Organelle Preparations Research Articles

Filtering Centrifugation Through Two Layers of Silicone Oil: A Method for the Kinetic Analysis of Rapid Metabolite Transport in Organelles.

Kinetic studies of ATP uptake in amyloplasts from sycamore (Acer pseudoplatanus L.) have been performed with a newly developed method of centrifugation through a double layer of silicone oil; the results are compared with the frequently used method of centrifugation through a single layer. The present technique employs two separate silicone layers: the upper one prevents mixing of the organelle preparation with the incubation layer, which contains the metabolites, and the lower one prevents mixing of the incubation and pelleting layers. Incubation of the organelles takes place in the incubation layer after the upper layer of silicone inverts during centrifugation. Depending on the speed of centrifugation, incubation periods as short as 1 sec and as long as 2 min can be achieved. High reproducibility and accuracy, acquisition of multiple data in a single centrifugation, and maintenance of structural integrity and metabolic activity of the organelles make the technique advantageous for the analysis of metabolite transport in amyloplasts and other kinds of organelles.

Selective inactivation of peroxisomal and cytosolic 3-ketothiolase IB by 2-chloro-6-phenylhexanoate in intact hepatocytes.

Rat liver mitochondria and cytosol contain two types of 3-ketothiolases, namely 3-ketothiolases IA and IB, which cleave 3-ketoacyl-coenzyme A (CoA) esters containing four or more carbons and 3-ketothiolases IIA and IIB, which cleave 3-ketoacyl-CoA esters containing four carbons, i.e. acetoacetyl-CoA (Aragon, J.J., and Lowenstein, J.M. (1983) J. Biol. Chem. 258, 4725-4733). We now report that rat liver peroxisomes also contain 3-ketothiolases IA and IB and show that incubation of hepatocytes with 2-chloro-6-phenylhexanoate causes the selective inactivation of peroxisomal and cytosolic 3-ketothiolase IB, while mitochondrial 3-ketothiolases are not appreciably affected. The basis of the selectivity of the inhibitor for peroxisomal and cytosolic 3-ketothiolases can be accounted for in terms of the specificities of the enzymes in the different pathways of beta-oxidation. Evidence is presented that 2-chloro-6-phenylhexanoate is metabolized to 2-chloro-3-keto-6-phenylhexanoyl-CoA, which then alkylates 3-ketothiolase and thereby inactivates the enzyme. Evidence is presented which suggests that cytosolic 3-ketothiolases IA and IB are not artifacts of homogenization and organelle preparation.

Isolation of nuclear, chloroplast and mitochondrial DNA from the moss Physcomitrella patens

In this report efficient, rapid and easy methods for the isolation of nuclee, chloroplasts and mitochondria and their corresponding DNAs from a large scale culture of the moss Physcomitrella patens are described. Microscopical controls of organelle preparations revealed single and intact nuclei, chloroplasts and mitochondria. Per 10 g fressh weight average yields of 15 μg nuclear chloroplast DNA and 1 μg mitochondrial DNA were obtained. The purity of organelle DNA was tested by hybridization with non-corresponding organelle specific clones. Isolation of nuclei and mitochondria from mosses and characterization of their DNAs are presented for the first time.

Studies on catalase compartmentation in digitonin-treated rat hepatocytes

The cellular compartmentation of catalase was studied in digitonin-permeabilized rat hepatocytes. A biphasic dose-response curve was observed for the unmasking of catalase activity by digitonin in latency studies. About 40–60% of the total catalase activity was seen in the range of 5–200 μM digitonin compared to 13% free activity in control preparations without digitonin. The free catalase activity began to increase again above 300 μm digitonin, and all latency was lost around 500 μM and above. These results indicate that there exists in rat hepatocytes catalase with two levels of crypticity to digitonin, only one of which was seen in a mixed organelle preparation containing peroxisomes.

Light‐mediated changes in the plastidic phosphorylase patterns in shoots of Pisum sativum

α‐1,4‐Glucan phosphorylase (EC 2.4.1.1) forms from light or dark grown shoots of Pisum sativum L. cv. ‘Kleine Rheinländerin’ have been studied using various electrophoretic techniques. The phosphorylase patterns of green and etiolated shoots differed. Etiolated shoots contained two enzyme forms, one residing inside and the other outside the etioplast; this was shown by electrophoresis of extracts of isolated etioplasts. Purity and intactness of the organelle preparation were ascertained by electron microscopy. Light‐grown shoots contained, in addition to these two enzyme forms, a third phosphorylase which appears to be chloroplast‐specific. The two plastidic phosphorylase forms differed slightly in their apparent molecular masses (as determined by non‐denaturing polyacrylamide gel electrophoresis) and in their affinities towards branched polyglucans (as revealed by affinity electrophoresis). The apparent affinity of the extrachloroplastic phosphorylase form to these polyglucans was orders of magnitude higher than that of the two plastidic enzyme forms. The development of the chloroplast‐specific phosphorylase pattern is under photocontrol. Investigations performed with red or far‐red illuminated wild‐type plants and with a pale mutant which has a highly reduced pigment and thylakoid content suggest that this photocontrol is mediated by phytochrome.

