A phosphatidyl choline exchange protein isolated from germinated castor bean endosperms

A phospholipid exchange protein (PLEP) functioning between theendoplasmic reticulum and the mitochondrion was purified fromthe cytosolic fraction of germinated castor bean endosperms.In the protein fraction eluted from Sephadex G-100 column, theexchange rate reached 7.3µg phospholipids exchanged/mgprotein/15 min, which was 60-fold that of pota to tuber PLEP.The lipid transfer by this protein was specific for phosphatidylcholine and the transfer rate from microsomes to mitochondriawas as high as that from mitochondria to microsomes. Castorbean PLEP transferred phospholipid from castor bean microsomesto mitochondria from other sources such as potato tubers, cauliflowerinflorescences, pumpkin hypocotyls and rat livers, and to liposomes,but not to Avena etioplasts. In addition, it transferred phospholipidfrom potato microsomes to potato mitochondria. (Received November 17, 1978; )     

Recent studies of the enzymic synthesis of ricinoleic Acid by developing castor beans

Oleate Δ¹²-hydroxylase activity was measured in extracts of developing castor bean seeds. Most of the hydroxylase activity is associated with microsomes. However, when microsomes are washed, the activity is completely lost. Some (50%) of the activity can be restored by addition of the 100,000g supernatant to the washed microsomes. Supernatant extracts (100,000g) of developing safflower seeds are able to restore all (100%) of the hydroxylase activity to the washed castor bean microsomes. In addition, purified mammalian catalase can restore some (25%) of the activity to the microsomes but is not as effective as either castor bean or safflower 100,000g supernatants. The K_m of the hydroxylase for oxygen is 4 micromolar. Inasmuch as the activity was not inhibited by high concentrations of either carbon monoxide or cyanide, neither the involvement of cytochrome P450 nor other cytochrome systems is suggested. The enzyme system was not saturated by oleoyl-CoA, even at concentrations as high as 200 micromolar. When [¹⁴C]oleoyl-CoA is supplied as a substrate, the acyl component is rapidly transferred to phosphatidylcholine (PC). Hydroxylation may occur on PC or on a lipid which receives its acyl component from PC. However, exogeneously added 2-[1-¹⁴C]oleoyl-PC was hydroxylated at a much lower rate than was [1-¹⁴C]oleoyl-CoA added as the primary substrate.     

Evidence that a Proliferation of the Endoplasmic Reticulum Precedes the Formation of Glyoxysomes and Mitochondria in Germinating Castor Bean Endosperm

Excised castor bean endosperm halves incubated with CDP-[Me-¹⁴C]cholineactively incorporated this compound into membrane phosphatidylcholine.The capacity of the tissue to synthesize phosphatidyl-[¹⁴C]cholineincreased during the first 3 d of germination and subsequentlydeclined. At the onset of germination phosphatidyl-[^l4C]cholinewas exclusively recovered in the ER membrane fraction. The rateof incorporation into the ER membranes increased strikinglyduring the first 24 h of germination while that into mitochondriaand glyoxysomes remained low. At later developmental stagesan increasing proportion of the newly synthesized phosphatidyl-[¹⁴C]cholinewas present in mitochondria and glyoxysomes; the rate of incorporationinto the membranes of these organelles increased while thatinto the ER membrane began to level off. The kinetics of CDP-[¹⁴C]cholineincorporation into membrane phosphatidylcholine of the majororganelle fractions of 3-d-old endosperm tissue showed thatthe ER was immediately labelled, whereas a lag period precededthe labelling of mitochondria and glyoxysomes. Assuming that the incorporation of CDP-[¹⁴C]choline into phosphatidylcholineserves as a reliable indicator of membrane synthesis, the resultsobtained suggest that a proliferation of ER membranes precedesthe formation of glyoxysomes and mitochondria in germinatingcastor bean endosperm. A comparison of developmental changesin (a) total ER and glyoxysomal phospholipid content and (b)ER and mitochondrial NADH cytochrome c reductase activity providedadditional evidence supporting this conclusion.     

