4 ‘s 74 iv...“ ga—‘wiiz SSOLATION OF IHE MEMBRANE SYSTEMS OF ACANIHAMOEBA PALESTWENSISI STUDIES OF LINE: SYNTI'ESIS AND ASSEMELY 0F LEHDE 3M0 MEMBRANES “to“: for “to Deg?“ 05 pk. D. MICHIGAN STATE UNWERSITY Francis Joseph Chlap-owski I 969 ABSTRACT ISOLATION or THEMDIBRANE srsmas wmw: STUDIES or LIPID smmrsszs mu ASSMY or LIPIDS INTO mamas By Francis Joseph Chlapowski A fractionation procedure is described for the isolation of mem- tranes and mem‘u'ane-bomd organelles of W W. Nuclei . mitochondria. rough endoplasmic reticulum. elongate smooth endoplasmic reticulum. small cisternal smooth membranes. Glogi mem- branes and a fraction containing plasma and digestive vacuole mem- tranes were isolated using this procedure. Electron micrographs. chemical analysis and enzyme assays were used to characterize these fractions. Utilizing cm-oholine as a specific marker of pmsphaucm choline. it was demonstrated that an exchange reaction occurred be- twen free choline and the choline moiety of phosphatidyl choline. This exchange reaction occurred only on membranes in a cell free system. A time sequence study of Ila-glycerol incorporation . turn- over and intracellular movements in lipids is reported. Most lipids are synthesised in the rough endoplasmic reticulum and transferred to the non-menu'ane lipids recovered in the post-microsomal super- natant. The nuclear membranes and rough endoplasmic reticulum are ilplicated as sites of lipid assembly into membranes. Cyclical pat- terns in the pulse and chase studies indicated a recycling of lipids among the different membrane types. These results suggest the possible recycling of membranes. involving interconversions among memtu'anes. ISOLATION OF THE MEIBRANE SYSTEMS 01“ W W: STUDIES OF LIPID SYN'IHESIS AND ASSMY OF LIPIDS INTO mamas By Francis Joseph Chlapowski A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of mC‘rOR OF PHILOSOPHY Department of Zoology 1 969 1b Dad and Mom. Rev. Henry S. hnach and Susan ii. ACKNOWLEDGfliENTS This thesis would not have been possible. if it were not for the untiring work and consideration of Dr. R. Neal Band on my behalf. I shall always remember and appreciate his original and dynamic manner. which has often served as a guide in my own approach to scientific problems. Thank you for everything Dr. and. I wish to express my gratitude to Dr. 0. S. Thornton for making my sojourn at nichigan State an extremely pleasant and intellectually satisfying experience. I thank Ir. J. Shaver for cultivating nor interest in developmental biolon: 11'. B. Sadoff for his sound suggestions: 11'. H. Slattis for helpful and enjoyable conversation: and Sharon Morhlok for all her excellent assistance. I extend my appreciation to Dr. B. Anderson. who was responsible for stimulating my interest in scientific research. Life would not have been the same without the warm friendship of Josb Ortis and AJovi Soott-Dauakpor. For their help and advise. I thank 'Ibm Connolly. John Defazio. Lonnie Eiland. Dr. Nick Shuraleff. Dr. Roy Tassava. Charles Needle and 11'. Peter Weber. I thank u brothers - Albert. Michael and William - and my sisters - Jean. Dorothy and later - for all their help. This work was conducted under the tenure of a National Institute of Health fellowship (1-P1-0i-37.252-01) and a grant n‘om the National Institute of Health to Dr. 3. Neal End (A1-06117-06-TMP). iii INT! m m 01' CONTDJTS (a) Kinetics of Incorporation: (b) Kinetics of Turnover: (0) Call Free Incorporation: (d) Call Free Turnover: (a) comparison at WW“ W W: (f) Tun-Dover of (in-Choline in Phospholipid of Cell Fractions: iv Page OQOVVVVVQ 1o 11 11 11 12 12 12 13 J Page W3: 13 (a) Kinetics of Inoomoration: 13 (b) Kinetics of Tumover: 13 (c) Cell Free Incorporation: 114 (d) Incorporation into Phospholipids of Cell Fractions: 114 (e) Turnover of Phospholipids in Cell Fractions: 111 (f) Pulse and Chase of Phospholipids in Cell Fractions: 1!: RADIOACTIVE ASSAIS: 15 W3 15 W3 15 W‘ 15 W' 15 IDID EXTRACTION AND mm: 16 16 16 16 16 16 16 17 17 17 17 18 18 HIMOWY: 1 8 REM magnum Eli uds¥( P880 RBULTS mmnw or W W: W: m: CELL FRAGTIONATION : (a) RNA/protein ratios: (b) Phospholipid composition of cells and microscmes: mm'rs mm c‘“.cnOLIna: Wins: (a) neasurement of the radioactivity of the medium with growing cells: (b) 002 trapping: (c) Recovery of radioactivity in lipid extracts: (d) Cinematography of radioactive lipid extracts: (e) Monument of lipid radioactivity of medium: (a) Kinetics of incorporation into TCA soluble and insoluble fractions: (b) Kinetics of turnover in TCA soluble and insoluble fractions: 19 19 19 P380 11:1?st me DB-carcslm: 68 MW= 68 (a) Measurement of the radioactivity of medium with 68 growing cells: (b) Recovery of radioactivity in lipid extracts: 68 (c) Chromatograplw of radioactive lipid extracts: 70 W 71 m: (a) Kinetics of incorporation into TCA soluble and 71 insoluble fractions: (b) Kinetics of turnover in TCA soluble and insoluble 71 fractions: 7:. 74 80 83 84 DISCUSSION 88 cm. FRACTIONATION: 88 W: 88 W8 90 W: 91 A mouse aromas REACTION: 92 W: 92 W8 91: LIPID SINTHEIS AND 1133111er or LIPIDS INTO 11mm 95 95 96 when: «n EEG: LIST! viii 103 105 Table 11 iii iv Vii Table ii iii iv vii viii LIST OF TABLES RNA/protein ratios of membrane fractions. Specific activities of enzymes in cell fractions. 1h Recovery (:1 of c -radioactivity in lipid extracts of cells. Localization of C‘urcholine in phosphatidyl choline by chromatography. Recovery (f) of Ha-radicactivity in lipid extracts of cells. localisation of H3-glyoerol in pho spholipids by chromatography. Comparison of the specific activities of whole mitochondria and nuclei to the particulate matter of mitochondria and nuclei. Comparison of relative specific activity ratios (specific activity of nuclear fraction) at temporal intervals after the inoculation of radioactive glycerol. Decrease (fl) in specific activity over a 21: hr chase period. Relative specific activity ratios (specific activity of nuclear fraction) at u” ”d of a 2n hr chase period. Cbmparison of 12 hr'labeling specific activities of nuclear and post-microsomal supernatant fractions. Page \‘8 ESTRS B 74 83 84 til LIST OF FIGURES Figure ii iii iv viii xii Standardized cell fractionation scheme. Values are average g forces at the middle of the tube. Phase contrast micrograph of multinucleate W minim- X 6.000- Phase contrast micrograph of a group of amoeba cells. Note the binucleate cell undergoing synchronous nuclear mitosis. X 6. $0. Electron micrograph of portion of cell illustrating nuclear enve10pe (N). mitochondria (M). digestive vacuole (D). small. cisternal smooth membranes (C) and cell surface membrane (cm). X 32,000. Electron micrograph of portion of cell. Note the edge of a lipid droplet (I. . glycogen particles (0). rough endoplasmic reticulum (REE). Profiles and cross sections of elongate. tubular smooth endoplasmic reticulum (E-SER). mitochondria (11) and cell surface membrane ((24). x 68.000. Growth of W in shaker flasks. The broken‘line --- represents cells/ml and the unbroken line (—) represents mg cells/ml. Diagramatic representation of some centrifugal steps in the fractionation procedure. Phase contrast micrograph of nuclear fraction. X £1.90. Electron micrograph of the nuclear fraction. 1: 211.000. Electron micrograph of the plasma and digestive vacuole membrane fraction. X 67.500. Electron micrograph of the mitochondrial fraction. 1 m '0000 Electron micrograph of the elongate type of smooth endo- plasmic reticulum from the pellet of band 1. 1 60.000. Page 21 21 23 25 27 31 35 37 Figure xiii Electron micrograph of the small. cisternal smooth membranes. Section was from the middle of the pellet of band 2. 1110.000. xiv mectrcn micrograph of the Golgi membrane contaminants at the bottom of the pellet of band 2. X 60.000. xv Electron micrograph of the Golgi. membrane fraction. Section as from the middle of the pellet of band 3. x 60 .000. xvi Electron micrograph of the bottom of the pellet of band 3 demonstrating the rough endoplasmic reticulum contamination. K 67. $0. xvii Electron micrograph of the rough endoplasmic reticulum composing band it. I 82.$0. xviii Cinematography of phospholipids on silica gel plate. xix Kinetics of incorporation into TCA fractions. (mt. 1) xx Kinetics of incorporation into TCA fractions. (Expt. 2) mid. Kinetics of turnover in TCA fractions. ($01K chase) xxii Kinetics of turnover in TCA fractions. (equal chase) xxiii Kinetics of turnover in TCA fractions. (1001 chase) xxiv Kinetics of turnover in TCA fractions. ($01 chase) xxv Cell free incorporation into cell fractions as a function of protein concentration. xxvi Cell free incorporation into microscmes as a function 01‘ “we xxvii Cell free incorporation into microscmes as a function of time and the effects of chase. xxviii The effects of equal chase on an W. “in W and lipid extractable an. W xxix The effects of 100! chase on W. mm W and lipid extractable all We an Turnover of c:1 u—choline in pho sphatidyl choline of cell fractions. xi Page 51 53 55 61 61 61 63 63 63 611 611 67 6? 69 m m Figure xxxi Kinetics of incorporation into TCA fractions. (Knit. 1) lentii Kinetics of incorporation into TCA fractions. (upt. 2) xxxiii Kinetics of turnover in NA fractions. (6.6x chase) xxxiv Kinetics of turnover in TCA fractions. (6601 chase) xxxv Incorporation Hinto lipids of cell fractions. (110 pc/ ml ofH -g1ycerol) xxxvi Incorporaticnaintc lipids of cell fractions. (3) pc/ml of H ~glycercl) xxxvii Incorporation into lipids of cell fractions. (20 pc/ml of H -)glycerol xxxviii Tumover of 113- glycerol in lipids of cell fractions. (mt- 1) :ccdx vaer of 83- glycerol in lipids of cell fractions. (luat.2 xxx: ”long pulse" experiment. Cells labeled for 12 hr prior to bein washed and chased in medium containing 3.3 )1 moles ml of non-radioactive glycerol. maxi 'Short pulse” experiment. Cells labeled for 10 min Prior to being washed and chased in medium containing 3.3 )1 moles/ml cf non-radioactive glycerol. Pisa 73 73 76 81 82 85 INTRODUCTION In the evolution from procaryotic cells to eucaryotic cells. mem- brane development plays a singularly important role. In the smallest unit capable of genetic continuity - the virus - no membrane struc- tures are present. with the exception of the myxovirus. whose envelope seems to be derived from the cell surface membrane of the infected cell (Evie. Dulbecco. Eisen. Ginsberg and Wood. 1967). Most pro- caryotes have only a cytoplasmic membrane underlying the cell wall. Some Gram-positive bacteria contain mescsomes. These invaginations of the cytoplasmic membrane might be involved in respiratory functions. similar to the mitochondria of eucaryotic cells. Whereas procaryotic cells are essentially devoid of membranous components. eucaryotic cells contain a complex array of membranes and membrane-bound entities. A general higher cell type is encompassed by a plasmalemma and is com- posed cf a double-membrane nuclear envelope . granular endoplasmic re- ticulum. smooth endoplasmic reticulum. Golgi-complex membranes. mito- chondria constructed of inner and outer membranes and various vesicular components (e.g. lysosomes. peroxisomes. pinocytotio or phagocytctic vacuoles. etc.). Photosynthetic eucaryotic cells also contain chloro- plasts surrounded by two membranes. Thus. more intricate functions at the cellular level are accompanied by an amplification of the membrane systems. The mnielli-mvson model (1935) suggests that cell membranes are composed of a bimolecular layer of lipid covered with a monomolecular layer of protein on each side. Electron micrographs (Robertson. 19 59). 1 38 pr 1: t1 1‘; Vi 2 as well as X-ray diffraction patterns (Finean. 1953: Finean. Coleman. Green and Limbrick. 1966). have been interpreted as support for this concept. The ultrastructural resemblance among membranes derived from different sources has led to the term ”unit membrane“ (Robertson. 1960). The unit membrane theory suggests that all membranes are lamellar in construction due to the predictions of the Danielli-Ihvson model. Korn (1960) has recently criticized the unit membrane theory as lacking in proof (see also Curtis. 1967: Weiss. 1967). An alternative to the lamellar ccuposition proposed for membranes might be micelles of a hengonal character (Luzzati and Husson. 1962). Room temperature nega- tive staining of plasma membranes has illustrated such properties (Benedetti and helot. 1965). It is not known whether the observed micelles are an intrinsic property of the membrane or a fixation arti- fact. Creen and Perdue (1966) reject the mnielli-Davson model. as well as the unit membrane theory. and emphasize that the basic frame- work of membranes is protein. These authors propose that membranes are basically repeating units of linroteins joined by hydrOphobic bonds. One of the principal reasons for their contention is the fact that mitochondrial membranes retain their ultrastructural characteristics after severe lipid extraction. This is also true in muslin and other membranes (Napolitano. Iehron and Scaletti. 1967). Ebwever. the most imortant reason put forth by Green and his colleagues for the emphasis on protein in membrane systems is their isolation of a structural pro- tein from mitochondrial membranes (Ch'een. T‘isdale. Griddle. Chen and Dick. 1961: see also ween. Heard. lanes and Silman. 