Duplicated chromosome segments in maize (Zea mays L.): further evidence from hexokinase isozymes

The genetic control of hexokinase isozymes (ATP: d-hexose-6-phosphotransferase, E.C. 2.7.7.1, HEX) in maize (Zea mays L.) was studied by starch gel electrophoresis. Genetic analysis of a large number of inbred lines and crosses indicates that the major isozymes observed are encoded by two nuclear loci, designated Hex1 and Hex2. Five active allozymes and one null variant are associated with Hex1, while Hex2 has nine active alleles in addition to a null variant. Alleles at both loci govern the presence of single bands, with no intragenic or intergenic heteromers visible, suggesting that maize HEX's are active as monomers. Organelle preparations demonstrate that the products of both loci are cytosolic. All alleles, including the nulls, segregate normally in crosses. Vigorous and fertile plants were synthesized that were homozygous for null alleles at both loci, suggesting that other hexosephosphorylating enzymes exist in maize that are undetected with our assay conditions. Linkage analyses and crosses with B-A translocation stocks place Hex1 on the short arm of chromosome 3, 27 centimorgans from Pgd2 (phosphogluconate dehydrogenase) and Hex2 on the long arm of chromosome 6, approximately 45 centimorgans from Pgd1. It is suggested that the parallel linkages among these two pairs of duplicated genes reflects an evolutionary history involving chromosome segment duplication or polyploidy.

Soybean root response to infection by Rhizobium japonicum: mannoconjugate turnover in effective and ineffective nodules

Crude organelle preparations from uninfected Glycine max root tissue, or from root tissue infected with the nod +/nif + (effective) or the nod +/nif − (ineffective) strains of Rhizobium japonicum all assembled mannose from GDP-mannose into trichloroacetic acid-precipitable forms. Cellular fractionation studies showed that the enzyme responsible for the initial event in the mannoconjugate assembly process was located exclusively in the endoplasmic reticulum (ER) ofthe host cell. Exogenous 14C-labelled mannose, when supplied to intact tissue for short periods, tended to accumulate at this location. After longer labelling periods the product was found to be present in both heavier and lighter membrane fractions, which corresponded with the subcellular sites of the enzyme α-mannosidase. The mannose-containing material assembled by the ER in vivo acted as a substrate for the α-mannosidase-containing fractions. During nodule development α-mannosidase activity showed two maxima, the early concomitant with, and the latter preceded by, an increase in ER marker enzyme activity. In these changes tissue infected with ineffective strains of R. japonicum showed parallel, but less pronounced biochemical trends. These data suggest that in nod +/nif + symbioses mannoconjugate assembly is stimulated and that this extra product is transferred through the cell where structures formed only in nod +/nif + symbioses may be responsible for its disassembly or modification.

Methods using tissue preparations and isolated biomolecules.

The possibility to use organs, organelle preparations and biologically active chemicals in toxicity tests and in toxicology will be reviewed. Examples are perfused liver preparations, tissue slices and homogenates, isolated nerve preparations, nerve-muscle preparations, membrane preparations, microsomes, mitochondria, synaptosomes, antibodies and isolated chemical compounds (receptors, enzymes).

Antibody inhibition of external aldolase activity in spinach chloroplast preparations

Abstract Antibodies (rabbit) have been prepared against total stroma from isolated spinach (Spinacia oleracea L. cv. Viking II) chloroplasts. These antibodies inhibited most of the aldolase activity present outside the chloroplasts in preparations of intact (80–95%) chloroplasts. They also reduced the amount of labelled fructose‐1,6‐bisphosphate found in the medium after 14CO2 fixation with such preparations. Both intact and broken chloroplasts were strongly agglutinated by the antibodies. The results indicate that the external fructose‐1,6‐bisphosphate was formed from excreted dihydroxyacetone phosphate by the action of aldolase and triose phosphate isomerase present outside the chloroplasts. The contamination of organelle preparations with free enzymes or enzymes adsorbed on the outer surface of the organelles is probably a general phenomenon. It is suggested that antibodies can be used as a tool to detect and selectively inhibit such contaminating enzyme activities.