Long chain fatty acid synthesis in developing castor bean seeds IV. The synthetic system in proplastids

The proplastid fraction containing no cytosol and mitochondrionwas isolated from developing castor bean endosperm by stepwisesucrose density centrifugation. This fraction possesses thecapacity to synthesize LFAs from [u-14C]sucrose, [u-¹⁴C]-glucose,[u-¹⁴C]G-1-P, [u-¹⁴C]G-6-P, [2-¹⁴C]pyruvate and [1-¹⁴C]acetate.Little was incorporated from [1-¹⁴C]pyruvate into LFAs, butmuch into ¹⁴CO_a. Addition of cytosol to the proplastid fractiondid not enhance the LFA synthesis. From these data, the wholepath from sucrose to LFAs through glycolytic path and pyruvatedecarboxylation seems to be located within the proplastid indeveloping castor bean endosperm. The difference in utilizationof substrates indicates that the rate of LFA synthesis in castorbean proplastids is limited at a step between sucrose and hexosephosphate. In addition, experiments with CO₂ output and LFAsynthesis from [1-¹⁴C]glucose, [6-¹⁴C]glucose and [u-¹⁴C]G-6-Pstrongly suggest that the path flow branches actively throughG-6-P to the pentose phosphate path and little through acetylCoAto the TCA cycle. (Received May 12, 1975; )     

Glutamine synthesis in germinating castor bean endosperm

The pathway of glutamine synthesis in germinating castor beanendosperm was investigated by feeding experiments with (2,3-¹⁴C)succinateand by determining enzyme activities related to pyruvate formationand utilization. ¹⁴C of (2,3-¹⁴C)succinate was rapidly and sequentiallyincorporated into amino acids in the following order: aspartateor alanine, glutamate and glutamine. ¹⁴CO₂ was slowly released,especially during the early hours of incubation. Fluorocitrateinhibited ¹⁴CO₂ release while aminooxyacetate stimulated itslightly. Fluorocitrate inhibited the incorporation of ¹⁴C intoglutamate and glutamine. Aminooxyacetate inhibited ¹⁴C incorporationinto aspartate, alanine, glutamate and glutamine. Glutaminesynthetase activity was detected in a soluble fraction. NAD-malicenzyme activity was detected in mitochondria by sucrose densitygradient centrifugation. Activities of pyruvate decarboxylaseand aldehyde dehydrogenasewere detected. Aldehyde dehydrogenasewas partially purified about 60-fold by ammonium sulfate fractionationand the DEAE-cellulose chromatography. The Km values of theenzyme were 0.71 miu for NAD and 0.43 mM for acetaldehyde. Basedon these results and properties of pyruvate kinase reportedpreviously (9), the metabolism of pyruvate in cytosol and mitochondriawas discussed in connection with glutamine synthesis in germinatingcastor bean endosperm. (Received August 25, 1978; )     

Oxidative phosphorylation and the electron transport system of castor bean mitochondria

The difference spectrum (reduced minus oxidized) of castor bean(Ricinus communis L.) mitochondria showed the presence of cytochromeoxidase (cytochromes a+a₃), b-type cytochromes and cytochromec. The mitochondria actively oxidized succinate, -ketoglutarate,pyruvate and exogenous NADH, and oxidations of these substrateswere stimulated by added ADP, as in mammalian mitochondria.Values for the P/O ratio obtained for succinate, pyruvate and-ketoglutarate were the same as those reported for mammalianmitochondria, indicating that theoretical values are 2, 3 and4, respectively. The theoretical P/O ratio for exogenous NADHseemed to be 2. Oxidations of succinate and exogenous NADH instate 3 were almost completely inhibited by 0.3 mM cyanide and10 µM its antimycin A, while those of NAD⁺-linked substratesin state 3 were not completely suppressed even by excess concentrationsof these inhibitors. There seem to be two types of pathway forelectron transfer in the oxidation of NAD⁺-linked substratesin castor bean mitochondria, i.e. pathways which are sensitiveand insensitive to these inhibitors. Oxidation of exogenousNADH in state 3 was not inhibited by rotenone. Transitions of redox levels of the respiratory components fromstate 4 to state 3 on addition of ADP and from state 3 to state4 on exhaustion of added ADP were observed with a dual-wavelengthspectrophotometer. Effects of inhibitors on redox levels ofthe respiratory components in state 3 were investigated. Cytochromesof b-type and cytochrome c were fully reduced on addition ofcyanide. Cytochromes of b-type were also fully reduced on additionof antimycin A, but cytochrome oxidase (cytochromes a + a₃)and cytochrome c changed to the oxidized forms. The redox levelof the component(s) with an absorption maximum at 465 mµshifted further, but not completely, to the reduced side onaddition of antimycin A. However, this component(s) was oxidizedon addition of cyanide. Cyanide-, or antimycin A-resistant oxidationof NAD⁺-linked substrates seems to occur via an alternate electrontransfer pathway branching from NAD⁺-linked flavoprotein(s)in the mitochondria, not via the normal pathway through thecytochromes-cytochrome oxidase system. (Received June 8, 1970; )     

