Lecithin synthesis during microbody biogenesis in watermelon cotyledons

During the normal development of watermelon seedlings, leaf peroxisomes succeed glyoxysomes as the major microbody component in the cotyledons. The possibility has thus been raised that the two organelles are ontogenetically related; that leaf peroxisomes are derived from glyoxysomes. The behavior of lecithin, an important constituent of the membranes of both kinds of organelle was examined in this study. Using labeled choline as a precursor of lecithin, its incorporation into various membrane fractions was followed during the period when glyoxysomal activity was declining and that of leaf peroxisomes increasing after exposure to light. The results showed that glyoxysomal membrane was selectively destroyed during this period. Furthermore, from double-labeling experiments using [¹⁴C]- and [³H]choline it was shown that newly synthesized lecithin was incorporated into the membranes of the developing leaf peroxisomes. These results support the thesis that leaf peroxisomes are not derived from glyoxysomes and instead represent two distinct microbody populations.     

ACYLATION OF LYSOPHOSPHATIDES BY PLASMA MEMBRANE FRACTIONS OF RAT LIVER

The plasma membrane fraction of rat liver was isolated and incubated with labeled lysophosphatides in the presence of cofactors; the acylation of lysolecithin to lecithin by the fraction was compared to that of the rough and smooth microsomes. The purity of the isolated fractions was ascertained by enzyme markers and electron microscopy, and the maximal contamination of the plasma membrane fraction by microsomes did not exceed 20%. Under conditions at which the reaction was proportional to the amount of enzyme used, the plasma membrane had a specific activity similar to that of the smooth and rough microsomes. With doubly labeled lysolecithin (containing palmitic acid-¹⁴C and choline-³H) it was shown that the lecithin formed retained the same ratio of the two labels, which indicated that lysolecithin was converted to lecithin through an acylation reaction. The newly formed lecithin was shown to be bound to the plasma membrane fraction; this suggested that it is incorporated into the structure of the membrane itself.     

LOCALIZATION OF ENZYMES WITHIN MICROBODIES

Microbodies from rat liver and a variety of plant tissues were osmotically shocked and subsequently centrifuged at 40,000 g for 30 min to yield supernatant and pellet fractions. From rat liver microbodies, all of the uricase activity but little glycolate oxidase or catalase activity were recovered in the pellet, which probably contained the crystalline cores as many other reports had shown. All the measured enzymes in spinach leaf microbodies were solubilized. With microbodies from potato tuber, further sucrose gradient centrifugation of the pellet yielded a fraction at density 1.28 g/cm³ which, presumably representing the crystalline cores, contained 7% of the total catalase activity but no uricase or glycolate oxidase activity. Using microbodies from castor bean endosperm (glyoxysomes), 50–60% of the malate dehydrogenase, fatty acyl CoA dehydrogenase, and crotonase and 90% of the malate synthetase and citrate synthetase were recovered in the pellet, which also contained 96% of the radioactivity when lecithin in the glyoxysomal membrane had been labeled by previous treatment of the tissue with [¹⁴C]choline. When the labeled pellet was centrifuged to equilibrium on a sucrose gradient, all the radioactivity, protein, and enzyme activities were recovered together at peak density 1.21–1.22 g/cm³, whereas the original glyoxysomes appeared at density 1.24 g/cm³. Electron microscopy showed that the fraction at 1.21–1.22 g/cm³ was comprised of intact glyoxysomal membranes. All of the membrane-bound enzymes were stripped off with 0.15 M KCl, leaving the "ghosts" still intact as revealed by electron microscopy and sucrose gradient centrifugation. It is concluded that the crystalline cores of plant microbodies contain no uricase and are not particularly enriched with catalase. Some of the enzymes in glyoxysomes are associated with the membranes and this probably has functional significance.     

Compartmental utilization of carboxyl-C-tripalmitin by tissue homogenate of pine seeds

