Phospholipid-synthesizing Enzymes Associated with Golgi Dictyosomes from Pea Tissue

Golgi dictyosomal membranes isolated from pea (Pisum sativum) stem tissue, using a combination of rate zonal and isopycnic sucrose density centrifugation, were shown to bear cytidine diphosphate-choline:diglyceride phosphorylcholinetransferase, CDP-ethanolamine:diglyceride phosphorylethanolaminetransferase, and CTP:phosphorylcholine cytidyltransferase activities. Although the majority of the activity of the phospholipid-synthesizing enzymes was associated with the endoplasmic reticulum, the activity found in the Golgi system was about 25% of the total activity. These results suggest that Golgi dictyosomes probably synthesize at least part of the membrane phospholipids that they may need for their secretory function and for dictyosomal proliferation during cell growth, rather than importing this material entirely from the endoplasmic reticulum.     

Structural Development during Germination of Different Populations of Mitochondria from Pea Cotyledons

The crude mitochondrial fraction from pea cotyledons can, from days 1 to 7 of germination, be separated into three fractions by sucrose density gradient centrifugation. When seeds were grown in water (control) or cycloheximide (120 micrograms per milliliter of medium) for 4 days, the originally different populations of mitochondria acquired a uniform density and separated together in band 1 (density, 1.205 grams per milliliter). The oxidative and phosphorylative activities of mitochondria obtained from 4-day-old control and 4-day-old cycloheximide-treated pea seeds were the same. However, mitochondria from pea seeds that were grown in d-threo-chloramphenicol (1.5 milligrams per milliliter of medium) or erythromycin (0.5 milligram per milliliter of medium) for 4 days separate into three bands (fully developed mitochondria in the top band [band 1] and partially developed mitochondria in the lower two bands [bands 2 and 3]). Separation patterns and oxidative and phosphorylative activities were the same for mitochondria separated from 4-day-old cotyledons treated with d-threo-chloramphenicol or erythromycin and from 1-day-old cotyledons grown in water. This indicated that these inhibitors prevented the partially developed mitochondria originally in bands 2 and 3 from developing further. In contrast, cycloheximide did not seem to interfere with the mitochondrial structural development. These results along with those obtained from the experiments on the effects of d-threo-chloramphenicol, erthromycin, and cycloheximide on ¹⁴C-leucine incorporation into mitochondrial membrane proteins suggest that the increase in mitochondrial activity during germination may be a result of structural development (membrane synthesis) in pre-existing mitochondria.     

ADP/ATP Translocator from Pea Root Plastids (Comparison with Translocators from Spinach Chloroplasts and Pea Leaf Mitochondria)

The kinetic properties of the adenosine 5[prime]-diphosphate/adenosine 5[prime]-triphosphate (ADP/ATP) translocator from pea (Pisum sativum L.) root plastids were determined by silicone oil filtering centrifugation and compared with those of spinach (Spinacia oleracea L.) chloroplasts and pea leaf mitochondria. In addition, the ADP/ATP transporting activities from the above organelles were reconstituted into liposomes. The Km(ATP) value of the pea root ADP/ATP translocator was 10 [mu]M and that for ADP was 46 [mu]M. Corresponding values of the spinach ADP/ATP translocator were 25 [mu]M and 28 [mu]M, respectively. Comparable results were obtained for the reconstituted ATP transport activities. The transport was highly specific for ATP and ADP. Adenosine 5[prime]-monophosphate (AMP) caused only a slight inhibition and phosphoenolpyruvate and inorganic pyrophosphate caused no inhibition of ATP uptake. With pea root plastids and spinach chloroplasts, Km values >1 mM were obtained for ADP-glucose. Since the concentrations of ATP and ADP-glucose in the cytosolic compartment of spinach leaves have been determined as 2.5 and 0.6 mM, respectively, a transport of ADP-glucose by the ADP/ATP translocator does not appear to have any physiological significance in vivo. Although both the plastidial and the mitochondrial ADP/ATP translocators were inhibited to some extent by carboxyatractyloside, no immunological cross-reactivity was detected between the plastidial and the mitochondrial proteins. It seems probable that these proteins derive from different ancestors.     

Age-Dependent Variations in Peroxide-Utilizing Enzymes from Rat Brain Mitochondria and Cytoplasm

Abstract: The location of peroxide-utilizing enzymes has been studied in rat brain. Glutathione peroxidase and glutathione reductase distributions indicate that both enzymes are located in the cytoplasm and in the matrix space of "synaptosomal" and "free" mitochondria. On the other hand, catalase distribution parallels that of NADH-cytochrome c reductase (rotenone-insensitive), and appears to be associated with the outer membrane of brain mitochondria. Whereas no gross age-dependent changes in various marker enzymes were found, a gradual but significant increase in glutathione peroxidase from the soluble fraction of free mitochondria was detected. The consequences of such increase are discussed with regard to the reducing potential of the cell.     

