Nuclear-Chicago Corporation, Chicago. Dinitrofluorobenzene (DNFB), CfP, was obtained from California Foundation for Biochemical Research, Los Angeles. Cesium chloride (or carbonate which was 59 on A uust 2, 2006 w w w .jc.org D ow nladed fom 60 LEUCINE-C 14 IN MICROSOMAL PARTICLES converted to chloride) 1 was obtained from American Potash and Chemical Corporation, Los Angeles, and was used without further purification. Analyses.--The Nessler method (9) was used for total nitrogen determination, and the biuret method (10) for protein. The modified method of Allen (IT) was used for determination of phosphorus. The arginine content of the protein was measured by the method of Albanese et al. (12), using free arginine as the standard. The per cent of arginine in the protein was calculated by dividing the arginine weight by the protein weight as determined by the biuret method. The value 8 to 8.5 per cent agrees reasonably well with the value of 9.2 per cent determined by microbiological assay on the protein hydrolysate (13). Equipment.--All fractional centrifugations were done in the Spinco ultracentrifuge model L with No. 40 rotor. The centrifugation of the verseneand ribonucleasetreated microsomal particles was done in an International clinical centrifuge, International Equipment Company, Boston. The ultraviolet absorption measurements and colorimetric determinations were done in the Beckman spectrophotometer DU, or the Beckman recording spectrophotometer, DK-2. Plant Tissues.--Pea seedlings, Pisum sativum, were grown in vermiculite in the dark for 7 days at 25°C. The apical 2.5 to 4.5 cm. of the stems were collected. These are referred to below as shoots. In certain experiments, only the apical 1.5 to 2.0 cm. sections (tip section) or only the 2 cm. long subapical stem sections (stem section) were harvested. The tissue was ground in a mortar at 2--4°C. with 0.4 M sucrose solution (0.5 ml. per gram of tissue). Before fractionation, the homogenate was filtered through a fabric (Nu-fab, Essentials Products, Inc., Los Angeles) with a suction flask aided by pressure applied to the top. Fractionation of Subcelhdar Components.Nuclei: Pellet from centrifugation at 4,000 g, 15 minutes. This was washed with 0.2 M sucrose and recentrifuged at 24,000 g for 10 minutes. This fraction contained more than 93 per cent of the total DNA in the homogenate. Mitochondria: Pellet from centrifugation at 42,000 g, 72 minutes after the removal of nuclear fraction, washed once with 0.2 M sucrose, and recentrifuged at 82,000 g, 12 minutes. Microsomes: Pellet from centrifugation at 110,000 g, 90 minutes after removal of the mitochondrial fraction. Supernatant: Supernatant after removal of the microsomal fraction. Purification of Microsomal Particles.--The procedure above is for the quantitative separation of the subcellufar components. The microsomal particles were, how1 The chloride salt is 97 per cent pure and the carbonate salt is 93 to 95 per cent pure, according to the analysis of the producer. Major contamination is rubidium. ever, further purified as described below. In addition, ribonucleoprotein particles from the nuclear fractions were also purified. Nuclear fraction, nuclear ribonucleoprotein particles, and nuclear supernatant: The pellet obtained by centrifugation at 2,000 g, 15 minutes, was washed with 0.2 M sucrose, 0,001 ~a CaCI2, pH 6.5, and 5 mg./ml, of leucine. This was recentrifuged at 4,000 g, 15 minutes. The supernatant which contained little RNA or protein was discarded. The pellet was then homogenized in potassium phosphate buffer, 0.05 /z, MgSO4, 5 X 10 -4 M, pH 6.5 and was twice frozen and thawed. The resulting solution was next centrifuged at 24,000 g for 15 minutes. The pellet so obtained is designated washed nuclei. The supernatant was centrifuged at 100,000 g to yield a ribonucleoprotein pellet which is designated nuclear ribonucleoprotein particles (5). A small amount of material which was left behind in the supernatant after sedimentation of nuclear ribonucleoprotein particles is designated nuclear supernatant. Microsomal pellet 1 (MP 1): After the removal of mitochondria (42,000 g, 15 minutes), the supernatant fraction was centrifuged at 110,000 g, for 75 minutes. The pellet was dispersed in water for 60 minutes at 0°C. This suspension was centrifuged at 16,000 g for 15 minutes. The pellet so obtained is designated microsomal pellet 1. Microsomal pellet 2 (MP 2) and microsomal particles: The clarified microsomal solution prepared by the first cycle differential centrifugation was sedimented again at ll0,000 g for 60 minutes, and the pellet resuspended in a small volume of water at 0°C. for 60 minutes. The suspension was centrifuged at 42,000 g for 12 minutes. The pellet obtained is designated microsomal pellet 2 (Fig. 1). The clarified solution contains the microsomal particles referred to earlier as the DC-2 preparation (4). Incubation Procedure.--Stainless steel baskets containing 5 to 8 gm. of plant tissue were submerged in leucine-C 14 solution buffered with potassium phosphate, 0.05/z, pH 6.5, and aerated continuously with oxygen. The temperature was maintained at 25-26°C. At the end of the incubation periods the tissues were rinsed, blotted, and placed in cold 0.4 M sucrose with nonradioactive leucine for homogenization at 2-4°C. Washing and Radioactivity Measurement.--Proteins of all the subcellular fractions were washed, counted, and analyzed by the following procedure. Resuspended pellets or supernatants were treated with 0.5 ~ TCA (trichloroacetic acid) at 0°C. overnight, and then the resulting precipitate was washed twice with 12 per cent TCA, cooled to 0°C. rapidly, and more 12 per cent TCA added. After 30 minutes, the precipitates were washed twice again with TCA, followed by an ethanol wash (50°C.), an ether wash (30°C.), and finally an ethanol wash (50°C). The precipitates were dissolved in 1 N NaOH by heating at 95°C., 1 to 4 minutes, and were reprecipitated by addition of TCA with a large amount on A uust 2, 2006 w w w .jc.org D ow nladed fom PAUL O. P. TS'O AND CLIFFORD S. SATO 61