1968). Recently. Woodward. Kubic. fleece and hbodward (1968) have isolated 3 electrophoretically similar structural proteins from membranes of W mitochondria. corn mitochondria and com chloroplasts. This finding establishes the notion of a membrane structural protein more firmly. Newer concepts of membrane structure and function place more aphasia on the role of proteins. macromolecular complexes and hydro. phobic bonding. Less emphasis is placed on the role of electrostatic attractions between polar groups of lipids and proteins. However. independent of the importance of proteins in the structure of membranes . all biological membranes of eucaryotes are lmcwn to contain phospho- lipids and sterols. although the specific amounts of each may vary from one source to another (Ashworth and Green. 1966: Finean. 1967: Korn. 1966: Takeachi and Terayama. 1965). The principal impediment in analyzing the membrane systems of cells is the isolation of these systems from the cell. In the case of membrane- bound organelles. the problem is simplified. The isolation of the or- ganelle separates its membranes from other cellular membranes. Ole of the first cellular organelles isolated was the nucleus (Dounce. 19113: Allfrey. Stern. Mirsky and Saetren. 1952). More recent advances have yielded nuclear preparations of high purity in aqueous media (Chaveau. Meals and Rouiller. 1956: Maggic. Siekevitz and Palade. 1963: Blobel and Potter. 1966). Widnell and Siekevitz (1967) have reported a method for obtaining preparations of nuclear membranes from rat liver nuclei. Many methods have been developed for the isolation of mitochondria (Schneider. 19W: Hogeboom. Schneider and Palade. 19MB). mplan and Coeenawalt (1966. 1968) have succeeded in separating the inner and outer membranes of mitochondria. The isolation of lysosomes and w. when a peroxisomes has been accomplished by the work of deDuve and his colla- borators (deDuve. Pressman. Gianetto. hattiaux and Appelmans. 1955: Ioighton. Poole. Beaufay. &udhuin. Coffey. Fowler and demve. 1968). In animal cells the isolation of Golgi material has essentially been restricted to cells in which the Golgi system is extensively elabora- ted. such as the rat epididvmis (Schneider. miton. Kuff and Felix. 1953: Schneider and Kuff. 1951:). An exception to this has been the recent successful isolation of the Golgi apparatus from rat liver cells (Morrb. Mollenhauer. Hamilton. Mahley and Cunningham. 1968). Microsomes are generally isolated by high speed centrifugation. with subsequent density gradient fractionation (kitten and Roberts . 1960) to resolve granular and agranular microscmes (Palade. 1955: Palade and Siekevitz. 1955: Rothschild. 1961: Meter. Siekevitz and Palade. 1962: Minor and Nilsson. 1966: Illlner and Ernster. 1968). With the exception of red blood cell ghosts (Ponder. 1955). the most difficult cell membrane to isolate is the cell surface membrane. Neville (1960) first isolated the cell surface membranes of rat liver cells. Hallach and Kamat (1964) claim to have isolated the plasma membranes of marlich ascites carci- noma cells (see also Kamat and Wallach. 1965; vallach. 1967). The me- thods of Warren. Glick and Mass (1967) have been used to isolate fixed cell surface ghosts from mouse L cells. The isolation of a biologically active plasma memtx'ane ghost preparation from HeLa cells has been described by Bosmann. Nagopisn and aylsr (1968). The biosynthesis of lipids and proteins. the enzymatic properties of membranes (e.g. muster. Sickevitz and Palade. 1962: Novikoff. Essner. Goldfischer and Heus. 1962) . the intracellular movements of membranes 5 (e.g. Friend and Parquhar. 1967) and the role of membranes in trans- port. storage and secretion (e.g. Jamieson and Palade. 1967a. 1967b. 1968a. 1968b: Bowers and Kern. 1967: Beams and Kessel. 1968) are areas under intensive investigation. Although the literature is replete with hypotheses and research concemed with membrane biosynthesis and degra- dation. the actual events in the process by which the molecular compo- nents of membranes are united into a macromolecular complex can not be defined at this time. Since different membrane systems are quite similar with respect to pass chemical concosition. but operate in different structural and functional situations. the assembly of membranes is a fascinating area of research. ficause of the fundamental resem- blance of various cellular membranes. the origin of one type of mem- brane by modification of a pre-e:d.sting. but different. type. is not an unreasonable thought. In fact. some evidence has been assembled which shows the fusion of plasma membranes. or their derivatives. with membranes derived from the Golgi complex (e.g. Friend and Parquhar. 1967: Bowers and Kern. 1967). Considering all the latter facts. an investigation into the movement of radioactive precursors into and out of the phospholipid molecules of membranes might be expected to shed some light on the biogenesis of membranes. The organism chosen for this research was the soil amoeba W W. Ultrastructural evidence will be given to demonstrate that W W represents a typical eucaryotic cell type containing a full complement of membranous organ- elles. For this reason. and because growth of homogeneous populations under controlled amenic conditions is possible. this organism is 6 particularly suitable for the investigation of membrane systems at the cellular level. A prerequisite to examining the incorporation and tumover of radioactive tracers in membranes was the development of procedures for the isolation of various membrane systems and membrane- bound organelles from the same population of cells. The characteriza- tion of several sumellular fractions by chemical. enzymatic and electron microscopic procedures will be presented. Utilizing these fractions. data will be put forth concerning the role of lipid precursors in membrane biosynthesis. years Band‘ 18 g pH 0: coat atur 196: don] Er MATERIALS AND METHODS ORGANISiS: These experiments utilized the soil amoeba W pglggtingngig maintained in Dr. R. N. Band's laboratory for several years. ‘ggltggg_uggigmg Cells were cultured under axenic conditions in Band's Amoeba Medium (1959). The medium contained 3 mg MgClZ.6H20. 3 mg CaClz. 3 mg PeSQu.7H20. 120 mg NaCl. 136 mg KH PO 2 h 18 g glucose and 15 g Protease Peptone (Difco) to 1 L 320 at a final . 1&2 mg NaZHPQu. pHTof 6.8. The complete medium was sterilized by autoclaving for 20 min. .ininzs.§andiiisnss Cells were routinely cultured in 1 L silicone- coated Erlenmyer flasks containing 500 ml of medium. A constant temper- ature. retaty shaker was used at 100 rpm and 29°C (Band and Machemer. 1963). Under the conditions described. cells grew with a dry weight doubling time of 27 hr. Only exponentially growing cells were utilized in experiments. ‘Q[axinggg1g_ggg§gggggn§§; Cell mass increase was determined by washing cells from 1 ml of medium once in cold distilled H20 followed by desiccation over P O and under vacuum in tared vials. The brief 2 5 HZO'wash produced no cytolysis. CELL FRACTIONATION: Aliquots of cells (3 to 5 ml wet. packed vol- ume) were harvested. homogenized and fractionated according to a stand- ardized scheme given below (Figure 1). in which TKM stands for 0.005M Tris. 0.025M K01 and 0.002M MgSO : and TM is a similar solution minus 8 flash cells in 0.2514 sucrose-m ‘125g X 5 min Homogenize in 2 vol of 0.25M sucrose-TM with 7 strokes of Teflon pestle 2.000g X 15 min in HB-h sediment supernatant (nuclei + plasma and food (microscmes + mitochondria) vacuole membranes) 12. 500g X 20 min in 55-34 resuspend in 0.2524 sucrose-T104 mitochondrial --- pellet 12.00% x 15 min in Hs-u summ‘mt --discard supernatant (microscmes) sediment 100.000g X 70 min in type 30 (washed nuclei 4* plasma and u-post-microsomal supernatant food vacuole membranes) loose sediment above glycogen pellet resuspend in 1.3M sucrose-T101 layer over sucrose-TKM gradient (“lifts-2:03:23; in 0 25M sucrose-TM 130.0008 X 3° “'1“ 1" 501* 1100,0003 x 50 min in type 30 ---remove plasma and food ---discard supernatant vacuole membrane band loose sediment sediment (washed microscmes) (nuclear pellet) resuspend in 0.2511 sucrose-TM llayer over sucrose gradient 200.000g x 5: hr in 50L 1: membrane bands mitochondrial pellet (continued from above) resuspend in 0.2514 sucrose-0.00114 EDTA wash 3 times at 10.000g x 15 min in 33-34 washed mitochondria post-microsomal supernatant (continued from above) {100.000g X 50 min in type 30 post-micro somal supernatant with residual membranes removed plasma and food vacuole membranes (continued from above) make up to 0.25M sucrose-0.00m EDTA wash 3 times at 1.100g X10 min in PIS-1+ washed plasma and food vacuole membranes Figure 1. Standardized cell fractionation scheme. Values are average forces at the middle of the tube. m below cacke in 3 Co.) than eithe it fc with aembz Pena: 9031- 30C!!! 9 KC]. (cf. Blobel and Potter. 1966). All steps were carried out at pH 7.5. below 1:00. W: Cells were washed once in 0.25)! sucrose-TM and resuspended in the same solution to yield a final cell concentration of 1:2 (wet, packed volumexsolution volume). W: The cell suspension was homogenized with 7 strokes in a 30 ml capacity Potter-Elvehjem type Teflon grinder (A. H. Thomas Co.) with a clearance of 0.125—0.175 m. driven by motor at 2.500 rpm. Wm: For relative centrifugal forces less than 20.000g. a Sorvall RC-Z refrigerated centrifuge was used with either an HB-l: swinging bucket rotor or an 38-34 fixed-angle rotor. At forces exceeding 20.000g. a Spinco LZ-S) ultracentifuge was used with either a 50L swinging bucket rotor or a type 30 fixed-angle rotor. (a) The initial nuclear and plasma and food (digestive) vacuole membrane fraction was isolated from the 0.251! sucrose-m homogenate and quickly transferred to. and washed once in. 0.2514 sucrose-Tim to remove residual mitochondria. (b) The mitochondrial fraction was immediately isolated from the post-nuclear supernatant. resuspended and washed 3 times in 0.25M sucrose-0.00m ethylenediamine-tetra-acetic acid (EDTA). According to and and Morhlok (1969). the latter washings remove lysosomes and per- oxisomes. as Judged by the loss of acid hydrclase and catalase activi- ty. (c) Microsomes were separated from the post-mitochondrial superna- tant by centrifugation at 100 .000g X 70 min. The resulting microsomal fraction was very loosely layered above a large. almost transparent. glycogen pellet. The microsomes were removed from under the 10 post-microsomal supernatant witha pipette. gently resuspended in 0.25M sucrose-TM. and washed at 100.0003 X 50 min. At the end of the run. the loosely layered microsomes were once again removed with a pipette. (d) The post-microsomal supernatant with its floating non-membrane lipid layer (Stein and Shapiro. 1959) was recentrifuged at 100.0003 X 50 min to remove any residual membranes. W: All linear sucrose density gradient centrifugations were performed in the Spinco L2-50 using a 50L swing- ing bucket rotor. (a) The washed nuclear and plasma and food vacuole membrane pellet was gently resuspended by pipetting in 1 ml of 1. 3M sucrose-T101 and layered over a 4 ml continuous sucrose-T104 gradient extending from 1.311 to 2.011 (1.17-1.27 8/ cc at 11°C). The preparations were centrifuged at 130.000g X 30 min (cf. Blobel and Potter. 1966). At the end of this brief run. the microsomal contaminants were floating at the top of the tube. the plasma and food vacuole membranes formed a band in the gradient and the nuclei were pelleted at the bottom of the tube. The plasma and food vacuole membrane band. which extended from 1.18 to 1.195 g/cc was removed with a J-shaped needle attached to a syringe (Jamieson and Palade. 1967a). The residual supernatant. including the floating microsomal layer. was poured off. leaving the translucent pellet of pure nuclei. The plasma and food vacuole membrane prepara- tion was diluted to 0.2514 sucrose-0.00m EDTA. homogenized with 3 strokes of a 10 ml capacity Potter-Elvehjem type Teflon grinder (A. H. Thomas Go.) with a clearance of 0.10-0.15 m at 1.000 rpm. This homogenate was washed twice in 0.2514 sucrose-0.00111 EDTA at 1.1003 Bl 11 X 10 min to yield the final plasma and food vacuole membrane pellet. (b) Subfractionation of microscmes recovered from the differential centrifugations was accomplished by layering the microsomes. in 0.25 ml of 0.25M sucrose-TH (5 to 10 mg protein). over a “.5 m1 continuous sucrose density gradient (Britten and Roberts. 1960) extending from 1.01111 to 2.011 (1.11.4.2? glee at u°c). and centrifuging for 5} hr at 200.000g (cf. Jamisson and Palade. 1967a). Upon termination of the run. the a distinct bands in the gradient (see Figure 7) were removed with a J-shaped needle attached to a syringe. The isolated fractions were either fixed for electron microscopy. washed for chemical analysis or precipitated with 10%.trichloroacetic acid (TCA) prior to lipid extraction. For cell free incorporation experiments. mitochondria. microscmes and the post-microsomal supernatant were isolated in the same manner. exeept that 0.25M sucrose-0.1M phosphate buffer (pH 7.h) was used in all steps. RADIOACTIVE LABELING: We“: (7.6 mole mole - New England Nuclear): (a) Kinetics of Incorporation: In experiments invoving the kinetics of C1u¥choline incorporation into TCA soluble and insoluble cell frac- tions. 10 ml of cells in culture medium (106 cells/ml) were incubated with 1 pc/ml (0.132 p mole/ml). At appropriate time intervals. 1 ml aliquots of the medium were removed. diluted with ice-cold 0.25M sucrose- THQ and centrifuged to remove the cells. The cells were immediately resuspended and washed in the same solution. The washed cells were 12 brought to 1 ml (original volume) with cold 10% TCA.and stored for a minimum of 2 hr below 14°C. (b) Kinetics of Turnover: Turnover in TCA soluble and insoluble fractions was examined by “labeling" cells with 0.25 pc/ml (0.033 p moles/ml) for 12 hr. The cells were then harvested. washed and re- suspended in Amoeba Medium containing non-radioactive choline. The quantity of non-radioactive choline used was either 0.033 p moles/ml (equal chase). 