The Phosphate Transporter from Pea Mitochondria (Isolation and Characterization in Proteolipid Vesicles)

The phosphate transporter from mitochondria will exchange matrix phosphate for cytosolic phosphate and facilitate either phosphate/proton symport or phosphate/hydroxyl ion antiport. The phosphate transported into the matrix by this carrier is either used for ATP synthesis or exchanges back out to the cytosol on the dicarboxylate transporter, permitting entry of malate and succinate into the matrix. The phosphate transporter was solubilized from etiolated pea (Pisum sativum L. cv Alaska) mitochondrial membranes with Triton X-114, purified approximately 500-fold by hydroxylapatite chromatography, and reconstituted into azolectin vesicles that were preloaded with 0.1 or 10 mM phosphate. Phosphate transport was measured as the exchange of preloaded phosphate for external [32P]phosphate. Phosphate/phosphate exchange occurred for over 40 min at room temperature with an apparent K0.5 of 1.6 mM and a maximum velocity of over 700 nmol (mg protein)-1 min-1. Diethyl pyrocarbonate was used as an inhibitor-stop reagent. Transport was inhibited by p-hydroxyphenylglyoxal, p-hydroxymercuribenzoate, pyridoxal 5-phosphate, and dansyl chloride but was insensitive to sulfate, nitrate, and N-ethylmaleimide, the standard inhibitor for the mammalian phosphate transporter. Phosphate/hydroxyl exchange was stimulated when the proton gradient was collapsed with carbonyl cyanide m-chlorophenylhydrazone, but phosphate/phosphate exchange was unaffected by the uncoupler.     

NADP-Utilizing Enzymes in the Matrix of Plant Mitochondria

Purified potato tuber (Solanum tuberosum L. cv Bintie) mitochondria contain soluble, highly latent NAD⁺- and NADP⁺-isocitrate dehydrogenases, NAD⁺- and NADP⁺-malate dehydrogenases, as well as an NADPH-specific glutathione reductase (160, 25, 7200, 160, and 16 nanomoles NAD(P)H per minute and milligram protein, respectively). The two isocitrate dehydrogenase activities, but not the two malate dehydrogenase activities, could be separated by ammonium sulfate precipitation. Thus, the NADP⁺-isocitrate dehydrogenase activity is due to a separate matrix enzyme, whereas the NADP⁺-malate dehydrogenase activity is probably due to unspecificity of the NAD⁺-malate dehydrogenase. NADP⁺-specific isocitrate dehydrogenase had much lower K_ms for NADP⁺ and isocitrate (5.1 and 10.7 micromolar, respectively) than the NAD⁺-specific enzyme (101 micromolar for NAD⁺ and 184 micromolar for isocitrate). A broad activity optimum at pH 7.4 to 9.0 was found for the NADP⁺-specific isocitrate dehydrogenase whereas the NAD⁺-specific enzyme had a sharp optimum at pH 7.8. Externally added NADP⁺ stimulated both isocitrate and malate oxidation by intact mitochondria under conditions where external NADPH oxidation was inhibited. This shows that (a) NADP⁺ is taken up by the mitochondria across the inner membrane and into the matrix, and (b) NADP⁺-reducing activities of malate dehydrogenase and the NADP⁺-specific isocitrate dehydrogenase in the matrix can contribute to electron transport in intact plant mitochondria. The physiological relevance of mitochondrial NADP(H) and soluble NADP(H)-consuming enzymes is discussed in relation to other known mitochondrial NADP(H)-utilizing enzymes.     

Azelaic Acid-Induced Enzymes of Phenolic Defense in Pea Roots

The treatment of pea roots with azelaic acid for 72 h led to a change in the content of 28 proteins: the content of 20 proteins decreased, and the content of 8 proteins (including the phenylpropanoid metabolism enzymes, which are involved in the synthesis of phytoalexins) increased.     

The Effect of Carnitine on Palmitate Oxidation by Pea Cotyledon Mitochondria

The conditions for maximum O₂ uptake by pea cotyledon mitochondriaoxidizing palmitate were established. It was found that CoASH,Mg²⁺, ATP, malate, and carnitine were necessary additions tothe incubation medium. It is suggested that carnitine combineswith palmitoylCoA to produce palmitoylcarnitine with the releaseof CoASH. The palmitoylcarnitine thus formed may penetrate themembranes of the mitochondria with greater ease than palmitoylCoA.     