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[(14)C]galactose into chloroform/methanol (2:1)-soluble glactolipids. At least two galactolipids were formed. Most of the [(14)C]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 [(14)C]galactose into the microsomal galactolipid with a concomitant increase in microsomal [(14)C]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.

Responses of nuclear glucose-6-phosphatase to diabetes and to hydrocortisone administered to normal and diabetic rats differ from those of the microsomal enzyme

The effect of alloxan-diabetes, and of pharmacological doses of hydrocortisone administered to normal and diabetic rats, on carbamyl phosphate:glucose phosphotransferase and d-glucose-6-phosphate phosphohydrolase (EC 3.1.3.9) activities of isolated hepatic nuclei and microsomes were studied by assay at pH 7 in the absence and presence of deoxycholate. Hormonally related alterations both in activity levels and in the activation by the detergent (i.e. latency) of activities of the two cellular structural elements differed significantly. Most strikingly, (a) a 3–4-fold increase in the levels of activities of nuclei was seen in response either to diabetes or to hydrocortisone administered to normal rats whether or not detergent was added to preparations prior to assay; (b) the normally low degree of stimulation by detergent of activities of nuclei was unaltered in diabetes, and (c) administration of the glucocorticoid to diabetic rats decreased activity levels and increased their activation by detergent. Directly contrasting responses were noted with isolated microsomal preparations. Fundamental differences in the enzymes in these two organelle preparations are thus demonstrated. It appears that both synthetic and hydrolytic activities of this enzyme of nuclei may be manifest in the presence of requisite substrates, and that activities of this organelle may become increasingly prominent under certain hormonally perturbed conditions.

Intracellular localization of β-aspartate kinase in spinach ( spinacea oleracea)

..beta..-Aspartate kinase is a key regulatory enzyme catalyzing the first step in the aspartate pathway leading to the synthesis of lysine, threonine and methionine. As such, its presence in a particular cellular compartment or organelle would suggest that control of the aspartate pathway resides in that organelle. The present work reports results of initial efforts to demonstrate the presence of ..beta..-aspartate kinase (ATP: L-aspartate 4 phosphotransferase, EC 2.7.2.4) in organelle preparations from spinach leaf tissues.

Formation of lipid-linked mono- and oligosaccharides from GDP-mannose by castor bean endosperm homogenates

A crude organelle preparation from germinating castor bean endosperm catalysed the incorporation of mannose from GDP[(14)C]mannose into acid-labile mannolipids. Solubility and chromatographic properties have identified the most rapidly synthesized products as mannosyl-phosphoryl-polyisoprenol, while the more polar lipid formed was shown to contain oligosaccharide. Little radioactivity from GDP[(14)C]mannose accumulated in insoluble product in the cell-free system, but supplying GDP[(14)C]mannose to intact endosperm tissue has shown that the major incorporation product in vivo is glycoprotein. This product was readily solubilized by either pronase or sodium dodecyl sulphate treatment suggesting it was membrane bound glycoprotein. Incorporation of mannose into mannosyl-phosphoryl-polyisoprenol during the cell-free assay was stimulated by the addition of dolichol monophosphate. This enzymic activity was optimal at pH 7.5 and in the presence of 10 mM Mg(2+). The Km for GDP-mannose was estimated to be 5×10(-7) M. Cellular mannosyl transferase activity changed markedly during early post-germinative growth; from being absent in the dry seed, enzyme activity increased to peak between the second and third days of growth and subsequently declined.

Organelle membranes from germinating castor bean endosperm

Endoplasmic reticulum, mitochondria, and glyoxysomes were obtained from germinating castor bean endosperm,Ricinus communis, by sucrose gradient centrifugation. When each of the three organelle preparations was diluted in 150 mM KCl and centrifuged, all of the component membrane material, measured as phospholipid, was sedimented. Also, the respective membrane enzymes, phosphorylcholine-glyceride transferase, cytochrome c oxidase and alkaline lipase were recovered. The endoplasmic reticulum retained most (60%) of its protein. The mitochondria lost almost no protein while the glyoxysomes lost much of their soluble contents.