3.3 u moles/ml (100x chase) or 16.5 p moles/m1 (500x chase). At appropriate time intervals. 1 m1 aliquots of cells were removed and processed as described above. (c) Cell Pres Incorporation: Mitochondria. microscmes and post- microsomal supernatant. isolated in 0.25M sucrose-0.1M phosphate buf- fer. were brought to 1 mg protein/ml in 0.1M phosphate buffer (pH 7.11) with 1 u mole CaClzlml. Either 2 or u mg of protein from these suspen- sions was brought to 5.25 ml with a final mixture containing 5‘u moles 01:61 and 0.1)! phosphate buffer. At time 0. 0.25 ml of phosphate buf- 1uncholine was introduced into 2 for containing 1 uc (0.132 )1 mole) of C the reaction mixture. After incubation at 29°C in a water bath with agitation. the reactions were terminated at specified time intervals by adding an equal volume of ice-cold 20% TCA (Vendor and Richardson. 1968). The 10% TCA solutions were stored at 2°C prior to lipid extrac- tion. Methods for lipid extraction will be given in a subsequent sec- tion. (d) Call Free Turnover: The same methods described above were used. except that at certain times. concentrated. non-radioactive cho- line was introduced into the reaction mixture in 0.1 m1 of phosphate buffer. As a control. 0.1 ml of phosphate buffer devoid of choline 13 was added simultaneously to identical mixtures. The concentration of non-radioactive choline was 66 p moles (500X chase). (0) 00an 01‘ MW“ WW8 In experiments to determine the loss of radioactivity from cells into the medium. cells were labeled. washed and resuspended in medium con- taining non-radioactive choline as dexcribed for the kinetic studies of turnover. At sampling intervals. the total radioactivity of washed cells from 1 ml of medium (921]. W was determined by re- suspending the cells to 1 ml (original volume) with distilled water and counting aliquots directly in scintillation fluid. In addition. the total radioactivity of 1 ml of medium. from which cells had been removed by centrifugatien (mm W . was ascertained by directly counting aliquots of the medium in scintillation fluid. u-Choline in Phospholipid of Cell Fractions: (f) Turnover of C1 Cells were labeled as described above. In time sequence. aliquots of cells were fractionated into mitochondria. microscmes and post-micro- somal supernatant. The fractions were precipitated with 10% TCA. washed and lipid extracted. W3 (5)0 mc/m mole - New England Nuclear): (a) Kinetics of Incorporation: 113 -glycerol incorporation into TCA soluble and insoluble fractions was examined by labeling and processing cells in a manner identical to the c1 “-choline studies. The only dif- ference was that 5 pc/ml (0.01 p mole/ml) of Til-glycerol was used. (b) Kinetics of Turnover: The kinetics of [TB-glycerol turnover in TCA soluble and insoluble fractions was monitored in a manner identi- cal to the separable studies with C1 “-choline. except that 2.5 pc/ml (0.005 p mole/ml) was used to label the cells. The non-radioactive 11: glycerol concentrations were 0.033 u mole/ml (6.6x chase). or 3.3 u mole/ml (6601 chase). (c) Cell Free Incorporation: The conditions for cell free incor- poration utilising H3-glycerol were as previously described for c1 “- choline. 1.25 no (0.0025 )1 mole) of Tia-glycerol was present in the reaction mixture. (d) Incorporation into Phospholipids of Cell Fractions: For these experiments. cells were ccncentated in 20 ml of Amoeba Medium at a ratio of 1:3 (cells:medium). Concentrations of 20 pc/ml (0.0!: )1 mole/ ml). and in some cases £10 pc/ml (0.08 p mole/ml). were added at time 0. In time sequmce. aliquots of cells were fractionated according to the scheme in Figure 1. The cell fractions were precipitated with cold 10% TCA prior to lipid extraction. (e) Turnover of Phespholipid in Cell Fractions: The experiments involving turnover of Tia-glycerol in phospholipid of cell fractions were carried out in a manner identical to similar studies with C1 l:_ choline. cells were labeled for 12 hr with 2 pc/ml (0.001: p mole/m1) of Ila-glycerol. washed and resuspended in medium containing 3.3 u mole/ ml (825! chase) of unlabeled glycerol. (1') Pulse and Glass of Phespholipid in Cell Fraction: Concentrated cell suspensions were suspended in medium containing 30 pc/ml (0.06 p mole/ml) for 10 min. Then. the cells were removed from the medium by centrifugatien. washed once in Amoeba Medium. and finally. resuspend- ed in medium with 16.5 p mole/m1 (275x chase) of non-radioactive gly- cerol. At suitable time intervals. aliquots of cells were fractionated as described. ti 15 RADIOACTIVE ASSAYS: W: All radioactive counting was done in a scin- tillation fluid containing 7.81; g 2.5-diphemrlonsole (FPO). 0.16 g p-bis-(o-mettwlstyryl)-benzene (bis-MSB) and 120 g naphthalene to 1 L of p-dioauane. All counting rates were corrected for background and quenching to disintegrations per minute (dpm). Counting was done on a Beclcnen Mark III refrigerated liquid scintillation counter. W: Gases derived from the radioactive medium in which cells were growing was passed through a 0.1N NaOH trap over a 3 day period. At the end of this time interval. aliquots of the NaOH solution with trapped 002 were counted directly in the scintillation solution. W: Aliquots of radioactive lipid extracts in chloroformmethanol (2:1) were placed in scintillation vials and allomd to evaporate to dryness. The scintillation fluid was added and the vials counted as described. cells from 1 ml of medium. which had been brought to exactly 1 ml with 10$ m, were left in this suspension for at least 2 hr at 2% (see sections on kinetic studies). The TCA insoluble fraction was centri- fuged down at 16.000g X 5 min. Aliquots of the clarified cold TCA sol- uble fraction were removed and counted directly in the dioxane-base fluid. The TCA precipitate of the cells from 1 ml of medium was washed twice in cold 5% TCA and once in cold H20 to remove am residual insol- uble radioactivity. The T'CA insoluble material was dissolved in 88% formic acid and transferred to counting vials. Thus.values can be re- lated to 1 ml of medium and are expressed as dpm/cells/ml of medium. 3cm Siake tats: with at n trad E81 1 3111.11 coun‘ dehe: with em. 16 LIPID EXTRACTION AND CHRQIAT'OGRAPHY: W: Tb insure complete removal of insoluble radio- activity. lipids were extracted from washed TCA precipitates (mllner. Siekevits and Palade. 1966a). The original 10% T‘CA insoluble precipi- tates were washed by centrifugation twice with cold 5% TCA and once with cold distilled water. The precipitates were then lipid extracted at room temperature for 2 hr in chloroformmethanol (2:1). The ex- tracts were separated from the insoluble residue and washed with 0.111 H0]. (5 MM ml extract) to remove any protein remaining. Separate aliquots were used for lipid phosphorus determinations . scintillation counting or chromatographic analysis. W: Lipids in the chloroform :methanol extract were spotted on activated silica-gel G plates and developed in chloroform:methancl:acetic acid:water (25:15:l+:2) for 90 min (Randerath. 1966). The separated phospholipids were detected by iodine vapors and scraped into scintillation vials for counting. CHMCAL ANALYSIS: m: Using bovine serum albumin as a standard. protein was determined by the Folin-Ciocalteau test (1927). W: RNA was determined on washed TCA precipitates with the 'Hejbaum (1939) orcinol tectmique. utilizing hot (90%) TCA extraction of nucleic acids as proposed by Schneider (19146). Purified yeast RNA was used as a standard. W: Lipid phosphorus was measured according to Marinetti (1962) using 70% perchlorate at 150°C to digest the lipid phos 3583 17 phosphorus in dried lipid extracts. W: ibnophosphates liberated in the phosphatase assays were measured by the Fiske and Subbarow method (see Lindberg and buster. 1956). mm ANALYSIS: Allenzymes examined were phosphatases and were assayed by the liberation of inorganic phosphates. Values are expressed as )1 moles Pi/mg protein/hr. W: This enzyme was assayed by a modification of the method of Swanson (1955). The reaction mixture contained 250 4} ug cell fraction protein. 2 X 10 moles disodium g1ucose-6-phosphate (Sigma medical Co.). 1 x 10"5 moles histidine and 1 x 10'6 moles EDTA in 1 ml of 1120 at pH 6.5. After incubation at 29°C for 30 min. the reaction was terminated by the addition of 0. 5 ml of cold 20% TCA with rapid cooling of the tubes. The TCA precipitate was centrifuged down and the liberated inorganic phosphorus in the supernatant deter- mined. Blanks were processed similarly. except that no substrate was present. W: The method of Novikoff and Heus (.1963) was used to determine this enzyme' s activity. The assay mixture con- tained 250 ug cell fraction protein. 6 x 10"7 moles of thiamine pyro- phosphate chloride (Sigma Chemical Co.). 2.11 x 10'5 moles of Tris-BC]. and 1.25 X1O-6 moles of MgCl2-6H20 in a volume of 1 ml at pH 7.11. The other aspects of the assay were identical to those described for glucose-6-phosphatase. h‘ . Wallach and Ullrey's (1961;) method was used to analyze this enzyme as well as sodium and potassium-activated. magnesium-dependent adeno sine triphosphatase. The reaction mixture contained 250 ug cell fraction protein. 5 X 10"? moles n-is-ademsinc triphosphatase (Sigma Chemical Co.). 1 x 10"5 moles Tris-HCl. 5 x 10'8 moles EDTA and 5.5 X 10'6 moles MgClZ-6Hzo in a volume of 1 m1 at pH 8.11. The other parts of the assay procedure were identical to the other phosphatase methods described. W: The reaction mixture was the same as for magnesium- dependent adenosine triphosphatase. except that it contained 1.02 X 10-5 moles NaCl and 5 X 10-6 males KCl. The analysis procedures were the same. MICROSCOPY: Pellets of cell fractions were fixed in £1123 in 5% glutaraldehyde with 0.114 phosphate buffer (pH 7.2) at 4% for 12 hr. These samples were rinsed in cold phosphate buffer and post-fixed for 115 min at 11°C in Zetterqvist's osmium fixative (see Pease. 1961+). Pellets of cells were fixed in osmium without pro-fixation in glutar- aldehyde. All specimens were dehydrated in ethanol and embedded in Araldite. Sections were cut with glass knives on a Porter-Elum MT-Z micrctome. mounted on carbon coated or uncoated grids and stained with uranyl acetate . followed by lead citrate. Sections were examined and micrographed in a Hitachi Till-11B operated at 75kv with a double condenser. Preparations were seamed and micrographed at direct magnifications ranging n-cm 10.000 to uo.ooox. REULTS DESCRIPTION OF AW W: W: Populations of cells grown in a state of continuous suspension in Ameba Medium are heterogeneous with respect to cell size. nuclear size and the presence or lack of multinuclearity (Band and Machemer. 1963). As can be seen in Figures 2 and 3. nuclei with associated nucleoli. numerous vacuoles and small particles (mostly mitochondria) are visible utilizing phase contrast microscopy. Ultra- structurally (Figures £1 and 5). W appears to contain a typical array of cytoplasmic organelles (see also Bowers and Kern. 1968. for a complete analysis of the fine structure of W. Fine structural observations reveal no extracellular coat like those of m m and M m. The ultrastructurally distinct membanes are: (1) the inner and outer portions of the nuclear enve- lope. (2) the inner and outer mitochondrial membranes. (3) the large vacuole membranes (e.g. food (digestive) and contractile vacuole mem- branes). (14) the small'vesicle membranes (e.g.pinocytotic vacuoles. lysosomes and peroxisomes). (5) the Golgi complex membranes. (6) the smooth endoplasmic reticulum (primarily in the form of elongate cisternal elements.“ well as. tubular membranes). (7) the rough endoplasmic reticulum and (8) the cell surface membrane. Osmiophilic lipid drop- lets. which probably represent mcst of the non-membrane lipids. are evident in these cells. Copious amounts of glycogen particles are also visible in the cytoplasmic matrix. 19 Figure 2. Phase contrast micrograph of multinucleate Aganthgnggbg Wm X 6-000- Figure 3. Phase contrast micrograph of a group of amoeba cells. Note thz binucleate cell undergoing synchronous nuclear mitosis. X .500. n .31. z... I 3" \.. . . ll 22 Figure 1+. Electron micrograph of portion of W cell illustrating nuclear envelope (8) . mitochondria (H) . di stive vacuole (D). small. cisternal smooth membranes (C).and cell surface membrane (QT). I 32.000. 215 21 1U '1. Figure 5. Electron micrograph of portion of cell. Note the edge of a lipid droplet (L . glycogen particles (0). rough endoplasmic reticulm (RER). profiles and cross sections of elongate. tubular smooth endoplasmic reticulum “688‘:ch mitochondria (14) and cell surface membrane (01). I . . 27 hr call in ch weigh began 26 m: Cells grown in silicone-coated shaker flasks exhibited a 2? hr generation time (Figure 6). Due to the variations in cell size. cell number counts were not as precise as dry weight determinations in checking growth rates. The average number of cells per mg dry weight was 0.64 t 0.05 X 106. Cells inoculated into Amoeba Medium began exponential growth without my noticeable lag phase. GEL FRACTIGTATION: ' W: The primary criteria employed in deve- loping the fractionation procedure (Figure 1) were the preservation and purity of the isolated membrane systems and membrane bomd organ- elles. Since all of the work with these fractions will be presented in terms of specific activities. total recovery was not considered to be an important factor. Homogenization was a critical step in the fractionation scheme. The procedure used produced the breakage of a meadmum number of cells. while preserving the released cellular organelles and membrane sys- tems. The homogenization medium of 0.291 sucrose-TM was used as a com- promise to allow all fractions to be isolated from the same homogenate. It was found that nuclei were most easily isolated from cells homogen- ized in 0.2511 sucrose-n01 (cf. alobel and Potter. 