Effect of Cycloheximide on Development of Mitochondria in Germinating Pea Cotyledons

Polyethylene glycol (PEG) mediated transfection of Lactobacillus casei ATCC 27092 protoplasts by phage PL-1 DNA was done. The protoplasts were obtained by treatment with purified PL-1 phage N-acetylmuramidase in the presence of citrate. Optimum conditions for transfection were 50% PEG 4,000, 15 µg protamine sulfate/ml, 0.15 m sucrose, and 10 m m MgSO₄ in MR medium (pH 6.0). The extent of transfection was proportional to the amounts of DNA added, and the greatest efficiency of transfection after a 10-min incubation was about 3.3 × 10⁵ PFU/µg DNA. The eclipse period of growth of progeny phages in the transfectants was 3 hr and the average burst size was 200.     

Biochemical Characterization of Soluble Acid and Alkaline Invertases from Shoots of Etiolated Pea Seedlings

Soluble invertase was purified from pea(Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation,DEAE-Sepharose column,Con-A-and Green 19-Sepharose affinity columns,hydroxyapatite column,ultra-filtration,and Sephacryl 300 gel filtration.The purified soluble acid(SAC) and alkaline(SALK) invertases had a pH optimum of 5.3 and 7.3,respectively.The temperature optimum of two invertases was 37 ℃.The effects of various concentrations of Tris-HCl,HgCl2,and CuSO4 on the activities of the two purified enzymes were examined.Tris-HCl and HgCl2 did not affect SAC activity,whereas 10 mM Tris-HCl and 0.05 mM HgCl2 inhibited SALK activity by about 50%.SAC and SALK were inhibited by 4.8 mM and 0.6 mM CuSO4 by 50%,respectively.The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis.The Kms of SAC and SALK were determined to be 1.8 and 38.6 mM,respectively.The molecular masses of SAC shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were 22 kDa and 45 kDa.The molecular mass of SALK was 30 kDa.Iso-electric points of the SAC and SALK were estimated to be about pH 7.0 and pH 5.7,respectively.     

NAD-Linked Isocitrate Dehydrogenase: Isolation, Purification, and Characterization of the Protein from Pea Mitochondria

The NAD⁺-dependent isocitrate dehydrogenase from etiolated pea (Pisum sativum L.) mitochondria was purified more than 200-fold by dye-ligand binding on Matrix Gel Blue A and gel filtration on Superose 6. The enzyme was stabilized during purification by the inclusion of 20% glycerol. In crude matrix extracts, the enzyme activity eluted from Superose 6 with apparent molecular masses of 1400 ± 200, 690 ± 90, and 300 ± 50 kD. During subsequent purification steps the larger molecular mass species disappeared and an additional peak at 94 ± 16 kD was evident. The monomer for the enzyme was tentatively identified at 47 kD by sodium dodecyl-polyacrylamide gel electrophoresis. The NADP⁺-specific isocitrate dehydrogenase activity from mitochondria eluted from Superose 6 at 80 ± 10 kD. About half of the NAD⁺ and NADP⁺-specific enzymes remained bound to the mitochondrial membranes and was not removed by washing. The NAD⁺-dependent isocitrate dehydrogenase showed sigmodial kinetics in response to isocitrate (S_0.5 = 0.3 mm). When the enzyme was aged at 4°C or frozen, the isocitrate response showed less allosterism, but this was partially reversed by the addition of citrate to the reaction medium. The NAD⁺ isocitrate dehydrogenase showed standard Michaelis-Menten kinetics toward NAD⁺ (K_m = 0.2 mm). NADH was a competitive inhibitor (K_i = 0.2 mm) and, unexpectedly, NADPH was a noncompetitive inhibitor (K_i = 0.3 mm). The regulation by NADPH may provide a mechanism for coordination of pyridine nucleotide pools in the mitochondria.     

Interactions Between Soluble Enzymes and Subcellular Structur

Abstract

Soluble enzymes contribute significantly to the metabolic capabilities of living organisms, but it is becoming increasingly clear that the activities of these enzymes are significantly modified by their interactions with structural components of the cell, and that these interactions may make important contributions to metabolic regulation. In the past, specification of these interactions has been limited by the availability of suitable experimental techniques, but this deficiency is now being rectified and our understanding of these processes is advancing rapidly. Research in this area is moving into a second phase, with the emphasis no longer being focused on demonstrations of the biological reality of these interactions, but directed more towards quantitative aspects of binding, the determination of the characteristics of binding domains, and the theoretical basis of regulatory involvements. All of these aspects are discussed in the present review.     