Echanges d'acides gras in vitro entre mitochondries, microsomes et surnageants cytoplasmiques de cellules de pommes de terre et de chou-fleur

When potato tuber or cauliflower buds mitochondria, containing fatty acids labelled from [1-¹⁴C]acetate, are incubated in a small volume of cytoplasmic supernatant with unlabelled microsomes isolated from the same species, the specific radioactivity of the mitochondrial fatty acids decreases and the fatty acids of the microsomes and supernatant become radioactive. The same result is obtained when labelled microsomes are mixed with unlabelled mitochondria. These data suggest an exchange of fatty acids from microsomes to mitochondria and in the opposite way. All types of fatty acids are transferred, the major ones being the most actively exchanged. There is no breakdown of fatty acids and no subsequent synthesis during the transfer since the distribution of the total radioactivity remains constant among the various fatty acids. The same transfers are observed when potato mitochondria are incubated with cauliflower microsomes or reciprocally.

The cytoplasmic supernatant plays an important role in these phenomena; the labelled fatty acids of the supernatant can be transferred to unlabelled microsomes or mitochondria from the same tissue. The transfer is less active from a supernatant of one species to organelles of another species.     

Particulate cytochromes of mung bean seedlings

Efforts have been made to solubilize cytochrome components from particulate fractions of etiolated mung bean seedlings. Low temperature spectrophotometry reveals that the cytochrome composition of mitochondria isolated from whole seedlings is the same as that reported by Bonner for mung bean hypocotyls. On the basis of the identity in position of the α-bands in low temperature difference spectra for mitochondria, for a partially purified haemoprotein from mitochondria, and for purified cytochrome b-555, it is suggested that cytochrome b-555 is an intrinsic component of mung bean mitochondria. Difference spectra show that both the mitochondrial and microsomal fractions contain at least 2 b-type cytochromes. Cytochrome b-555 is almost certainly present in the microsomes, since the low temperature difference spectrum for the cytochrome is identical with the spectrum for this particulate fraction.

By freezing and thawing mung bean mitochondria in 4% cholate and centrifuging, cytochrome oxidase activity can be concentrated in the supernatant fraction, although it is not completely solubilized. The oxidase is inhibited by high concentrations of cytochrome c. A particle-bound cytochrome c can be obtained from mitochondria by digestion with snake venom. However, the autoxidizability of the preparation indicates that the cytochrome has been solubilized in a modified form. A CO-binding pigment can be obtained from mung bean microsomes by digestion with snake venom.

     

Properties of a soluble ATPase from castor bean endosperm mitochondria

An ATPase was extracted and purified from castor bean endospermmitochondria. The enzyme is stable at 60°C only in the presenceof ATP in the incubation medium. It is less stable at 0°Cthan at 30°C but is stabilized by ammonium sulfate or glycerol.Activity is dependent on the presence of Mg⁺⁺, and in the presenceof Mg⁺⁺ is enhanced by 2,4-dinitrophenol, but is not inhibitedby oligomycin. The enzyme hydrolyzes ITP in addition to ATP,but ITPase activity is hardly enhanced by 2,4-dinitrophenol.This preparation has many properties in common with the ATPase(coupling factor 1) from beef heart mitochondria. (Received November 8, 1969; )     

Electron transport dependent adenosine triphosphatase activity in castor bean endosperm mitochondria

The release of inorganic phosphate from ATP by mitochondriaisolated from endosperms of castor bean (Ricinus communis) wasstimulated by Mg⁺⁺, but not by Ca⁺⁺. EDTA, succinate, NADH₂or oligomycin depressed the reaction. The depression by succinatewas removed by KCN, antimycin A or anoxia. DNP alone did notaffect activity but did stimulate the Pi release in the presenceof succinate under aerobic conditions. Enhanced Pi release inthe presence of succinate and DNP was cancelled by KCN, antimycinA, oligomycin or anoxia. On the basis of these results, themechanism of ATPase action in castor bean endosperm mitochondriais discussed. (Received January 27, 1969; )     