1966): but the 1:" ions destropd the mitochondria and affected the densities of the microscmes (of. humor and Nillscn. 1966). Mitochondria. on the other hand. could have been isolated intact. from the post-nuclear superna- tant of cells homogenized in 0.234 sucrose-0.00111 EDTA. However. the lack of divalent catios and proper ionic strenght would have caused 1O‘QIq-IL 1 0 o ?\I HHIO Q... mg cells/ ml 27 2.0- - 20.0 1.0- 10.0 .° Y‘1111 0e1- 1e0 d .051, , - 0.5 / - fl d P -1 l- .01. g - . , 0.1 0 25 50 75 100 hours post-inoculation Figure 6. Growth of W in shaker flasks. The broken line -- represents 4' cells/ ml and the unbroken line (__) represents mg cells/ml. Im/§0t X SIIGO # .m WM.“ m .m 28 the nuclei to aggregate. swell and break (of. Unbleit. Burris and Stauffer. 19 57). The compromise homogenization medium settled upon (0.25M sucrose-TM) was isotonic enough to allow the nuclei to remain in it briefly. and contained a small amount of divalent cations. which prevented the nuclei from clumping. The presence of divalent cations did cause some adherence of mitochondria to membrane fragments: but this process was reversed by the eventual washings of the mitochondria in 0.25M sucrose-0.00114 EDTA. The washings caused many mitochondria to swell and lyse. Imediately following homogenization. the nuclei. along with the plasma and food vacuole membranes. were separated from the homogeni- zation medium by centrifugatien. The light colored top layer of the resulting pellet. containing most of the nuclei and plasma and food vacuole membranes. was quickly resuspended and washed in 0.2 5H sucrose- m (Figure 7). The bottom of the initial pellet. containing unbroken cells. was discarded. In isolation medium containing TTCM. the nuclei were quite stable. However. this medium caused the plasum and food vacuole membranes to shrink greatly. After the centrifugal washing. the nuclear and plasma and food vacuole membrane pellet was resuspend- ed in 1.3M sucrose-T104 and subjected to gradient centrifugation as described in the methods. The passage of nuclei down the gradient was sufficient to rip off and float up any cytOplasmic rough endoplasmic reticulum attached to the nuclei. Hence. a pure. translucent nuclear pellet was obtained (Figure 8). The linear gradient was steep enough to allow the plasma and digestive vacuole membranes to band above the nuclear pellet at 1.18 to 1.19 g/cc at 11°C (pigm 7). This band was .omseooomm 20330308.“ on» 5 human Hewsfimpmoo been no 533280.53 capesmnmfio .m 35mg aw... . 1: +933 .3303 98a .7 -11.: ease .I III: 1 111Hn~m wuss-m . 2: inhommmneoa .8539 t .1114 -‘0'F fig ,IIJfi lungs... .. c. .2 mm x mooo.oo~ can on N wooo.omp 80.83 551883» 5 modpmwsfimpmoo on a— 5 mogwgfimpmoo Induce moxosnms oneness + an .1 c . scenes + an .. £3 5.1-33.3...3 m m m n m. a m, i Home manhood m, m... 3a: massed 2 iii! + assessmensn cm 18.1 sec: 338G... on .1... cinnamon 0. Homoeomoaalunoa 0 ghe§o3dslouoa amoaommtenon Figure 8. Phase contrast micrograph of nuclear fraction. X 14,500. 32 removed. rehomogenized and washed in 0.25M sucrose-0.001M EDT . This washing, in addition to removing mitochondrial and microsomal contami- nants. allowed the membranes to return to a sac-like shape and caused vesiculation. Mitochondria were separated from the post-nuclear supernatant by high speed centrifugatien (Figure 7) which. while insuring the removal of the mitochondria from the post-mitochondrial supernatant, also in- creased the sedimentation of contaminating microsomes. These contami- nants were removed by the subsequent. three low speed washings at 10.000g in 0.25M sucrose-0.001M EDTA. In addition. the washings of the mitochondrial fraction removed peroxisomes and lysosomes (Band and Morhlok, 1969). which are common contaminants of mitochondrial prepa- rations (deDuve. 1967). The duration of the differential centrifugations involving separa- tion of the microsomes from the post-mitochondrial supernatant. and their subsequent washing. was a critical factor in the preservation of the membrane systems. It was essential that the lenght of these 100.000g centrifugations be adjusted such that the microsomes were gently layered over the glycogen pellet at the end of each run (Figure 7). If the microsomes were packed tightly over the glycogen pellet at the end of each differential centrifugation. not only were microscmes lost into the glycogen pellet; but distinct banding patterns were not obtained in the subsequent gradient centrifugation. It was observed that the presence or lack of Mg++ ions in the gradient had no effect on equilibrium densities. Figure 7 illustrates the banding pattern routinely obtained using the described methods. 33 W: When viewed with a phase microscope (Figure 8) the nuclear fraction appeared homogeneous. Electron micro- graphs (Figure 9) demonstrated that no cytoplasmic rough endoplasmic reticulum remained adhering to the outer rough nuclear membrane. In most eased the inner and outer nuclear memh‘anes were left intact. Some nuclei were observed with portions of the outer rough nuclear mem- brane sheared off. Phase contrast scanning of the plasma and digestive vacuole mem- trane fraction in 0.2514 sucrose-m revealed a highly shrivelled state induced by the medium. At this time it is uncertain whether this was a contractile phenomena or an osmotic shrinking. However. the same event was encomtered when cells were homogenized directly in 0.2514 sucrose-m. In the latter case the ionic strength inside and outside the membrane vesicles would be expected to be the same. If these same membranes were washed several times in 0.294 sucrose-0.00m EDTA. they regained a sac-like shape and vesiculated. It is interesting that the equilibrium density of the membranes was unaltered by the presence or lack of the 15' ions. Ultrastructurally. plasma membranes could not be distinguished from the food vacuole membranes (Figure 10). Chly trilamellar structures of the membrane vesicles were observed when the membranes were cut at normal angles. ‘ ihe mitochondrial pellet recovered after 3 washings in 0.2514 sucrose-0.001)! EDTA contained intact and swollen mitochondria (Figure 11). Intact mitochondria exhibited a highly characteristic structural organisation. The matrix of the mitochondria stained intensely. Vesi- cles of swollen mitochondria usually retained this matrix. indicating Figure 9. Electron micrograph of the nuclear fraction. X 2h,000. -4-— - a . .- 4 e .d' _,..,- 'Qfi.m‘ifl 5.. 36 Figure 10. Electron micrograph of the plasma and digestive vacuole membrane fraction. X 67.500. 38 Figure 11. Elggtron micrograph of the mitochondria fraction. X .000. V 8 "ft T—l —— gr-“ their origin. Band 1. recovered from the continuous gradient centrifugation of the microscmes (see Figure 7). was the least dense (1.14-1.15 g/cc at #90) and was composed almost entirely of smooth surfaced elements (Fig- ure 12). These smooth membranes appeared to be in a multitude of con- figurations. ranging from large vesicles. usually containing a smaller vesicle. to curved.cisternal structures and long. narrow; tubular structures. It is suggested that the images of a vesicle within a vesicle are. in reality. tangential cuts across one of the two primary structures composing this fraction. This structure can be likened to a thin-surfaced. hollow. sphere. which had been folded in upon itself. The curved. cisternal structures are. in fact. different profiles of these same structures. These elements fit the current cytological description of the intact. elongate. smooth endoplasmic reticulum. Membranes are also present in narrow tubular configurations. Bowers and Kern (1968) have described this type of smooth endoplasmic reticu- lum in Acanthamoeba (see also Figure 5). The majority of membranes in band 211.175 g/cc at QPC) were in a small cisternal configuration (Figure 13). However. these smooth memp brane elements. unlike those of band 1. were only about 1.500 i,in length. small particles were also present. The chemical analysis (see hble 1) suggests that these particles are most likely ribonucleo- protein. The bottomnmost edge of the pellet of this band (Figure 14). in addition to containing the small. cisternal elements. also contained larger vesicles in distended shapes. It is suggested that these membranes might be damaged Golgi membrane contaminants from the top of band 3. 1+1 -W T‘F‘ “, Figure 12. Electron micrograph of the elongate type of smooth i endoplasmic reticulum from pellet of band 1. X 60.000. ‘ I “3 We 13. Electron micrograph of the small. cistemal smooth membranes. Section was from the middle of band 2. 1 110.000. 1&5 Figure 14. Electron micrograph of the Golgi membrane contaminants at the bottom of the pellet of band 2. X 60.000. \l 1&7 Band 3 (1.183 g/cc at 0°C) was composed primarily of Golgi complex membranes sectioned at various angles. Some vesicular rough endOplas- mic reticulum (Figure 15) was also present. The distended ends of the Golgi profiles were often filled with an electron dense material. The amount of rough surfaced vesicles increased at the bottom of the pellet of this band (Figm'e 16). and h (1.19h-1.208 g/ec at 10°C) was composed almost entirely of rough surfaced membranes (Figure 1?). In some instances matrix mate- rialwas seen. Very few free ribosomes were present. The rough endoplas- mic reticulum was usually in the form of large. distended. shapes. rather than small. round vesicles. W: (a) MIA/protein ratios: uble 1 gives the RNA/ protein ratios of Table 1. RNA/protein ratios of membrane fractions."I Membrane Fraction" Expt. 1 kpt. 2 kpt. 3 Average a-sna 0.05 0.05 0.05 0.05 C-SM 0.09 0.09 0.11 0.10 0 0.10 0.10 0.19 0.13 am 0.20 0.22 0.28 0.25 P114 0 0 0 0 ‘valuee expressed in pg RNA/pg protein "Iv-SEE =-' elongate smooth endoplasmic reticulum from band 1. C-SM = small. cistemal smooth membranes from band 2. G 8 Golgi membrane fraction of band 3. BER = rough endoplasmic reticulum of band it and P114 3 plasma and digestive vacuole membrane fraction Figure 15. Electron micrograph of the Golgi membrane fraction. Section was from the middle of the pellet of band 3. X 60.000. Figure 16. Electron micrograph of the bottom of the pellet of band 3 demonstrating the rough endoplasmic reticulum contamination. I 67. 500 . Figure 16. Electron micrograph of the bottom of the pellet of band 3 demonstrating the rough end0plasmic reticulum contamination. 1 67.500. Figure 1?. Electron micrograph of the rough endoplasmic reticulum composing band a. X 82.500. 5“ the membrane fractions isolated. The values of three separate deter- minations on the fractions isolated from 3 different cell populations are given. (b) Phospholipid composition of cells and microscmes: Both whole cells and rushed microsomes were lipid extracted and chromatographed as described in the methods section. The phospholipids found in each case were identical. Phosphatidvl ethanolamine. phosphatidyl choline . phosphatidyl eerine. and phosphatidvl inositol were identified (Figure 18). Triglycerides. fatty acids. phosphatidic acid and sterols would not be detectable by the chromatographic procedure used. and would all appear in the spot at the solvent front. the enzymes of W have not been extensively investigated. no 1 m method was available to ensymatically classify the mem- brane fractions. Several enzymes oomonly used as membrane markers in mamlian cells were employed. The specific activities of the enzymes in each fraction are given as u moles of inorganic phosphorus twdro- lyaed per hour per mg protein 0: moles Pi/hr/mg protein) in some 2. The results of 2 experiments are given. The specific activity of glucose-6-phosphataee was enhanced in the Golgi and rough endoplasmic reticulum fractions. relative to the specific activity observed in the homogenate. The elongate type of smooth endoplasmic reticulum and the small. oistemal smooth membranes contained no activity. be highest specific activity (101 higher than the homogenate) was observed in the plasma and digestive vacuole membrane fraction prepared by the described methods. If the plasma 55 __ solvent U --triglycerides- 6 front t7 «gamma-- W 0 WU O Wigwam- O g «game, e a __start cells microscmes Figure 18. Chromatography of phospholipids on silica gel plate. Thble 2. Specific activities of enzymes in cell fractiona.‘ Fraction“ Expt. 0 0.6.9». TPPase 148309;” 118/ K+-ATPase a 1 0.002 0.038 0.151 0.181 2 0.009 0.036 0.109 0.123 a 1 0.028 0 0.233 0.217 2 0.017 0 0.233 0.228 n 1 0.100 0.039 0.025 0.201 2 0.090 0.032 0.076 0.210 1 0 0.261 0.013 0.555 3.333 2 0 0.109 - - 1 0 0.039 0.610 0.696 0.311 2 0 0.017 0.566 0.613 0 1 0.177 0.006 0.572 0.686 2 0.177 0.389 0.502 0.028 m 1 0.168 0.103 0.271 0.259 2 0.153 0.192 0.222 0.321 2111 1 0.080 0.076 0.773 0.776 mm 1 0.270 0.312 0.659 0.509 1 0.105 0.009 0.100 0.100 ”‘3 2 0.116 0.039 0.102 0.100 *values expressed in pg Whrfeg protein "B =3 homogenate. N . nuclear fraction. )1 =- mitoohondrial fraction. E-SER ' elongate smooth endoplasmic reticulum. G-S'I = small. oisternal membranes. 