Soluble Cell Wall Polysaccharides Released from Pea Stems by Centrifugation : I. EFFECT OF AUXIN

The metabolism of polysaccharides by pea stem segments treated with and without auxin was investigated using a centrifugation technique for removing solution from the free space of the cell wall. Glucose is the predominant sugar in both the ethanol-soluble and ethanol-insoluble fractions of the cell wall solution extracted with water. In the water-soluble, ethanol-insoluble polysaccharides, arabinose, xylose, galactose, and glucose make up 9.5, 23.8, 23.9, and 39.9%, respectively, of the neutral sugars, while rhamnose, fucose, and mannose are present at concentrations between 0.5 and 2.0%.     

Soluble Cell Wall Polysaccharides Released from Pea Stems by Centrifugation : II. EFFECT OF ETHYLENE

The effect of ethylene on cell wall metabolism in sections excised from etiolated pea stems was studied. Ethylene causes an inhibition of elongation and a pronounced radial expansion of pea internodes as shown by an increase in the fresh weight of excised, 1-cm sections. Cell wall metabolism was studied using centrifugation to remove the cell wall solution from sections. The principal neutral sugars in the cell wall solution extracted with H₂O are arabinose, xylose, galactose, and glucose. Both xylose and glucose decline relative to controls in air within 1 hour of exposure to ethylene. Arabinose and galactose levels are not altered by ethylene until 8 hours of treatment, whereupon they decline in controls in air relative to ethylene treatment. When alcohol-insoluble polymers are fractionated into neutral and acidic polysaccharides, xylose and glucose predominate in the neutral fraction and arabinose and galactose in the acidic fraction. Ethylene depresses the levels of xylose and glucose in the neutral fraction and elevates arabinose and galactose in the acidic fraction. Ethylene treatment does not affect the level of uronic acids extracted with H₂O; however, the level of hydroxyproline-rich proteins in this water-extracted cell wall solution is increased by ethylene. Extraction of sections with CaCl₂ results in an increase in the levels of neutral sugars particularly arabinose. Ethylene depresses the yield of arabinose in calcium-extracted solution relative to controls in air. Similarly, extraction with CaCl₂ increases the yield of extracted hydroxyproline in ethanol-insoluble polymers and ethylene depresses its level relative to controls. Metabolism of uronic acids and neutral sugars and growth in response to ethylene treatment contrast markedly with auxin-induced polysaccharide metabolism and growth. With auxin, sections increase mostly in length not radius, and this growth form is associated with an increase in the levels of xylose, glucose, and uronic acids. With ethylene, on the other hand, stem elongation is suppressed and expansion is promoted, and this growth pattern is associated with a decrease in xylose and glucose in the ethanol-insoluble polysaccharides.     

Complex Coacervation and Precipitation Between Soluble Pea Proteins and Apple Pectin

Complex formation (leading to either coacervation or precipitation) offers a tool to generate plant-based novel food structures and textures. This study investigated the formation of complexes between soluble pea proteins and apple pectin upon varying the protein-to-pectin ratio (r?=?2:1 to 10:1), pH (3–7), and temperature (25 and 85 °C) with a total biopolymer concentration set to 1% (w/w). The results showed that predominantly soluble biopolymer complexes were formed at pH 5, and at low ratio (r?=?2:1), whereas lowering the pH to more acidic condition, and to higher ratios (r?=?4:1–10:1) induced the formation of more insoluble biopolymer complexes. In general, the mean particle sizes of the biopolymer complexes ranged between approximately 20 and 100 μm. Upon heating to 85 °C, the amount of insoluble biopolymer complexes increased at pH 3–5 at all ratios, except at r?=?2:1. In addition, the complex sizes became somewhat larger at r?=?2:1 to 6:1 upon heat treatment, whereas only trivial size changes were observed at higher ratios (r?=?8:1 to 10:1). Overall, electrostatic and hydrophobic interactions played a major role in the complex formation between the soluble pea proteins and apple pectin. These findings are important for designing solely plant-based food structures.

     

Rapid Development of Mitochondria in Pea Cotyledons during the Early Stage of Germination

Rapid increases in activities and components of mitochondrial particles isolated from cotyledons of Pisum sativum var. Alaska during the early stage of germination are described. Respiratory rate of the cotyledons increased rapidly as hydration proceeded. A similar but slightly delayed increase in respiratory activity of the isolated mitochondrial fraction was observed. The respiratory control ratio and adenosine 5′-pyrophosphate/oxygen ratio rose during imbibition. Cytochrome oxidase and malate dehydrogenase activities in the mitochondrial fraction increased during the initial phase of imbibition. The increase seemed to precede that in respiratory activity. A significant activity of cytochrome oxidase and most of the malate dehydrogenase activity in the cotyledons were present in the postmitochondrial fraction in the case of the dry seeds. Mitochondrial protein and phospholipid also increased during imbibition, and the rise in the components seemed to concur with that in respiratory activity. The mechanism of mitochondrial development during imbibition is discussed.