A tissue homogenate of megagemetophyte of germinating seeds of Jeffrey pine (Pinus Jefferii Grev. and Balf.) was incubated with sonication-dispersed and albumin-carried ¹⁴C-tripalmitin in order to elucidate the sequential and quantitative role of cellular organelles in utilizing lipid reserve in seeds. After 5 minutes at 30 C, 25% of the tracer was localized in the fat body fraction, 9% in the pellet containing mitochondria and glyoxysomes, 14% in the supernatant, and 2% was found as CO₂. Radioactivity increased with time of incubation in the latter three fractions indicating the forward direction of utilization. Fat bodies contained mainly lipases and hydrolyzed the tracer to palmitate with diglyceride and monoglyceride as intermediates. About two-thirds of the palmitate had left the fat bodies in 5 minutes and entered the pellet fraction within which the tracer was distributed 1:2 in mitochondria and glyoxysomes, respectively. Longer incubation reduced the ratio to 1:3 while both organelles acquired more radioactive intermediates. Labeled acetyl-CoA and intermediate of β-oxidation were found in both organelle-containing fractions. The supernatant fraction contained radioactive diglycerides, monoglycerides, palmitate, sterol esters, and phospholipids, indicating lipase activity and direct utilization of fatty acid for the synthesis of sterol esters and polar lipids.     

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.     

LECITHIN SYNTHESIS, INTRACELLULAR TRANSPORT, AND SECRETION IN RAT LIVER : IV. A Radioautographic and Biochemical Study of Choline-Deficient Rats Injected with Choline-3H

Injection of choline-³H into choline-deficient rats resulted in an enhanced incorporation of the label into liver lecithin, as compared to the incorporation of label into liver lecithin of normal rats. The results obtained with the use of different lecithin precursors indicate that in the intact liver cell, both in vivo and in vitro, exchange of choline with phosphatidyl-choline is not significant. The synthesis and secretion of lecithins by the choline-deficient liver compare favorably with the liver of choline-supplemented rats, when both are presented with labeled choline or lysolecithin as lecithin precursors. Radioautography of the choline-deficient liver shows that 5 min after injection of choline-³H the newly synthesized lecithin is found in the endoplasmic reticulum (62%), mitochondria (13%), and at the "cell boundary" (20%). The ratio of the specific activity of microsomal and mitochondrial lecithin, labeled with choline, glycerol, or linoleate, was 1.53 at 5 min after injection, but the ratio of the specific activity of phosphatidyl ethanolamine (PE), labeled with ethanolamine, was 5.3. These results indicate that lecithin and PE are synthesized mainly in the endoplasmic reticulum, and are transferred into mitochondria at different rates. The site of a precursor pool of bile lecithin was studied in the intact rat and in the perfused liver. Following labeling with choline-³H, microsomal lecithin isolated from perfused liver had a specific activity lower than that of bile lecithin, but the specific activity of microsomal linoleyl lecithin was comparable to that of bile lecithin between 30 and 90 min of perfusion. It is proposed that the site of the bile lecithin pool is located in the endoplasmic reticulum and that the pool consists mostly of linoleyl lecithin.     

Lecithin biosynthesis in liver mitochondrial fractions

The pretreatment of rat liver mitochondrial fractions with phospholipase C preparations enhanced the incorporation of cytidine diphospho-[¹⁴C]-choline into phospholipids several-fold. Similar pretreatment of the microsomal fraction produced a similar stimulation. When the extent of microsomal contamination in the mitochondria was determined, and increments of pretreated microsomes were added to the mitochondria, the incorporation values extrapolated to zero for zero microsomal contamination. It was concluded that lecithin biosynthesis from endogenous diglycerides in the mitochondrial fractions could be ascribed to contaminating microsomes.     

Tomato Fruit Cell Wall Synthesis during Development and Senescence : In Vivo Radiolabeling of Wall Fractions Using [C]Sucrose

The pedicel of tomato fruit (Lycopersicon esculentum Mill., cv `Rutgers') of different developmental stages from immature-green (IG) to red was injected on the vine with 7 microcuries [¹⁴C(U)]sucrose and harvested after 18 hours. Cell walls were isolated from outer pericarp and further fractionated yielding ionically associated pectin, covalently bound pectin, hemicellulosic fraction I, hemicellulosic fraction II, and cellulosic fraction II. The dry weight of the total cell wall and of each cell wall fraction per gram fresh weight of pericarp tissue decreased after the mature-green (MG) stage of development. Incorporation of radiolabeled sugars into each fraction decreased from the IG to MG3 (locules jellied but still green) stage. Incorporation in all fractions increased from MG3 to breaker and turning (T) and then decreased from T to red. Data indicate that cell wall synthesis continues throughout ripening and increases transiently from MG4 (locules jellied and yellow to pink in color) to T, corresponding to the peak in respiration and ethylene synthesis during the climacteric. Synthesis continued at a time when total cell wall fraction dry weight decreased indicating the occurrence of cell wall turnover. Synthesis and insertion of a modified polymer with removal of other polymers may produce a less rigid cell wall and allow softening of the tissue integrity during ripening.     