Phospholipid Synthesis and Exchange in Castor Bean Endosperm Homogenates

Crude organelle preparations from castor bean (Ricinus communis L.) endosperm rapidly incorporate CDP-((14)C)choline and CDP-((14)C)-ethanolamine into phosphatidylcholine and phosphatidylethanolamine, respectively. Separation of organelles by sucrose density gradient centrifugation following incubation with these substrates demonstrated that most of the (14)C phospholipids thus formed were present in the endoplasmic reticulum membranes, although label was also found in mitochondria, proplastids, and glyoxysomes. The phospholipid-synthesizing enzymes, cholinephosphotransferase and ethanolaminephosphotransferase, are exclusively confined to the endoplasmic reticulum membrane fraction, suggesting that the appearance of (14)C-phospholipid in other organelles was due to phospholipid exchange. Phospholipid synthesis was inhibited by the cytoplasmic supernatant fraction. The active inhibitor in this fraction was not identified, but the inhibition was not significantly relieved by either dialyzing or boiling the supernatant. Phosphatidylcholine synthesis showed an absolute requirement for Mg(2+); the Michaelis constant was 1 mm. Ca(2+) was a potent inhibitor of Mg(2+)-stimulated phospholipid synthesis and enhanced the decay of (14)C-phospholipids from pre-labeled membranes, particularly when the membranes were resuspended in the cytoplasmic supernatant.The data are consistent with the concept that the endoplasmic reticulum is a major site of membrane proliferation where structural lipids, and possibly proteins, are inserted into, and thus expand, a pre-existing membrane fraction. Other organelle and cellular membranes could therefore originate from the proliferating endoplasmic reticulum by a process of membrane flow and differentiation.

Kaurene synthetase from plastids of developing plant tissues

Summary Kaurene synthesis from copalyl pyrophosphate is present in the plastid fraction from all three sources of developing plant tissue studied. Isolated plastids from Echinocystis macrocarpa endosperm have kaurene synthetic capacity from either geranylgeranyl pyrophosphate or copalyl pyrophosphate. However, organelle preparations from etiolated pea shoot tips or developing castor bean endosperm have either no or a very limited ability to synthesize kaurene from geranylgeranyl pyrophosphate. These results suggest that the first committed step in gibberellin biosynthesis, geranylgeranyl pyrophosphate conversion to copalyl pyrophosphate, may be limited under certain stages of plant development.

Evaluation of silicon and germanium retention in rat tissues and diatoms during cell and organelle preparation for electron probe microanalysis.

Chemical, radiochemical and x-ray microanalysis assays were used to define parameters of silicon (Si) retention during preparation og biologic samples (rat liver, spleen, kidney, lung, diatoms and cell organelles) for x-ray microanalysis, Due to its longer half-life 68-Fe was used in some cases to trace SI. Leaching of Si from cells and organelles by the aqueous preparation media was overcome by use of the freeze-substitution process. Cells were treated with 30% glycerol hypertonic sucrose medium to reduce ice damage. Embedment in Spurr's low viscosity epoxy resin medium caused no apparent Si loss. A semiquantitative evaluation showed 0.5 x 10-8 to 0.3 x 10-17 g detectable Si in isolated rat liver mitochondria in thin sections, which is within the instrument's range of detection. This study indicateds that the presence of Si in the mitochondria is not the rsult of contamination.

Cytochemical Localization of Catalase Activity in Glyoxysomes from Castor Bean Endosperm

Glyoxysomes isolated from castor bean (Ricinus communis L. var. zanzibariensis) endosperm have been stained by the cytochemical diaminobenzidine reaction. The reaction product obtained by preincubation with 3,3'-diaminobenzidine and incubation with the reagent and H(2)O(2) is distributed uniformly throughout the matrix of the organelles. Ricinosomes or dilated cisternae may be completely absent from the organelle preparation or are, at the most, a minor contaminant.

Malate Dehydrogenases of Pisum sativum

Mitochondria and leaf microbodies isolated from leaves of pea (Pisum sativum) by sucrose density gradient centrifugation were each shown to have a unique form (isoenzyme) of malate dehydrogenase (EC 1.1.1.37) based on chromatographic and kinetic properties. Root organelle preparations were shown to contain only a mitochondrial malate dehydrogenase with physical and kinetic properties similar to the leaf form. The absence of a detectable root microbody malate dehydrogenase similar to the leaf enzyme, which is intermediate in electrophoretic and chromatographic properties between the mitochondrial and soluble isoenzymes, was confirmed by diethylaminoethyl cellulose column chromatography and starch-gel electrophoresis of total homogenates from leaf and root tissue. These findings tend to support the role of the leaf microbody isoenzyme in a pathway unique to photosynthetic tissue.