Exoamylase activity in vacuoles isolated from pea and wheat leaf protoplasts

Vacuoles isolated from pea (Pisum sativum), and wheat (Triticum aestivum) leaf protoplasts contained considerable activities of electrophoretically highly mobile exoamylases. Vacuoles from spinach (Spinacia oleracea) leaf and photoautotrophic Chenopodium rubrum suspension culture cell protoplasts were devoid of amylolytic activity. Endoamylase activity was in all cases associated primarily with the chloroplast.     

Starch Degradation and Distribution of the Starch-Degrading Enzymes in Vicia faba Leaves (Diurnal Oscillation of Amylolytic Activity and Starch Content in Chloroplasts)

Subcellular localization of the starch-degrading enzymes in Vicia faba leaves was achieved by an electrophoretic transfer method through a starch-containing gel (SCG) and enzyme activity measurements. Total amylolytic and phosphorolytic activities were found predominantly in the extrachloroplastic fraction, whereas the debranching enzymes showed homogenous distribution between stromal and extrachloroplastic fractions. Staining of end products in the SCG revealed two isoforms of [alpha]-amylase (EC 3.2.1.1) and very low [beta]-amylase activity (EC 3.2.1.2) in the chloroplast preparation, whereas [alpha]- and [beta]-amylase exhibited higher activities in the crude extract. However, it is unclear whether the low [alpha]- and [beta]-amylase activities associated with the chloroplast are contamination or activities that are integrally associated with the chloroplast. Study of the diurnal fluctuation of the starch content and of the amylase activities under a 9-h/15-h photoperiod showed a 2-fold increase of the total amylolytic activity in the chloroplasts concurrent with the starch degradation in the dark. No fluctuation was detectable for the extrachloroplastic enzymes. The possible roles and function of the chloroplastic and extrachloroplastic hydrolytic enzymes are discussed.     

Adenosine triphosphatase of bean plastids: its properties and site of formation

Extracts of bean (Phaseolus vulgaris L.) etioplasts and chloroplasts contain a dithiothreitol-activated Ca²⁺-dependent adenosine triphosphatase which is inhibited by Dio-9. The chloroplast and etioplast enzymes have identical R_F values upon disc gel electrophoresis. Optimum extraction of the enzyme from either plastid preparation is accomplished with 1 mm ethylenediamine tetraacetic acid. Photophosphorylation capacity can be partially restored to depleted chloroplast preparations by addition of either the chloroplast or etioplast extract. These results suggest that the adenosine triphosphatase from etioplasts and chloroplasts represents a modified coupling factor for photophosphorylation.     

Characterization of d-Enzyme (4-alpha-Glucanotransferase) in Arabidopsis Leaf

Two major forms of d-enzyme (4-α-glucanotransferase, EC 2.4.1.25) were successfully separated from most of the amylase activity using FPLC-Mono Q column chromatography. Transfer of a maltosyl group was observed upon the incubation of d-enzyme with maltotriose and d-[U-¹⁴ C]glucose. About 4.5% of the radioactivity was transferred to maltotriose in 2 hours. End product analysis showed the accumulation of glucose and maltopentaose from maltotriose within the first 10 minutes of the reaction. Several other maltodextrins were also observed with longer incubation times, although maltose was never produced. A quantitative measurement of maltodextrin production from the reaction of [¹⁴ C]maltotriose with d-enzyme showed that the quantity of maltotriose decreased from 100% to 31% after 3 hours incubation, while glucose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose, and higher maltodextrins increased in amount. Glucose is the major product throughout the course of the reaction of d-enzyme with maltotriose. Maltotriose, in addition to glucose, are the major products in the reaction of d-enzyme with maltodextrins with a chain length greater than maltotriose. This study confirms the existence of a transglycosylase that disproportionates maltotriose and higher maltodextrins by transferring maltosyl or maltodextrinyl groups between maltodextrins resulting in the production of glucose and different maltodextrins, but not maltose, in leaf tissue with enzymic properties very similar to the previously reported d-enzyme in potato.     

Action of Corn and Rice-inactivating Proteins on a Purified Nitrate Reductase from Chlorella vulgaris

When nitrate reductase (NR) purified from Chlorella was incubated with NR-inactivating proteins purified from corn roots and rice cell suspension cultures or with trypsin there was a loss in NADH-NR and NADH cytochrome c reductase (NADH-CR) activities with time whereas the reduced methylviologen NR (MV-NR) remained active. When NADH-NR and NADH-CR activities were inactivated completely by the incubation with corn protein, the major protein band obtained by polyacrylamide gel electrophoresis shifted from an R_F value of 0.12 to an R_F of 0.25 and reduced MV-NR activity moved to the new position on the gel. When NADH-NR and NADH-CR activities were partially inactivated by the corn protein, NADH-NR activity was detected in an intermediate position (R_F value of 0.18). Incubation with trypsin also caused a change in the NR protein migration pattern (R_F value of 0.20). This protein band also had reduced MV-NR activity. Thus, the corn inactivator degrades NR in a fashion similar to but not identical with trypsin. The incubation of NR with rice inactivating protein resulted in a loss of NADH-NR but had no effect on the migration of NR protein or on the reduced MV-NR activity or mobility suggesting that the rice protein binds to Chlorella NR.     