0 a Golgi fraction. RER= rough endoplasmic reticulum. Pm (m) 8 plasma and digestive vacuole membrane fraction washed in 0.291 sucrose-0.00114 ETA. P111 (1101) 8 plasma and digestive vacuole membrane fraction washed in 0.2514 sucrose- mm and ms 1' post-microsomal supernatant 57 and digestive vacuole membranes had been washed in 0.2514 sucrose-T104. rather than 0.2511 sucrose-0.00114 EDTA. prior to the analysis. the specific activity was decreased by about 9%. A small enhancement of enzyme activity was found in the mitochondria}. fraction. As can be seen from the specific activity in the post-microsomal supernatant. some of the enzyme was soluble. While the elongate smooth and the rough endoplasmic reticulum exhibited some thiamine pyrophosphatase activity. the small. cisternal smooth memlranes. Golgi membranes and plasma and digestive vacuole memlranu were observed to contain the largest amount of this mayme. Again. no: washings of the plasma and digestive vacuole membrane frac- tion resulted in a decrease in activity. The nuclear and mitochondrial fractions demonstrated no activity. As can be Judged by the small amount of activity in the post-microsomal supernatant . very little of the enzyme was soluble. Magnesium-dependent adeno sine tripho sphatase activity was present in all the membrane containing fractions . indicating that practically all of the enzyme was particulate. The highest specific activities were found in the plasma and digestive vacuole membrane. Golgi mem- brane and small. cisternal membrane fractions. However. no increase in the latter values was produced by sodium and potassium ions in any of the fractions (compare lla+/K+-AT?ase values to Mgfi-Afi’ase values in Table 2). A 50$ decrease in activity was observed in the mitochondrial fraction. most likely due to potassium loading. Sodium and potassium stimulation of magnesium-dependent adenosine triphos- phatase is often used as a criterion for the detection of plasma 58 membranes (Novilcoff. Essner. Goldfischer and Hans. 1962). The remlts in hble 2 indicate that T101 washings had no deleterious effect upon adenosine triphospbatase activity. mmrs mm c1 “-mouns Wins: (a) heasurement of the radioactivity of medium with growing cells: To detect whether amr radioactivity was being lost from the growth medium to the atmosphere. 1 ml portions of radioactive medium were counted over an experimental period of 0 days. No changes in radioac- tivity were detected in 2 separate experiments. This indicated that no radioactivity was lost to the atmosphere from the culture of grow- ing 00118. (b) 00 trapping: As a check on the above results. the 00 gas. 2 2 111 from cells grown in 0.25 pc/ml of C -choline for 3 days. was trapped in 1N NaOB and counted. In 2 experiments. no radioactivity was detected. Thus. it is certain that none of the C}1 “-choline is metabolized down to the state of c1 “02 under the growth conditions described. (0) Recovery of radioactivity in lipid extracts: Three populations “-choline for 3 days were washed and of cells grown in 0.25 pc/ml of c lipid extracted as described. Tbtal radioactivity of the lipid extract and the lipid-extracted TCA precipitates was compared. The results in Thble 3 illustrate that essentially all of the TGA insoluble radioac- tivity was lipid extractable. 59 110 able 3. Recovery (t) of C --radioactivity in lipid extracts of cells. ‘ kpt. # Lipid Extract Extracted 1 Recovery of TCA Ppt. TCA Ppt. 1 1182.500 15.000 99.5 2 212.100 597 99.7% 3 358.500 1.354 99.5$ ’values given in dpm (d) Chromatography of radioactive lipid extracts: The lipid ex- tracts of whole cells. as wall as microscmes isolated from cells. labeled for 1 day in 0.25 pc/ml of Gui-choline were chromatographed as described (see Figure 18). The radioactivity of the spots was de- termined in 2 separate experiments. The results. illustrated in Thble 0. demonstrate that Gut-choline was a specific label for phosphatidyl choline. nor. 0. Localization of cm-‘oholine 1h phosphatin choline by chrontograptw.‘ Sample Expt. # PC PI PS PE 81" 1 1.282 0 0 0 0 “n" 2 020 0 0 0 0 1 19.000 0 0 0 39 2 6.890 0 0 0 0 *values given in dpm: ’20 - phosphaticbrl choline. T21 . phosphatidyl inoaitcl . PS - phosphatidyl serine . PE ' pbsphatidyl ethanolamine and SF 3 spot at solvent front. 60 (e) instrument of lipid radioactivity of medium: Radioactive me- dium (0.25 pc/ml cf Cm-choline). in which cells had been grown for 3 days. was cleared of cells by low speed centrifugation. The lipids extracted from the TCA insoluble fraction of the medium were counted as described. No lipid radioactivity was found in two experiments. It thus seems that all of the 01 “-choiino utilized by the cells was incorporated into phosphatidyl choline. was precipitatable with TGA and was extractable with lipid sovents. Therefore. direct count- ing of TCA precipitates was utilized to give an approximation of the 0‘ “-choline present as phosphatidyl choline. (a) Kinetics of incorporation into rat soluble and insoluble motions: By utilising washed cells derived from the same volume of medium (1 ml) and by determining the total radioactivity of both the TCA soluble and insoluble fractions of these cells. as described in the methods. no corrections for growth dilution of radioactivity were necessary. Figures 19 and 20 illustrate that incorporation into the TCA insoluble fractions of 2 different cell populations became linear about 05 min after introduction of 1 pc/ml of Cm-choline. Coinciding with the beginning of linear incorporation into the phosphatidyl choline of the TCA insoluble fraction. the soluble pool seemed to level off. However. the size of the soluble pool continued to increase after the initial leveling off (Figure 20). (b) Kinetics of turnover in TCA soluble and insoluble fractions: m cells. which had been labeled for 12 hr. were washed and resus- pended in meditmi containing 16. 5 p moles non-radioactive choline] ml 61 B .31 0-+ '8 E '8 \ 0) H '75 51L TCA.insoluble # 9.. N a 1 3,- TCA soluble O of incogporation 6 8 Figure 19. Kinetics of incorporation into TCA fractions. (Expt. 1) 5 300 3 TCA soluble a *1 ” 1 g .s 51’ 5?. >4 3. 1 '6 TCA soluble 0 h; of incorporation g 8 Figure 20. Kinetics of incorporation into TCA fractions. (mpt. 2) 3 5 "r? :32'1 \ '1 E4 0 TCA soluble :} $21 I N a . “0 TCA insoluble 0 50 hr postgéhase Figure 21. Kinetics of turnover in TCA fractions. (90X chase) 62 (m0! chase). radioactivity was rapidly lost from the TCA insoluble fraction (Figure 21). Since a stable label for membrane phospholipids was being sought. the rapid turnover of this label was investigated. A population of cells. which had been labeled for 12 hr. was devided into 3 equal volume groups. washed and resuspended in medium containing 0.033 11 moles/ml (equal chase). 3.3 u moles/ml (1001 chase) and 16.5 p moles/ml ($01 chase) of non-radioactive choline (Figures 22. 23 and 20). To make the comparison more meaningful. the values are pre- sented as percentages of the maximum T‘CA insoluble radioactivity. It can be seen. that as the concentration of non-radioactive choline was increased. the rate of loss of radioactivity from the TCA insoluble fraction increased. W8 deor and Richardson (1968) reported that etiolated pea seedling microscmes would incorporate 0‘ 11_ ethenolamine. 0"‘-scrinc and c:1 ”-choline into phospholipids. In light of the results with living W. it became important to deter- mine whether a similar system was operational. hicrosomes. mitochondria and post-microsomal supernatant were prepared as described in the me- thods. Figure 25 illustrates cell free incorporation as a function of protein concentration. As found by Vendor and Richardson (1968). only the membrane-containing fractions (i.e. mitochondria and microscmes) incorporated 0‘“-cholinc. Incorporation increased as a function of protein concentration. A divalent cation requirement (Gafi) is illus- trated by the low level of incorporation in the presence of EDTA. Cinematography of lipid extracts revealed that all of the radioactivity was incorporated specifically into phosphath choline. 1OOEF' f5 ‘1“ % maximum TCA insoluble radioactivity 63 1 TCA insoluble i;\c TCA soluble hr poié-chase 20 Figure 22. Kinetics of turnover in TCA fractions. (equal chase) o 53' H 8 g, TCA insoluble .5 *g’ ' -<-a 0 -1-" same. a o v—* - *‘ 9:8 ! ““‘r-l~\a\\\\\\‘ g 2 TCA soluble ha 0: 0 hr pole-chase 2“ Figure 23. Kinetics of turnover in TCA fractions. (100K chase) 100%?- 3 maximum TCA insoluble radioactivity a _L_ TCA soluble TCA insoluble Figure 20. Kineti 0 hr wit-chase 2“ es of turnover in TOA.fractions. (500X chase) 15. micropomes l . O I O 10- h =.\ 52 N O s mitochondria ' «8' 1 5‘ microscmes + 0.0114 ED’I‘A i post-microsomal summatant : I ¥ V . _._.___—_—.. mg cell fraction proteizn in reaction mixture Figure 25. Cell free incorporation into cell fractions as a function of protein concentration. 10 dpmx10u/2 mg microsomal protein “L c 0 minutes of iggorporation 120 Figure 26. Cell free incorporation into microscmes as a function of time. 65 Figure 26 demonstrates that cell free incorporation into microscmes was initially linear as a function of time. Between 30 and #5 min. in- corporation leveled off and exhibited no increase or decrease there- after. Figure 2? illustrates that the addition of concentrated non- radioactive choline. either before or after the leveling off. not only stopped incorporation. but effected a rapid turnover of label in the cell free nicrcsoaes. Thus. an equilibrium situation seemd to be the cause of the leveling off of incorporation in the cell me system. mine whether an equilibrium condition was reached by living cells and the free choline in the aediun. the mWwas compared to the “in W. is explained in the methods section. 9.911 mm was the total radioactivity of washed cells from 1 ml of medium: and mm W was the total radioactivity of 1 ml of medium from which cells had been renoved by centrifugatien. Figures 28 and 29 demonstrate that about 70 hr after the introduction of 0.033 p moles/ml or 3.3 p moles/n1 of non-radioactive choline. an equilibriul bot“!!! 9:11. W W mm W "88 reached. Figures 28 and 29 also illustrate that the attainment of this equilitrium coincided with the leveling off of the loss of lipid extractable 9.11 W. The results have been converted to percent-Leos of madma- aall W- In order to observe the effects of chase on the choline moiety of phosphatidyl choline in cell fractions. cells were labeled and resus- pended in medium containing 3.3 p moles/ml of non-radioactive choline 66 30-. . 5 - é a. a 20' 500 chase added ,a" 3 at minutes e 8 3 a O m - 5 i r' 5001 chase added >4 ,v'at 15 minutes 3.10- ,. '6 0 co 60 120 minutes of incorporation Figure 27. Cell free incorporation into microscmes as a function of time and the effects of chase. 1 maximum Cell Radioactivity 100%- \5 if 0, 67 Medium Radioactivity l I __‘ v i A 7 fi 1 Cell Radioactivity he 11 'id extractable Ce Radioactivity . so 100 150 hr post-chase Figure 28. The effects of equal chase on Cell Radioactivity. Medium % maximum Cell Radioactivity Radioactivity and lipid extractable Cell Radioactivity. 1002 Medium Radioactivity - I 50% Cell Radioactivity u liid extractable i 0' Ce 1 Radioactivity O 100 1 50 hr post-chase 50 Figure 29. The effects of 100x chase on Cell Radioactivity. Medium Radioactivity and lipid extractable Cell Radioactivity. 68 as described. The results. sumarized in Figure 30. illustrates that 2 equilibrium situations occurred. As anticipated. at about 60 to 70 hr post-chase. the loss of radioactivity from all cell fractions level- ed off and the decrease in specific activities thereafter was due to growth dilution. Thus. an equililrium was reached between free choline and the bond choline moiety. The other equilibrium attained was that reached among the cell fractions. Within 24 hr post-chase. all the cell fractions' specific activities had reached a stable position re- lative to each other. and decreased thereafter at the same rate. The non-membrane phosphatidyl choline in the post-micro somal supernatant also showed this equilibration effect. iherefore. although choline was found to be a specific label for phosphatin choline. a peculiar exchange reaction occurred. making it an unsuitable label for starving membrane assembly. murmurs wxm Riemann: W3 (a) masurement of the radioactivity of medium with growing cells: Ihe total radioactivity of medium with growing cells did not change over a 3 day experimental period. 'lhis indicated that no radioactivity was lost from the medium into the atmosphere. (b) Recovery of radioactivity in lipid extracts: Table 5 gives the results of 3 independent experiments on cells which had been grown for 3 days in 2.5 pc/ml of FIB-glycerol. The results indicate that essentially all of the TCA insoluble radioactivity was present in lipids. 69 .mcoapommm Haoo mo ocaaoso dhpapmnamonc ca onwaonouawo mo mo>ocmse .om madman on one 0 won as one oo. n no pcmvmcmmcmm Hegemomoaauvmoa eee mampmonooaas ooo monomomoaa . o d Ptdtt Bd/COt x wdp 70 table 5. Recovery (1) of Ila-radioactivity in lipid extracts of cells. it Empt. # Lipid Ektract Extracted i Recovery of TCA Ppt. TCA Ppt. ‘ 1 93609000 g a” 95. 6’ 2 1 .2162 .000 113.000 96. 5$ 3 1.206.000 51 .000 95.8% ‘values given in dpm (c) Chromatograplw of radioactive lipid extracts: The lipid extracts of cells incubated for 12 hr in medium containing 2. 5 pc/ml of Ila-gly- cerol were chromatographed (see Figure 18). The radioactivity of the spots of 2 experiments are given in Table 6. All detectable phospho- lipids demonstrated radioactivity. ‘lhe large amount of radioactivity Table 6. Localisation of Ila-glycerol in phospholipids by ctromatograplw.