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.     

Changes in the C-Labeled Cell Wall Components with Chase Time after Incorporation of UDP[C]Glucose by Intact Cotton Fibers

Intact, in vitro-grown cotton fibers will incorporate [¹⁴C]glucose from externally supplied UDP[¹⁴C]glucose into a variety of cell wall components including cellulose; this labeled fraction will continue to increase up to 4 hours chase time. In the fraction soluble in hot water there was no significant change in total label; however, the largest fraction after the 30 minute pulse with UDP[¹⁴C]glucose was chloroform-methanol soluble (70%) and showed a significant decrease with chase. The lipids that make up about 85% of this fraction were identified by TLC as steryl glucosides, acylated steryl glucosides, and glucosyl-phosphoryl-polyprenol. Following the pulse, the loss of label from acylated steryl glucosides and glucosylphophoryl-polyprenol was almost complete within 2 hours of chase; steryl glucosides made up about 85% of the fraction at that chase time. The total loss in the lipid fraction (about 100 picomoles per milligram dry weight of fiber) with chase times of 4 hours approximates the total gain in the total glucans.     

Subcellular localization of mannosyl transferase and glycoprotein biosynthesis in castor bean endosperm

Differential and sucrose density gradient centrifugation have shown that the mannosyl transferase present in germinating castor bean endosperm cells which catalyses the synthesis of mannosyl-phosphoryl-polyisoprenol is exclusively located in the endoplasmic reticulum membrane. This intracellular location was confirmed using both ribosome-denuded microsomes isolated in the presence of EDTA and rough-surfaced microsomes isolated in the presence of excess Mg²⁺ added to maintain ribosome-membrane attachment. Separation of organelles following the incubation of crude particulate fractions with GDP[¹⁴C]mannose demonstrated that most of the mannolipid thus formed remained associated with the microsomal fraction. When organelles were isolated from intact tissue which had previously been incubated with GDP[¹⁴C]mannose, [¹⁴C]glycoprotein was found to be associated with other cellular fractions in addition to the microsomes, in particular the glyoxysomes. The kinetics of radioactive labelling of these organelles suggest that [¹⁴C]glycoprotein appears initially in the microsomal fraction and subsequently accumulates in the glyoxysomes. Subfractionation of isolated, [¹⁴C]glycoprotein-labelled glyoxysomes established that over 80% of the total radioactivity was present in the membrane, while sodium dodecyl sulphate-polyacrylamide gel electrophoresis of solubilized glyoxysomal membranes showed that the [¹⁴C]sugar moiety was associated with several, but not all, constituent polypeptides.Abbreviations ER endoplasmic reticulum - TCA trichloroacetic acid - SDS sodium dodecylsulphate - GDP guanosine diphosphate     

Metabolism of three subfractions of myelin in developing rats.

Purified myelin, isolated from rat brain, was subfractionated into light, medium and heavy myelin. The metabolism of [³H] leucine in myelin subfractions was studied at intervals from 1 to 24 hours and from 18 hours to 85 days after the injection of 12-day-old rats. The metabolism of [¹⁴C] glucose in myelin subfractions was also examined during the 85 day interval. In addition, the development of each of these subfractions, as reflected by protein accretion, was determined.Between 13 and 97 days of age, the amount of the three myelin subfractions increased 10- to 44-fold. At 13 days of age the heavy subfraction accounted for the greatest percentage of myelin protein. However, beyond 13 days, light myelin predominated.The total ³H-radioactivity in the light, medium and heavy subfractions increased throughout most of the 85 day interval examined. The ³H specific radioactivity (³H dpm/μgram protein) of light myelin peaked at 12 hours after injection. The specific radioactivity of both ³H and ¹⁴C (¹⁴C dpm/μgram lipid) in light myelin declined beyond the initial time point in the long term (18 hour – 85 day) study. In contrast, the specific radioactivity of both ³H and ¹⁴C peaked in the medium and heavy subfractions at 4 days after injection of radioactive precursor.The possible existence of a membranous precursor to myelin is discussed.     

Characterization of deoxyribonucleic Acid species from castor bean endosperm: inability to detect a unique deoxyribonucleic Acid species associated with glyoxysomes

Three DNA buoyant density species (nuclear, 1.692 g cm⁻³; mitochondria 1.705 g cm⁻³; and proplastid, 1.713 g cm⁻³) can be detected in extracts from castor bean endosperm. No other buoyant density species can be identified. DNA extracts from sucrose density gradient purified glyoxysomes exhibit varying amounts of each of the three identified DNAs but no other distinguishable DNA species. RNA synthesized in vitro by Escherichia coli RNA polymerase using purified castor bean nuclear DNA as a template, hybridizes equally well with its template and with the 1.692 g cm⁻³ species from glyoxysome fractions. These results are discussed in terms of their relevance to microbody biogenesis.     