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : Changes in Enzyme Levels Induced by Fungal Infection and Intracellular Localization of the Pathway

Casbene is a macrocyclic diterpene hydrocarbon that is produced in young castor bean (Ricinus communis L.) seedlings after they are exposed to Rhizopus stolonifer or other fungi. The activities of enzymes that participate in casbene biosynthesis were measured in cell-free extracts of 67-hour castor bean seedlings (a) that had been exposed to R. stolonifer spores 18 hours prior to the preparation of extracts, and (b) that were maintained under aseptic conditions throughout. Activity for the conversion of mevalonate to isopentenyl pyrophosphate does not change significantly after infection. On the other hand, the activities of farnesyl pyrophosphate synthetase (geranyl transferase), geranylgeranyl pyrophosphate synthetase (farnesyl transferase), and casbene synthetase are all substantially greater in infected tissues in comparison with control seedlings maintained under sterile conditions. The subcellular localization of these enzymes of casbene biosynthesis was investigated in preparations of microsomes, mitochondria, glyoxysomes, and proplastids that were resolved by centrifugation in linear and step sucrose density gradients of homogenates of castor bean endosperm tissue from both infected and sterile castor bean seedlings. Isopentenyl pyrophosphate isomerase and geranyl transferase activities are associated with proplastids from both infected and sterile seedlings. Significant levels of farnesyl transferase and casbene synthetase are found only in association with the proplastids of infected tissues and not in the proplastids of sterile tissues. From these results, it appears that at least the last two steps of casbene biosynthesis, geranylgeranyl pyrophosphate synthetase and casbene synthetase, are induced during the process of infection, and that the enzymes responsible for the conversion of isopentenyl pyrophosphate to casbene are localized in proplastids.     

The Effects of Gibberellic Acid on Organelle Biogenesis in the Endosperm of Germinating Castor Bean Seeds

Mature seeds of castor bean (Ricinus communis L.) were imbibedin tap water or 0.3 mM GA₃, planted in vermiculite moistenedwith tap water or 0.3 mM GA₃, and incubated at 32 ?C. Duringthe course of germination, GA₃ promoted marked increases inthe activities of the glyoxysomal marker enzyme isocitrate lyaseand certain mitochondrial marker enzymes, but did not affectthe ER marker enzyme choline phosphotransferase. Glyoxysomaland ER protein and phospholipid were not increased in amountby GA₃, whereas mitochondrial protein and phospholipid were.SDS-polyacrylamide gels of the glyoxysomal matrix polypeptidesfrom GA₃-treated beans exhibited two polypeptides additionalto those found to be common to both GA₃-treated and controltissue. Incorporation of CDP-(methyl ¹⁴C)-choline into intactendosperm tissue and the distribution amongst the glyoxysomes,mitochondria, and ER of newly synthesized phosphatidyl-(methyl¹⁴C)-choline was unchanged by GA₃.     

Biosynthesis, Intracellular Transport and In Vitro Processing of 11S Globulin Precursor Proteins of Developing Castor Bean Endosperm

In vivo labeling experiments to study the biosynthesis of 11Sglobulin in developing castor bean (Ricinus communis) endospermdemonstrated that the subunit polypeptides of the 11S globulinwere synthesized as high molecular weight precursors with heterogeneousmolecular weights. These proglobulin species were not synthesizedconcomitantly during seed maturation. The largest proglobulinwas synthesized from 20 days after anthesis, whereas the smallerproglobulins were synthesized from 30 days after anthesis. Subcellularfractionation of the pulse-labeled endosperm showed that the[³⁵S]methionine label was present in proglobulins in both theendoplasmic reticulum (ER) and dense vesicles shortly afterthe pulse labeling. The label in the proglobulin in ER decreasedduring the chase and appeared in mature globulins associatedwith crystalloids of vacuoles (protein bodies). Proglobulinsin the ER fraction prepared from the pulse-labeled developingendosperm were processed in vitro into globulins by the matrixfraction of protein bodies isolated from the dry castor bean.Overall results indicate that precursor proglobulin moleculessynthesized on rough ER are transported to vacuoles via densevesicles, and are cleaved there by the matrix protease to yieldmature globulin. ¹Department of Botany, University of Maryland, Present address:CollegePark, MD 20742, U.S.A. ²Department of Biology, Faculty of Science, Kobe University,Present address:Rokkoudai, Nada, Kobe 657, Japan (Received June 1, 1987; Accepted December 16, 1987)     