‘ mt. 9 PC PI PS PE 3? 1 1.933 61 1.1188 2.1100 7.200 2 2.822 1.15 2.765 3.730 11.5110 I'values given in dpm: see Table 1+ for symbol abbreviations. recov'end in the'epot atlthe' 'eolvent'frcnt he not unexpected.’ since this spot should have contained triglycerides . diphosphatidyl glycerol and phosphatidic acid (Rack. Iaeger and McCaffery. 1962: Ralevy and Finkelstein. 1965: Elm. Avivi and Katan. 1966). 71 Since essentially all of the radioactivity of the TCA precipita- table material of cells was in lipid. direct counting of TCA insolu- ble fractions was used to give an approximation of the Ila-glycerol present in glycerides. (a) Kinetics of incorporation into TCA soluble and insoluble fractions: is in the c:1 “-choline experiments. values were determined on a basis of TCA soluble or insoluble radioactivity of cells from 1 ml of medium. Figures 31 and 32 illustrate that H3-glycerol linear incorporation into lipids began almost immediately after introduction of 5 pc/ml into the medium. In addition. the size of the soluble pool was very small and showed no increase over time. (b) Kinetics of turnover in TCA soluble and insoluble fractions: In light of the effects of varying concentrations of non-radioactive choline on the stability of the choline moiety of phosphatidyl choline. the effects of different concentrations of non-radioactive glycerol on the turnover of H3-glycerol were studied. Cells were labeled. washed and resuspended in medium containing 0.03 p moles/ml or 3. 3 p moles/ml of non-radioactive glycerol. The results of 2 experiments for each non-radioactive glycerol concentration are given in Figures 33 and 3b. Values were converted to percentages of the maximum TCA insoluble radioactivity to aid in the comparisons. is can be seen from the graphs (Figures 33 and 3t). about a 10% loss in radioactivity comm-red within the first 10 hr post-chase. Thereafter. the decrease in radioactivity was slight. No effect of the different concentrations of non-radioactive glycerol was observed. The size of the soluble pool 72 20- 5 «d 'g TFi.insoluble g : 5: 31w .3 $2 >4 E; Era soluble L o z. 3' hr of incorporation Figure 31. Kinetics of incorporation into TCA fractions. (Expt. 1) 20.. 5 3 8 1;“ insoluble g I H I .. ! 8 104 ¢ 9. N B 8' 50A soluble ' - 4: o 3’ a hr of incorporation Figure 32. Kinetics of incorporation into TCA.fractions. (Expt. 2) 73 100% .3. TCA ignsoluble 3. 2a- same 8 is 1! “ TCA sfluble of e— i I i 0 hr peg-chase 2“ Figure 33. Kinetics of turnover in TCA fractions. (6.6x chase) ‘ocnk e z i \ :3. TCA insoluble ' '3' e. 5 a: fig 50%“ O as g a ‘-"~ TCA syllable L 4' ; i j 0% O 12 214 hr post-chase Figure 34. Kinetics of turnover in TCA fractions. (660x chase) 7b was extremely small. W: Cell fractions. prepared exactly as des- cribed for the c‘ “-choline experiments . demonstrated no cell free incorporation. therefore . data from all of the preceding experiments with Ila-glycerol indicate that it is a relatively stable label for lipids. WW: Before experiments were considered utilising specific lipid activities of the cell fractions. it was important to determine whether the specific activities of the organelles bounded by membranes (i.e. mitochondria and nuclei) were equivalent to the particulate (i.e. membrane) or non- particulate (i.e. non-membrane) lipids of these organelles. To test this. nuclei and mitochondria from cells labeled for 12 hr in 2.0 pc/ml of Fla-glycerol were sonicated and washed in distilled water. This procedure should have removed the non-membrane lipids. The speci- fic activities of the sonicated and washed nuclei and mitochondria were about the same as the activities of the whole nuclei and mito- chondria (Table 7). Table 7. Comparison of the specific activities of whole mitochondria and nuclei to the particulate matter of mitochondria and 111101.," ’ Fraction Particulate Whole 1 Difference Nuclei 13.210 12.980 2% Mitochondria 9.670 9.100 2% ’values given in dpmhig lipid P 75 In the studies of incorporation of a3-glycerol into the lipids of cell fractions. cells were fractionated at time intervals after the inoculation of BB-glycerol according to the scheme in Figure . 1 . Figures 35. 36 and 37 show the pattern of incorporation over a 2 hr period. Incorporation into the non-membrane lipids of the post- microscmal supernatant was linear and occurred at a more rapid rate than incorporation into an of the other cell fractions. The only time this might not have been true was within the first 5 min of incorporation. Five minutes after the beginning of incorporation the nuclear and the supernatant fractions' specific activities were approx- imately the same. (if all the membrane fractions the nuclear fraction maintained the highest specific activity during all points of the incorporation experiments (Figures 35. 36 and 37). For this reason. the nuclear fraction' a specific activities were used as a reference for relative comparisons among the specific activities of other frac- tions. For these comparisons. relative specific activity ratios (i.e. ) were calculated. anction In the table (Table 8) of relative specific activity ratios. it can be observed that the ratios changed after 12 hr in medium contain- ing n3-glycerol. The ratios attained after 12 hr in labeling medium were designated as "labeling equilih'ium" ratios. These values accepted as the "labeling equilibrium” ratios were those that were the same (i.e. differed less than 1' 0.1 from the values of the other 2 experi— ments) in 2 out of the 3 experiments (see 0 time in Figures 38. 39 and no). is can be seen in Table 8. only 1 ”labeling equilibrium“ value of the elongate endoplasmic reticulum and 1 value for the 39805.38: we die: 05 .ncoapoanu 33 we age: 8.5 83303005“ .nn 09mg :oavahomhoofi Ho 55 .2 o. n n o k aooosoofisi\ ”ozone—nos mammal ssafihfimfit .n. .sséusafiv - p nonsense... “mace: .m / oasmaaaowmmzmmmm 4 fl 1 v. d m ”N “macs: d X m C Io. gowoemrsmoomfi 77 Aaonoohamnnm no Ha\o1 omv .mcoavomum HHoo no nuaaaa can“ coapouonnoonu .wn shaman moapmuonuooca no :Hs no on we mocunpaoe mandam-ill|l|IIIIll\\IIIIIIlllIIIIIIIIIIIIlIIIIIIIIII sszmwnmimmfi- a you» n n cannuanommmsmm mmnnnHHHHHHHHHHHHHV\\\\\\\QIV nocuuneos «mace: «vacant gomfiswmfioms ., o 0 \ln Co; I d PId'FI Sd/udp ow Aaonoohamu N nozannaos «anode! «anaconoOpdai ssasoapou odsnuamouco specs» ouemnoaoa .necennaoa cuooan Hacnounaou D noqeupsoe «maca- asfiso pen odamaamouco a soul doaonmi anon—echoes» gonaodauenoqu mm ’n Cot x d pldu fid/udp mo Ha\o: owv .mcoapoonm HHoo no nuaaaa owed :oapauomuoocH .mm enema» scapegoanooca mo us a o o \ ow 79 able 8. Comparison of relative specific activity ratios WW (specific activity of nuclear fraction) at temporal intervals after the inoculation of radioactive glycerol.* Tisheling equilibrium ratios“ Fraction 5 min 2hr W T2 hi" (1'18- 35) (Fig- 37) (Fiso 38) (Fig- 39) (Fig ‘10) u 1 1 1 1 1 m 0.83 0.87 0.90 0.93 0.86 0.3): 0.50 0.52 0.75 0.72 0.75 ml 0.20 0.31 0.66 0.511“ 0.74 c 0.59 0.68 0.611 0.70 0.70 1.31m 0.36 0.115 0.83“ 0.68 0.68 11 0.19 0.33 0.72" 1.00" 0. 56" 9118 1.10 2.21 12.65 11.98 3.00 *symbol definitions are given in Table 2. I”ratios differ w more than 0.1 from the other 2 experimental ratios at the end of 12 hr period plasma and digestive vacuole membrane fraction were not in agreement and are marhsd. 'nle mitochondrial fraction demonstrated no "labeling equilibrim" position. Within the first 5 min of incorporation . the rough endoplasmic reticulum was in its 'labsling equili‘u'ium' position (Table 8). By 2 hr the Golgi fraction had also reached its highest relative specific activity. Sometime between 2 and 12 hr after the introduction of radioactive glycerol into the medium. the plasma and digstive vacuole memh‘anes. the cisternal. smooth membranes and the elongate smooth endoplasmic reticulum reached their “labeling equilibrium" positions. 80 W: The primary objective of the turnover studies was to determine the relative changes in specific activities of the cell fractions. Thus. no corrections for growth dilution are shown on the graphs (Figures 38 and 39). The cells were growing exponentially in these experiments. The changes in speci- fic activities of fractions of cells. which had been labeled in 2.0 po/sl of HB-glycerol for 12 hr. washed and resuspended in medium con- taining 3.3 )1 mole/ ml of non-radioactive glycerol. are shown in Figures 38 and 39. Table 9 gives the actual decrease over the 211 hr chase per- iod in percentages. ’lhe expected decrease due to growth dilution would have been W. The nuclear and the rough endoplasmic reticulum fractions decreased at a more rapid rate than any of the other fractions. The non-membrane lipids of the post-microsomal supernatant decreased at a rate slower than all of the other fractions. Table 10 illustrates the relative specific activity ratios after the 211 hr period. In contrast to the uble 9. Decrease (f) in specific activity over a 211 hr chase period.‘ Fraction Figure 38 Figure 39 ll 70 74 RIB 76 67 0-91 52 66 Pm ‘53 61 0 53 62 E-SIR 57 57 H 52 62 P118 20 1&3 ‘symbol definitions are given in Table 2. 81 C .333 6.3333 .33 moan: 5 aaooaunm no .8355 .3 0.8»: am emanation .5 .3 o /., none pace EH8 , / nonsense-o «am can //l§.3am m du I 3 T. .m to— n. .d . a; 5.3.8 39330 " X 3.9a 3o." 333.335 33.8 ovuumode ... 5.93:.» 35133.8 swoon 0:. «0.7:: 1m . ans—Items» 138983138 82 AN .99an 65.33am 33 «3393” 5 HoaoomHMInm no 3358. .am 99mg em 3201.38 an a o 3.3.553 manna um , V oaanoaaopco npgagmwwfl / 35.5503 «mace . lop 35.338. 5085 H8533". I 59030.“ oaaneaaopno sun?" In w «sacs: fleeces—08.? 8. #788 E01 1: .1 UNIT fid/udp 83 Table 10. Native specific activity ratios W (specific activity of nuclear fraction) "t th" ““1 °f 3 211 hr chase period.‘ Faction Firm 38 Figure 39 u 1 1 m 0.72 1.19 0-311 1.18 0.93 9111 1.23 0.80 c 0.99 1.111 s-saa 1.18 1.111 n 1.16 1.118 as 33.05 26.58 ‘Iy'mbol definitions are given in Table 2. l'labeling equililrium" positions (see hble 8) . all cell fractions exceeded unity or approached it more closely than in the previous experiments. The ratios of the post-microsomal supernatant increased relative to all the other fractions. Wm: matmpointmmmw illustrates in graphical form the positions of the various fractions after 12 hr of labeling. The difference between this experiment and those shown in Figures 38 and 39 is that only 0.2 pc/ml of HB-glycerol was used to label the cells. whereas in the other 2 experiments 2.0 pc/ ml of B3-glycercl was used. By comparing the relative “labeling equilibrium" positions (see able 8) of the membrane fractions at the end of the 12 hr labeling period. it can be seen that they are 811 all the same with the qualifications previously mentioned. However. the non-membrane lipids of the post-microsomal supernatant in this experiment (Figure 110) has a very low ratio when compared to the other 2 experiments (Figures 38 and 39). In Table 11 . using the nuclear Table 11. Comparison of 12 hr labeling specific activities of nuclear and post-microsomal supernatant fractions. * Fraction Figure 110 Figure 38 Figure 39 (0.2 uc/ml) (2.0 pc/ml) (2.0 pc/ml) nuclei 11.380 12.980 (31) 16.120 (11x) Pcst-hicroscmal 3“,.th 12.9110 1611.100 (1311) 1911.000 (15x) ’values given in «1me pg lipid P: values in parenthesis show increase over value in Figure 110. fraction as a representative of the ”labeling equilibriuma positions of the membrane fractions. it can be seen that the increased amount of radioactive glycerol available in the medium. increased the speci- fic activity of the non-membrane lipids to a greater extent than the membrane's lipid specific activity. In both cases. the “labeling equilibrium" of the membrane fractions is unaffected by the amount of radioactive glycerol available (see ‘lable 8) . WW= Figure 110 ('lcng pulse” experiment) illustrates the effects of a 3. 3 11 moles/ ml non-radioactive glycerol I'chase'I on cells which had been labeled for 12 hr in medium containing 0.2 pic/ml of H3-glycercl. 35 40.303» 33000309120: «0 Hinges 1 m. m $533980 5.2002 5 vommno 0:0 030.03 9.309 3.8.29 .2 NF you 63090.” 938 555.898 nomda 9.3... 3 0.53m .5 N .. o o . L. n aw “ma—OW “H“III 1, gem» 30$ mmwfmw M 380.3503 030.31 ‘ % 35.3308 3H8...) m a m ouaneaaoewmsmwsmne? .e r I \ lo. (a «30.61 a. gafiwflfio - 86 .Houoohaw 0>300300hncoc no dimoaos 1 m.m mfifiapcoo 5:302 5 00050 30 0050.03 9300. 3 pain 53 0.. how 00.390.” 0.38 .pgfiuomxo gmmda upon? .5 09mg .E :00: N 00 o .0 F «a: r .309 H _ _ mauvconoofleu , 'lb III/d 00.8.8505 dmawm l £85 flaw»? 