Kinetics of entry of galactolipids and phospholipids into myelin.

Abstract— Brain slices from 17 day rats were incubated with [³H]galactose and [³⁵S]sulphate to label cerebroside and sulphatide. Myelin was isolated by centrifugation on a sucrose density gradient. Following lipid extraction and alkaline methanolysis, cerebroside and sulphatide were isolated by tic, and radioactivity was measured. Appearance of [³H]cerebroside and [³H]sulphatide in myelin showed a lag of less than 15min, while appearance of [³⁵S]sulphatide in myelin showed a longer lag of about 30min. In chase experiments, the rate of appearance of [³H]cerebroside and [^3SS]sulphatide in the non-myelin fraction and of [³H]cerebroside in the myelin fraction slowed markedly after the chase. In contrast, [³⁵S]sulphatide continued to increase in myelin at a normal rate for 30min after the chase, then stopped, while ³H from galactose continued to accumulate in myelin sulphatides for 60 min. These data are interpreted to demonstrate an interval of 30 min between synthesis of cerebroside and its sulphation in the non-myelin fraction, and another delay of 30 min between sulphation and appearance in myelin. The distribution of newly synthesized cerebroside and sulphatide between myelin and non-myelin fractions also supported the concept that a complex metabolic pool of cerebroside in the non-myelin fraction is precursor to sulphatide of myelin. For comparison, entry of phosphatidyl choline and phosphatidyl ethanolamine into myelin was followed with [2-³H]glycerol as precursor. Like cerebroside, both phospholipids showed little delay in their initial appearance in myelin, and prompt cessation of their addition after a chase with unlabeled precursor. These results are consonant with either rapid entry of these three lipids into myelin after synthesis at an extra-myelin site, or synthesis of the lipids within myelin itself.     

Isolation of microbodies from plant tissues

Specialized microbodies have previously been isolated and characterized from fatty seedling tissues (glyoxysomes) and leaves (leaf peroxisomes). We have now examined 11 other plant tissues, including tubers, fruits, roots, shoots, and petals, and find that all contain particulate catalase, a distinctive common enzyme component of microbodies. On linear sucrose gradients the catalase activity peaks sharply at a higher equilibrium density (1.20 to 1.25 gram per cm³ in the various tissues) than the mitochondria (1.17 to 1.20). Only small amounts of protein are recovered in the fractions containing catalase, although a definite band is visible in preparations from some tissues, e.g., potato. As in the preparations from castor bean endosperm and spinach leaves for which comparable data are provided, the distribution of glycolate oxidase and uricase follows closely that of catalase on the gradients. The preparations from potato lack glyoxylate reductase and the transaminases, typical enzymes of leaf peroxisomes, and the distinctive enzymes of glyoxysomes are missing. Nonspecialized microbodies with limited enzyme composition can thus be isolated from a variety of plant tissues.     

Comparative studies of glyoxysomes from various Fatty seedlings

The separation of various organelles from cotton cotyledon (Gossypium hirsutum L.), cucumber cotyledon (Cucumis sativus L.), peanut cotyledon (Archis hypogaea L.), pine megagametophyte (Pinus ponderosa Laws), and watermelon cotyledon (Citrullus vulgaris Schrad.) by sucrose density gradient centrifugation was found to be similar to that described for castor bean endosperm (Ricinus communis L.). Equilibrium densities were 1.12 to 1.13 g cm³ for endoplasmic reticulum, 1.17 to 1.19 g/cm³ for mitochondria, and 1.25 g/cm³ for glyoxysomes. Isolated glyoxysomes from different fatty seedlings have striking similar specific activities of individual enzymes. The only exception is alkaline lipase activity which, when assayed with an artificial substrate, varies some 10-fold in glyoxysomes from different fatty seedlings. The properties of individual enzymes in glyoxysomes from different fatty seedlings are qualitatively similar as regard to sub-organelle localization and behavior in the presence of KCl and Triton X-100. In pine megagametophyte, the glyoxysomes and not the mitochondria are the intracellular site for the breakdown of stored lipid.     