Deterioration of Mitochondrial Membranes from Castor Bean Endosperm by Hydrogen Peroxide

Loss of membrane integrity by hydrogen peroxide (H₂O₂) was studiedin isolated mitochondria of castor bean. Incubation of mitochondriawith H₂O₂ resulted in the release of fumarase with a concomitantloss of phospho-lipids. Degradation of membrane was found tooccur independent of lipid peroxidation. H₂O₂-treated mitochondriawere able to degrade exogenous radiolabelled phospho-lipids. ³Corresponding author; fax 82-42-821-2391     

Subcellular localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase and other membrane-bound enzymes in sweet potato roots

The subcellular localization of 3-hydroxy-3-methylglutaryl coenzymeA reductase and other membrane-bound enzymes in fresh, cut anddiseased sweet potato root tissues was resolved by differentialcentrifugation and sucrose density gradient centrifugation.In fresh, cut and diseased tissues, cytochrome c oxidase wasalmost localized in mitochondria, and NADH cytochrome c reductasewas in mitochondria in fresh and cut tissues, but in both mitochondriaand microsomes in diseased tissue. NADPH cytochrome c reductaseand antimycin A insensitive NADH cytochrome c reductase weremainly associated with microsomes. Catalase was dominantly foundin the mitochondrial fraction. 3-Hydroxy-3-methylglutaryl coenzymeA reductase was localized only in mitochondria and not in microsomaland supernatant fractions in both fresh and cut tissues. Indiseased tissue (infected with Ceratocystis fimbriata), in additionto being present in mitochondria, the enzyme was also localizedin microsomes. These results indicate that microsomal 3-hydroxy-3-methylglutarylcoenzyme A reductase whose activity rapidly increased in responseto the infection, predominandy participates in the formationof terpenes such as ipomeamarone. ¹ This paper constitutes Part 122 in the Series "The PhytopadiologicalChemistry of Sweet Potato with Black Rot and Injury." (Received March 1, 1976; )     

Role of Phospholipid Transfer Protein in the Exchange of Phospholipids between Microsomes and Chloroplasts

When microsomes containing phosphatidylcholine labelled with[1-¹⁴C]-linoleate were incubated with pea or spinach chloroplasts,active transfer of this phospholipid took place in the presenceof phospholipid transfer protein. This transfer also was demonstratedby incubating unlabelled microsomes, chloroplasts and the phospholipidtransfer protein in the presence of [1-¹⁴C]-acetate. The reconstitutedsystems could synthesize fatty acids which were acylated inmicrosomal phosphatidylcholine. The transfer of this phospholipidto chloroplasts is mediated by the transfer protein. Our resultssuggest a role for phospholipid transfer protein in the synthesisof chloroplast lipids. (Received October 25, 1983; Accepted July 18, 1984)     

Inattivazione dei Mitocondri Negli Endospermi di Ricino Durante la Maturazione

Abstract

inactivation of mitochondria in the castor bean seed endosperm during ripening. — The present research deals with the behaviour of mitochondria from the endosperm of castor bean seeds, during the last phases of seed maturation. The activities of citochrome oxidase, of malate dehydrogenase, of the succinate-citochrome c reductase system, and the phosphorylating activity, were chosen as tests of the state of mitochondria.

The results obtained show an increase of the first two activities up to the moment when some ovular tissues are still present, and, successively, a more or less rapid inactivation of the three enzymes investigated, which fall to extremely low values in the dry seed. Also the phosphorylating capacity, high during the first phases, drops quickly as the seed approches to dormancy.

A certain amount of citochrome oxidase is revealed in the supernatant from 20000xg centrifugation made to prepare the mitochondrial fraction; its activity gradually increases as the seed advances to ripeness. A further fractionation of the activity not sedimenting at 20000xg reveals that approximately one half of it sediments when centrifugated for 1 hour at 50000xg, while the other half remains in the supernatant. The particles sedimenting between 20000 and 50000xg show very little, if any, phosphorylating capacity (with succinate).