0 o :0: Essfiomaoficwa l— P 005.5903 om d adammaaowmmmmwwm I m a “30:: I m p d X m (9 IN pamfiogw I51»... I.” “nu... rm 87 Figure M ("short pulse' experimnt) demonstrates the effects of the sale concentration of non-radioactive glycerol on cells which had been labeled for 10 ain in medium containing 20 pc/ml of Ila-glycerol. more is one major difference between the two experiments. In tho ”long pulse" experiment. all the fractions were well labeled and had reached their ”labeling equilibrium" positions (see Table 8). This obviously was not true in the 'shcrt pulse“ experiment. With the lat- ter fact in lind. it may be predicted that some fractions would be affected by the chase differently in these two experiments. The Golgi membranes initially decreased in specific activity in the "short pulse' experiment. and increased in specific activity in the "long pulse“ experinmt. ihe lipids of the plasma and digestive vacuole newt-am fraction and the non-membrane lipids of the poet- nicrosonal supernatant fraction initially increased following the chase in both experiments. The nuclear. rough endoplasmic reticulum and cisternal smooth membrane fractions initially decreased in both emerimnts. The elongate smooth endoplasmic reticulum initially increased in the ''short pulse" experiment and showed a slight initial decrease in the ”long pulse” experiment. The mitochondrial n'acticn simply leveled off in both experiments. Notice in the ”long pulse' experiment. that we of the fractions that initially decreased 15 the first hr post-chase. increan in the next time interval. DISCUSSION CELL FRACTIONATION: W: In fixed cells. membranes occur in morpholo- gically and. most probably. in functionally distinct locations and configuration. Rough surfaced membranes are found as part of the nuclear envelope and as the rough endoplasmic reticulum in the cyto- plasm. Smooth membranes can be observed in the form of plasma membranes . vacuole membranes. Golgi complex membranes. smooth endoplasmic reti- culum. mitochondrial membranes. and cisternal smooth membranes in W (Bowers and Kern. 1968; this paper). Previous fractionation procedures have been developed for the iso- lation of a particular cell component or. at most. several cell com- ponents. from the same tissue or group of cells (e.g. Blobel and Potter. 1966; deDuve. Pressman. Manetto. httiaux and Appelmans. 1955; Bogebcom. Schneider and Palade. 1948: Jamieson and Palade. 1967a: Schneider and Ruff. 195a). Smooth surfaced and rough surfaced micro- scmes have been separated on the basis of density differences (e.g. Rothschild. 1961: Miner. Sielcevits and Palade. 1966a). Subfractiona- tion of these microsomal membranes were devised utilising decxycho- late solubiliaaticn (buster. Siehevits and Palade. 1962) . cation effects on sedimentation rates (mm:- and Nilsson. 1966) and density gradient centrifugation of rough endoplasmic reticulum elements (Mlner. Bergstrand and Nilsson. 1968). All of the latter procedures dealt with small membrane vesicles produced by homogenization and 88 89 fractionation . rather than intact membrane systems (see Dallner and Easter. 1968. for a review). More often then not. the parameters of isolation procedures were adjusted in such a way that marry of the organelles or membrane systems not being isolated. were destroyed in the course of fractionation. Since the eventual goal of the present research was the study of temporal events during membrane biogenesis. it was necessary that as many as possible of the membrane-bound orgamlles and membrane sys- tems be isolated from the same cell homogenate. Furthermore. an attempt was made to isolate the structurally distinct membrane sys- tems intact. In this manner. the complications of trying to subfrao- tionate a group of homogeneous-looking vesicles was avoided. In order to prevent vesiculation cf the membrane systems and organelles. it mas observed that careful control had to be exercised over homogeni- zation. isolation media and sedimentation. The intact cell components finally isolated were: (1) nuclei with intact envelopes. (2) rough endoplasmic reticulum. (3) elongate smooth endoplasmic reticulum. (4) small. cisternal smooth membranes. (5) Golgi complex membranes. (6) plasma and digstive vacuole membranes. (7) mitochondria and (8) pest-microsomal supernatant with the non-membrane up“: of the cell. line only major memlrane-bound elements not isolated were lyso- somes and peroxisomes. It has been reported (Bowers and Kern. 1968) that the smooth endo- plasmic reticulum in W my occur in the form of tubular elements. Observations on fixed cells used in this stucw confirmed the latter report (Figure 5). These tubular membranes. as well as 90 large oisternal membranes. were recovered after gradient centrifugatien in band 1 (Figure 7). lbs digstive vacuole membranes were observed to sediment with the plans membrane fragments. Ibis is not suprising in view of the fact that the digestive vacuoles are derived directly from the cell surface membranes (e.g. Kern and ”Heisman. 1967). Lyso- somes and psrozisomes. which normally sediment in the mioochondrial fraction (deDuve. 1967). were not apparent ultrastructurally. Pre- smava. these organelles were removed from the mitochondrial frac- tion by the extensive low-speed washings (and and Morhlok. 1969). With the exception of the mitochondria. all of the fractions illus- trated good morphological preservation and a relatively high degree of purity. w: lbs RNA/protein ratios (Table 1) of the elongate type of smooth endoplasmic reticulum and the rough endoplasmic reti- culum are in substantial agreement with published values of microscmes equilibrating at the same densities (mura. Sielcevits and Palade. 1967). 'me presence of RNA in the small. oistemal smooth menu-ans fraction and the Golgi membrane fraction correlated with the ultra- structural observations that free ribosome-liloe particles and rough endoplasmic reticulum vesicles. respectively were present in these fractions (Figms 13. 1b. 15 and 16). The negative chemical tests for 811A in the plasma and digestive vacuole membrane fraction also was in accord with electron microscope observations (Figure 10). m1. cells. as well as microsomes. contained phosphatin choline. phosphaticwl ethamlamins. phosphatim serine and phosphatichrl ino- sitol (Figure 18). 'h'iglyoerides. diphosphatiwl glycerol and sterols 91 also have been reported in W (Hack. Yaeger and McGaffery. 1962: Kalevy and Finlcelstein. 1965: Halevy. Avivi and Katan. 1966). W: Glucose-6-phosphatase is normally associated with microscmes in certain mam-alien organs (Swanson. 1955). The specific activity of this enzyme in the plasma and digstive vacuole membrane fraction was the highest observed. Some activity was also present in the Golgi and rough endoplasmic reticulum fractions. lhe presence of glucose-6-phosphatase activity in the mitochondrial fraction indicated a microsomal contaminant or activity in the mitochondrial membranes. Thiamine pyrophosphatase is nornlly used as a marker for Golgi membranes an! Gelgi associated membranes (Novilcoff. Essner. Goldfischer and nous. 1962). Very little of this enzyme was soluble (mule 2). High specific activities were found in the plasma and digstive vacuole membrane fraction. the Golgi membrane fraction and the small. cisternal smooth membrane fraction. The rough and elongate smooth endoplasmic reticulum contained a atelier. but considerable. amount of activity. Magnum-dependent adenosine tripho sphatase activity was ubiqui- tously distributed among the mantras-containing fractions (lable 2). This was not mooted. since this enzyme has been found in prac- tically all membranes (Novilooff. Essner. Goldfischer and Rene. 1962). What was suprising was the lack of stimulation of these activities in the plasma membrane-containing fraction by sodium and potassium ions. Sodium and potassium-stimtdated. magnesium-dependent adenosine triphosphatase is often used as a marker for cell surface membranes (Novilmff. Esmer. Goldfischer and Heus. 1962). The lack of monovalent 92 cation stimulation of the magnesia-dependent adenosine triphosphatase might have been due to the fact that the plasma and digestive vacuole membane fraction was isolated in a medium containing a high potas- sium content (0.02514 m). In contrast to the findings with glucose- 6—phosphatase and thiamine pyrophosphatase. 0.25M sucrose-Tm wash- ings had no deleterious effect upon adenosine triphosphatase activi- ties in the plasma and digestive vacuole membrane fraction. It is possible that the values observed with magnesium-dependent adenosine triphosphatase already include a cation stimulation. in alternative explanation would be that there is no sodium and potassium-stimulated. magnesium-dependent adenosine triphosphatase in the plasma membranes of M. Klein (1961;) observed that oubain. the classical in- hibitor of cation transport and sodium and potassium-stimulated. mag- nesium-dependent adeno sine triphosphatase. had no effect on potassium transport in W lhe enzyme analysis data ('Iable 2) indicated that the shame acti- vity in the plasma and digestive vacuole membrane fraction was en- riched for every phosphatase examined. It is not known whether the membrane-bound enzymes reside specifically in the plasma membranes. the digestive vacuole membranes or both. A mom EXCHANGE REACTION: W: The moms of C incorporated into phosphatidyl choline increased as a function of “-choline mitochondria or microsome protein concentration (Figure 25). 11c incor. poration into the non-membrane lipids of the post-micro somal 93 supematant fraction was observed. suggesting that membranes are the site of this reation. Incorporation of c:1 ”-choline into the phospha- tidyl choline of microscmes was linear as a function of time up to about 30 min. before leveling off (Figure 26). Vandor and Richardson. (1968). reporting on a virtually identical system in etiolated pea seed “microsomal suspensions”. offered 3 possible alternative reasons for the leveling off of incorporation: (1) depletion of an essential cofactor or substrate. (2) attainment of equilibrium by the reaction or (3) inactivation of the enzyme involved. As was demonstrated. addition of concentrated non-radioactive choline into the cell free system before or after the leveling off period. restllted in a rapid turnover of label (Figure 27). Thus. an equilibrium had been reached. where the amount of radioactive choline exchanging onto and off the microsomal phosphatidyl choline was the same. is in Vandor and Richardson's (1968) experiments. a as“ require. ment was indicated. The latter authors also gave evidence that the pea seed cell free system involved a single enzyme. Incorporation of choline into the membranes of isolated rat liver mitochondria has been reported (Kaiser and Bygrave. 1968: Bygrave and Kaiser. 1968). However. this was not a direct exchange reaction and required Optimal concentrations of ATP and 01?. indicating a synthe- tic pathway. ilthough a choline exchange reaction also has been re- ported in rat liver microsomes (Dils and Hdbscher. 1961). recent attempts to duplicate this finding have been unsuccessful (Nagley and Hallinan. 1968: Stein and Stein. 1969). : The data reported indicated that a choline exchange reation also occurred in living cells. If the concentrations of non-radioactive choline were increas- ed in turnover experiments. the loss of radioactivity from phospha- tifll choline was increased (Figures 22. 23 and 21.). 'nierefore. the turnover of the choline moiety of phosphatidvl choline was a function of the concentration of non-radioactive choline. Upon comparing 99],], W to mm W in tumover experiments. it we found that an equilibrium was eventually reached between the 99].], W and thMW (Figures 28 end 29)- file position of that equilibrium was dependent upon the concentration of non-radioactive choline in the mdium. With a larger concentration of non-radioactive choline. more radioactivity was lost. from the cells into the medium before leveling off occurred. Thus. in both the cell free and the living cell experiments. the exchange reaction was a function of the concentration of non-radioactive choline and reached an equilibrium. In the living cells. the attainment of an equilibrium coincided with the leveling off of loss of lipid extractable 291]. W (names 28 and 29). attending this to observations on cell fractions. the decrease in specific activities of cell fraction lipids also reached an equilitrium at the same time (Figure 30). In addition. it was observed that within 21+ hr post-chase. all the cell fractions' specific activities reached a stable position relative to each other. he non-membrane phosphatidvl choline of the post-micro somal superna- tant also showed this equilibration. If. in the living cells. as was 95 true in the cell free system. choline exchange only occurred with memlm'ane-bound phosphatidyl choline . than the non-memtrane pho spha- tidyl choline must somehow exchange with menu-ans phosphatim choline. before its choline moiety is exchanged. If this did not happen. the maximum decrease in specific activity that could be expected. would be that due to growth dilution (see Figure 30). Therefore. either the non-membrane phosphatidyl choline of the supernatant fraction exchanged (not necessarily a direct exchange) with membrane phospha- tidyl choline and had its choline moiety exchanged. or in contrast to the cell free system. a choline exchange reaction occurred in non- membrane phosphatim choline. It has been reported (Wirtz and Zilver- emit. 1968) that phospholipids. but not proteins. exchange between liver mitochondria and microscmes in m. It is possible. that an exchange between non-membrane and membrane phospholipids occurs in living W- LIPID SYNTHESIS AND ASSMBLY 01" LIPIDS INTO 1434380138: W: the results reported. indicated that H3-glycerol was a specific label for phospholipids and other glycerides (Table 6). Unlike (in-choline. variations in the concentration of non-radioactive glycerol had no effect on the rate of H3-glyoerol turnover in lipids (Figure 33 and 3b). In fact. glycerol-containing lipids exhibited a high degree of stability. For these reasons. Ila-glycerol was found to be a suitable label for studying the sites of synthesis of glycerol-containing lipids and assembly of these lipids into membranes. Incorporation of HB-glycerol into the non-membrane lipids of the post- mdcrosomal supernatant was linear and occurred at a rate faster than that found in any of the other cell fractions (Figures 35. 