Studies on the biosynthesis of protein and lipid components of rat liver mitochondria

1. Male rats were injected intraperitoneally with l-[³⁵S]methionine, [³²P]-phosphate and [2-¹⁴C]acetate. The animals were killed at various times up to 72hr. after injection, and liver mitochondria were prepared and fractionated into soluble protein, insoluble protein and lipid for assay of the radioactivity of each fraction. 2. The maximal specific radioactivity of total mitochondrial phospholipid with respect to both ³²P and ¹⁴C was attained after approx. 6hr. 3. ³²P was incorporated most rapidly into phosphatidylethanolamine, maximal incorporation being attained after approx. 6hr.; maximal incorporation into lecithin occurred after 6–12hr. The specific radioactivity of cardiolipin was still slowly increasing at the end of the experiment (72hr.). 4. There were no major differences between the rates of incorporation of ¹⁴C into the lecithin, phosphatidylethanolamine and cardiolipin fractions of mitochondrial phospholipid, maximal incorporation in each case occurring after approx. 6hr. 5. Maximal incorporation of ³⁵S into both soluble and insoluble protein fractions was attained less than 12hr. after injection, the maximal specific radioactivity of soluble protein being higher than that of insoluble protein.     

Effect of light on ribonucleic Acid metabolism in greening maize leaves

The effect of illumination on the incorporation of labeled precursors into RNA of dark-grown maize (Zea mays) leaves was studied using either ³²P-phosphate or double labeling with ¹⁴C- and ³H-uridine. In the dark, label was preferentially incorporated into etioplast ribosomal RNAs. Incorporation into this fraction and into lower molecular weight fractions was strongly and preferentially stimulated by light during the first 2 hours of illumination. The effect persisted after illumination was terminated. The possibility that light-induced alterations in plastid ribosomal RNA metabolism may not be required for chlorophyll accumulation in maize is discussed.     

In vitro stimulation of lecithin synthesis in rat liver mitochondria and microsomes after treatment with phospholipase C

Rat liver mitochondria in which diglycerides were generated by phospholipase C treatment were shown to incorporate labeled choline from cytidine-5′diphospho-[Me-¹⁴C] choline into lecithin to an extent which could not be ascribed to microsomal contamination. The response of this enzymatic activity to the extent of phospholipase C degradation was qualitatively different in microsomes and mitochondria, suggesting clearly different properties of this enzyme in the two subcellular fractions.     

Rapid Methylation of Cell Proteins and Lipids in Trypanosoma brucei

ABSTRACT. The fate of the [methyl-¹⁴C] group of S-adenosylmethionine (AdoMet) in bloodstream forms of Trypanosoma brucei brucei, was studied. Trypanosomes were incubated with either [methyl-¹⁴C]methionine, [U-¹⁴C]methionine, S-[methyl-¹⁴C]AdoMet or [³⁵S]methionine and incorporation into the total TCA precipitable fractions was followed. Incorporation of label into protein through methylation was estimated by comparing molar incorporation of [methyl-¹⁴C] and [U-¹⁴C]methionine to [³⁵S]methionine. After 4-h incubation with [U-¹⁴C]methionine, [methyl-¹⁴C]methionine or [³⁵S]methionine, cells incorporated label at mean rates of 2,880 pmol, 1,305 pmol and 296 pmol per mg total cellular protein, respectively. Cells incubated with [U-¹⁴C] or [methyl-¹⁴C]methionine in the presence of cycloheximide (50 μg/ml) for four hours incorporated label eight- and twofold more rapidly, respectively, than cells incubated with [³⁵S]methionine and cycloheximide. [Methyl-¹⁴C] and [U-¹⁴C]methionine incorporation were > 85% decreased by co-incubation with unlabeled AdoMet (1 mM). The level of protein methylation remaining after 4-h treatment with cycloheximide was also inhibited with unlabeled AdoMet. The acid precipitable label from [U-¹⁴C]methionine incorporation was not appreciably hydrolyzed by DNAse or RNAse treatment but was 95% solubilized by proteinase K. [U-¹⁴C]methionine incorporated into the TCA precipitable fraction was susceptible to alkaline borate treatment, indicating that much of this label (55%) was incorporated as carboxymethyl groups. The rate of total lipid methylation was found to be 1.5 times that of protein methylation by incubating cells with [U-¹⁴C]methionine for six hours and differential extraction of the TCA lysate. These studies show T. b. brucei maintains rapid lipid and protein methylation, confirming previous studies demonstrating rapid conversion of methionine to AdoMet and subsequent production of post-methylation products of AdoMet in African trypanosomes.