It is suggested that the gradual inactivation of mitochondrial efficiency during the ripening phase is due to a degradation of mithochondrial structures.     

Studies on NADP-isocitrate dehydrogenase from maturing castor bean seeds

NADP-specific isocitrate dehydrogenase from the soluble fractionof maturing castor bean endosperm was partially purified (approximately180-fold) and some of its enzymatic properties were studied.Mg⁺⁺, Mn⁺⁺, Cd⁺⁺, Ba⁺⁺, Co⁺⁺, Zn⁺⁺, and Sr⁺⁺ were activatorsof the enzyme reaction at a concentration of 6.7x10 M. The optimumpH of this enzyme was about 8.5. The enzyme was stable in thenarrow range from pH 7.0 to pH 8.0. Km values for isocitrateand NADP at pH 8.5 were 3.5x10^–6 M and 3.6x10^–6M, respectively. Enzyme stability was not affected by NaCl concentrationand enzyme reaction was inhibited at 5x10^–6 M PCMB (80%inhibition). It is suggested that the condensation product ofglyoxylate and oxalacetate also inhibits the reaction. NADP-IDHin the crude extract from maturing castor bean endosperm washeat-stable but the dialyzed enzyme preparation and the partiallypurified enzyme were labile against heat treatment at 57°C.When Mg⁺⁺ was added to the partially purified enzyme in thepresence of isocitrate or NADP, the enzyme was stabilized againstheat treatment. Mn⁺⁺, Ca⁺⁺, Co⁺⁺, Sr⁺⁺ or Ba⁺⁺ could be substitutedfor Mg⁺⁺. Addition of only one of the factors, Mg⁺⁺, isocitrateor NADP, had no effect on the heat stability. Moreover, a combinationof isocitrate and NADP did not establish stabilization. A divalentcation plays a central role, while adenine nucleotide, especiallyATP, may have an important part in stabilization. (Received August 14, 1972; )     

Involvement of a lipid-linked intermediate in the transfer of galactose from UDP [14C]galactose to exogenous protein in castor bean endosperm homogenates

A crude organelle preparation from germinating castor bean endosperm catalysed the incorporation of galactose from UDP[¹⁴C]galactose into chloroform/methanol (2:1)-soluble glactolipids. At least two galactolipids were formed. Most of the [¹⁴C]galactose was present in a galactolipid synthesized by the microsomal membranes, the remainder was present in a second galactolipid synthesized by other cellular membranes, possibly Golgi-derived. The addition of asialo-agalacto-fetuin reduced incorporation of [¹⁴C]galactose into the microsomal galactolipid with a concomitant increase in microsomal [¹⁴C]galactoprotein. Asialo-agalacto-fetuin did not affect galactolipid or galactoprotein synthesis by nonmicrosomal fractions. The results suggest that the endoplasmic reticulum is a major site of protein galactosylation in castor bean endosperm cells, and that galactose transfer from UDP-galactose to protein occurs via a lipid-linked intermediate.Abbreviations ER endoplasmic reticulum - ASGF asialoagalacto-fetuin - IDPase inosine diphosphatase - TCA trichloroacetic acid     

Comparative study of lipid transfer between cell organelles using lipid-transfer proteins in vitro

The transfer of de novo synthesized lipids from microsomes to lipid non-synthesizing membranes was studied in vivo and in vitro from the ratios of specific radioactivities of [14C]cholesterol, [14C] and [32P]phosphatidylcholine and [32P]phosphatidylethanolamine in the nuclei and mitochondria to that in microsomes. The radioactivity of lipids transferred from microsomes to mitochondria and nuclei was identical both in vitro and in vivo and when the lipid-exchange protein of the 105 000 g supernatant was used. Acceleration of lipid metabolism in the liver of gamma-irradiated rats was concomitant with the increase in the rate of labeled cholesterol transfer cation to liver cell nuclei and mitochondria, but remained unchanged in in vitro studies involving lipid-exchange protein. The reduction of phosphatidylethanolamine transfer to the nuclei in vitro and in vivo diminished in the same way. The existence in the cell of mechanisms of transfer of de novo synthesized cholesterol other than lipid-exchange protein is postulated.