36 and 37). Of all the membrane fractions. incorporation was most rapid into the lipids of nuclear msabranes and rough endoplasmic reticulum. The rela- tive specific activity ratios in Table 8 illustrate that the rough endoplasmic reticulum was in its ”labeling equilibrium" position at the earliest time measured (5 min). or all the smooth membranes. the Golgi membranes were the only ones to reach their “labeling equilibrium“ position within the first 2 hr of incorporation. The other smooth mem- brane fractions reached "labeling equilibrium“ positions between 2 and 12 hr after incubation in HB-glycerol-ccntaining medium. The mito- chondria. which incorporated radioactivity into their lipids at a slow rate. did not seem to reach a stable specific activity position rela- tive to the other membranes. Since incorporation was most rapid in the post-microsomal superna- tant's non-membrane lipids. this location must be either a site of lipid synthesis or an area where newly synthesized lipids are rapidly transferred. The nuclear membranes and rough endoplasmic reticulum are implicated as sites of synthesis of lipids and assembly of these lipids into membranes. This would agree with the finding that newly synthe- sised phospholipids are incorporated more rapidly into rough surfaced microsomes. then smooth surfaced microsomes. in rat liver (Dallner. Siekevits and Palads. 1966a). The Golgi membrane fraction did not in- corporate radioactivity’as quickly as the post-microsomal supernatant. 97 nuclear membranes and rough endoplasmic reticulum: but the Golgi mem- brane fraction did reach its “labeling equilibrium' within the first 2 hr of incorporation (see Table 8). Thus. it is likely that these membranes did not synthesise lipids. but did incorporate lipids derived from another location at a faster rate than the other smooth membrane fractions. or incorporated at the same rate as the other smooth mem- brane fractions. but turned over faster. The elongate smooth endoplas- mic reticulum. the small. cisternal smooth membrane and the plasma membrane fractions might be membranes which derive presynthesised lipids from another'fracticn. Since the micochondria seem to be inde- pendent of the other’membrane systems with respect to a ilabeling equilibrium“ position. these organelles might synthesize and utilize their own membrane lipids. derive their lipids from another source or both (Stein and Stein. 1969). W- W: The specific activity of the non-membrane lipid was found to be greatly affected by the concentration of radioactive glyb cercl used to label the cells for 12 hr. whereas the membrane frac- tions were not (see Tables 8 and 11). Using the nuclear fraction as representative of the membranes (Table 11). it can be seen that a 10- fold increase of the amount of Fla-glycerol in the medium. resulted in an increase in membrane specific activity of about 3x. On the other hand. the specific activity of the non-membrane lipid increased 13X to 151. The “labeling equilibrium“ positions of the membrane fractions were unaffected by the concentration of glycerol available (Table 8). It seems logical. that if membrane lipids were derived from the 98 non-membrane lipid. a more rigid relationship between the specific activities of the non-membrane and membrane lipids would be main- tained. The given results would imply that membrane lipids are not derived from the pool of lipids available in the non-membrane lipid fraction. a 2‘» hr chase period (Figures 38 and 39) the non-membrane lipids of the post-microsomal supernatant decreased in specific activity at a slower rate than amr other fraction (Table 9). The relative specific activity ratios (Table 10) of the non-membrane lipids increased with respect to all the main-ans fractions. All the membranes decreased at a faster rate than that expected due to growth dilution (Table 9). Thus. it seems that lipid radioactivity lost from the membranes goes to the non-membrane lipids of the post-microsomal supernatant fraction. Qt all the membranes. activity was lost most rapidly from the nuclear membranes and rough endoplasmic reticulum (Table 9). This correlates with the results of the incorporation studies and again suggests that these 2 fractions might be sites of lipid synthesis and incorporation of lipids into membranes. The relative specific activity ratios (Table 10) of all the other membrane fractions increased with respect to the nuclear fraction. Omura. Sieloevits and Palade (1967) and ‘d'idnell and Siekevitz (1967) have reported that the glycerol moiety of phospholipids in rough and smooth microsomes. nuclear entrance and plasma membranes of rat liver turnover at the same rate. There are several distinctions between the 99 latter authors experiments and those being presented. First. in adult liver. cells are not growing and there is a question as to whether one is stuhing newly elaborated membranes. molecular re- placement in stable membranes. or both. Secondly. and most impor- tantly. in the liver cell studies. no measurements were made before 23 hr post-injection of the isotope. In this manner. any pattern of equilibration of lipid radioactivity through. for instance . recycling of membranes or memlm'ane lipids would have been missed. In the experi- ments being considered. the cells were growing and an elaboration of new membranes was assured. Turnover did seem to be apparent. with loss of lipids from the membranes to the non-membrane fraction. In the W experiments. the snot rates of turnover were not being measured. but the relative changes in specific activity ratios were. and indicated that the nuclear membranes and the rough endo- plamaic reticulum were probable sites of lipid synthesis and assembly of lipids into membranes. In both the 'long" and the “short pulse“ experiments (Figures 110 and M) the non-membrane lipids of the post-microsomal supematant continued to increase in specific activity in the first hr post-chase. This. along With the ”Cults of the labeling experiments (hble 11) and the tumour experiments (Figures 38 and 39) is evidence that the non- membrane lipids are synthesised in and derived from memtranes. In the second half of the 'short pulse“ experiment the specific activity leveled off and in the second part of the "long pulse' experiment the specific activity decreased. This would imply that the non-membrane 100 lipids were finally being diluted by chase phopholipids. transfering some of their lipids back to the membranes or some combination of these 2 events. Several factors algae against. but do not rule out. reutilisation cf non-membrane lipids in membranes. First. the equi- librium ratios of the membrane fractions were not affected by the amount of radioactive glycerol available (Tables 8 and 11). Secondly. ever a 24 hr chase period (Figures 38 and 39; Thble 10) radioactivity was lost from the membranes to the non-membrane lipid. In the first hr immediately following both the I'Zlm‘lg" and the “short pulse" experiments. the nuclear membranes' specific activities leveled off. This would imply that if the nuclear memlranes are sites of lipid synthesis and incorporation of lipids into memtranes. these membranes are not transferred away from the nucleus very rapidly. Another alternative would be that the nucleus rapidly receives mem- branes with lipids mthesised elsewhere or non-neutrane lipids. and the chase effect is somewhat mashed. In the second hr of the “short pulse" experiment. the nuclear membranes rapidly decreased in specific activity. However. in the second half of the “long pulse" experiment. the nuclear membranes increased in specific activity. Thus. a source of presynthesised lipids for the nuclear membranes is ”hot“ in the 'long pulse" experiment and “cold“ in the “short pulse“ experiment. This source might be either recycled membrane lipids or non-membrane lipids. In both the "short" and the ”long pulse” experiments the non- seen-me lipids' specific activity .... latch higher then the nuclear memh'ane specific activity: but it was only in the “long pulse" 101 experiment that all the membranes were well labeled. Thus. it would have to be suggested that the post-chase increase. in the nuclear memlranes specific activity in the second half of the ”long pulse" experiment. was due to recycling of membrane lipids. I The rough endoplasmic reticulum decreased rapidly in specific ac- tivity in the first hr following the chase in both the “short" and the “long pulse“ experiments. This fact. together with all the other experimental data to this point. pinpoints this location as the major lipid synthesizing site in the cell. In addition. because the specific activity decreased rapidly. the lipids in the rough endoplasmic reti- culum met be rapidly transferred elsewhere. The only synthesized lipids could be transferred as non-membrane lipids. in the form of membranes. or both. In the second half of both the "long" and the ”short pulse“ experiments. the rough endoplasmic reticulum in- creased in specific activity. Again. a recycling of lipids is apparent. The call. cisternal smooth membranes decreased in specific acti- vity in the first hr of both the 'long" and the 'short pulse" experi- ments. The incorporation evidence does not implicate this type of meats-ans as a site of lipid synthesis. If it is not a site of lipid synthesis. then it must be an area which either rapidly incorporates newly sysnthesised lipids. rapidly transfers these lipids elsewhere or a combination of thesesultedhthe second hr of the 'short pulse” experi- ment. the specific activity of this fraction leveled off. In the se- cond part of the l'lcng pulse“ experiment this fraction increased slightly in specific activity. Again. a recycling effect is possible. 102 The Golgi membranes' specific activity decreased in the first hr of the 'short pulse” emeriment and increased in the first hr of the 'long pulse" experiment. From the incorporation data. it was suggested that the Golgi membranes were not sites of lipid synthesis but were sites which incorporated newly synthesized lipids. If. in addition. its memtranes were rapidly turning over (i.e. transferring these lipids elsewhere). this would fit into the rapid decrease in specific acti- vity observed in the first part of the "short pulse“ experiment. Since it increased in specific activity initially. following the chase in the I'long pulse” experiment. its source or sources of membrane lipids remain 'hct' for a considerable time following the chase. This frac- tion' s specific activity leveled off in the second half of the "short pulse” experiment and decreased rapidly in the second part of the "long pulse" experiment. One could envision that this was when the chase effect was finally felt. but since it was observed that other fractions were actually increasing in specific activity at this time. a recycling effect is more probable. in addition to the chase effect. Of all the memtranes. the plasma and digestive vacuole membranes incorporated radioactivity the slowest (Figures 35. 36 and 37) . even though their eventual I'labeling equilibrium" positibn was higher than. for example. the Golgi membranes (Table 8). .This fact. coupled with the knowledge that the plasma membranes continued to increase in both the 'long' and the ”short pulse.. experiments strongly suggests that these membranes are derived from another membrane type. The fact tint the elongate type of endoplasmic reticulum increased in the first part of the ''short pulse' experiment would suggest that 103 these membranes. like the plasma membranes were receiving prcsynthe- sized membranes. However. their decrease in specific activity in the first part of the ”long pulse“ experiment seems to contradict this conclusion. and at present can not be explained. (hoe again. the mitochondria seem to be the exceptions. In both the ”long" and the “short pulse' experiments. this fraction simply levels off. Therefore . mitochondria either synthesise their own lipids. utilize newly synthesized lipids. or both (of. Stein and Stein. 1969). The mitochondrial fraction’ s specific activity did not decrease at a rate much different than any of the other membranes in the turnover experiments (Figures 38 and 39: Table 9). This would suggest. that although the modes of incorporation are different . the rates of turnover are about the same. W: The rough endoplasmic reticulum is probably the major site of phospholipid and glyceride synthesis in W m m. The nuclear membranes might also be sites of lipid synthesis. but most certainly are sites where labeled membranes rapidly appear. All the other membrane fractions seem to receive prcsmthesized lipids. possibly in the for: of membranes. The post-microsomal. supernatant's non-membrane lipids are mostly derived from newly synthesized membrane lipids. However. lipids from membrane turnover also seem to be trans- ferred to this fraction. Since no membrane fraction completely lost its radioactivity in the turnover and the pulse and chase emeriments. one would have to predict a reutilisation or recyling of membrane lipids. Gyclio patterns observed in the pulse and chase experiments. suggest that there is a recycling of membrane lipids. It seems likely. 1015 particularly in the case of the plasma and digestive vacuole membranes. that this recycling occurs within the structure of the memtranes. However. in most of the cases. it can not be stated with certainty tint this recycling of membrane lipids involves the transformation of one membrane type to another. although it is strongly suggested. 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