Isolation and Characterization of a Starchless Mutant of Arabidopsis thaliana (L.) Heynh Lacking ADPglucose Pyrophosphorylase Activity

A mutant of Arabidopsis thaliana lacking ADPglucose pyrophosphorylase activity (EC 2.7.7.27) was isolated (from a mutagenized population of plants) by screening for the absence of leaf starch. The mutant grows as vigorously as the wild type in continuous light but more slowly than the wild type in a 12 hours light/12 hours dark photoperiod. Genetic analysis showed that the deficiency of both starch and ADPglucose pyrophosphorylase activity were attributable to a single, nuclear, recessive mutation at a locus designated adg1. The absence of starch in the mutant demonstrates that starch synthesis in the chloroplast is entirely dependent on a pathway involving ADPglucose pyrophosphorylase. Analysis of leaf extracts by two-dimensional polyacrylamide gel electrophoresis followed by Western blotting experiments using antibodies specific for spinach ADPglucose pyrophosphorylase showed that two proteins, present in the wild type, were absent from the mutant. The heterozygous F₁ progeny of a cross between the mutant and wild type had a specific activity of ADPglucose pyrophosphorylase indistinguishable from the wild type. These observations suggest that the mutation in the adg1 gene in TL25 might affect a regulatory locus.     

Purification of a beta-Amylase that Accumulates in Arabidopsis thaliana Mutants Defective in Starch Metabolism

Amylase activity is elevated 5- to 10-fold in leaves of several different Arabidopsis thaliana mutants defective in starch metabolism when they are grown under a 12-hour photoperiod. Activity is also increased when plants are grown under higher light intensity. It was previously determined that the elevated activity was an extrachloroplastic β-(exo)amylase. Due to the location of this enzyme outside the chloroplast, its function is not known. The enzyme was purified to homogeneity from leaves of both a starchless mutant deficient in plastid phosphoglucomutase and from the wild type using polyethylene glycol fractionation and cyclohexaamylose affinity chromatography. The molecular mass of the β-amylase from both sources was 55,000 daltons as determined by denaturing gel electrophoresis. Gel filtration studies indicated that the enzyme was a monomer. The specific activities of the purified protein from mutant and wild-type sources, their substrate specificities, and K_m for amylopectin were identical. Based on these results it was concluded that the mutant contained an increased level of β-amylase protein. Enzyme neutralization studies using a polyclonal antiserum raised to purified β-amylase showed that in each of two starchless mutants, one starch deficient mutant and one starch overproducing mutant, the elevated amylase activity was due to elevated β-amylase protein.     

Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana

Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic β-glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F₁ and F₂ progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F₁ progeny and most of the F₂ progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F₂ individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F₁ generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.     

Degradation of Glucan Primers in the Absence of Starch Synthase 4 Disrupts Starch Granule Initiation in Arabidopsis

Arabidopsis leaf chloroplasts typically contain five to seven semicrystalline starch granules. It is not understood how the synthesis of each granule is initiated or how starch granule number is determined within each chloroplast. An Arabidopsis mutant lacking the glucosyl-transferase, STARCH SYNTHASE 4 (SS4) is impaired in its ability to initiate starch granules; its chloroplasts rarely contain more than one large granule, and the plants have a pale appearance and reduced growth. Here we report that the chloroplastic α-amylase AMY3, a starch-degrading enzyme, interferes with granule initiation in the ss4 mutant background. The amy3 single mutant is similar in phenotype to the wild type under normal growth conditions, with comparable numbers of starch granules per chloroplast. Interestingly, the ss4 mutant displays a pleiotropic reduction in the activity of AMY3. Remarkably, complete abolition of AMY3 (in the amy3 ss4 double mutant) increases the number of starch granules produced in each chloroplast, suppresses the pale phenotype of ss4, and nearly restores normal growth. The amy3 mutation also restores starch synthesis in the ss3 ss4 double mutant, which lacks STARCH SYNTHASE 3 (SS3) in addition to SS4. The ss3 ss4 line is unable to initiate any starch granules and is thus starchless. We suggest that SS4 plays a key role in granule initiation, allowing it to proceed in a way that avoids premature degradation of primers by starch hydrolases, such as AMY3.     

Starch Degradation in Spinach Leaves: ISOLATION AND CHARACTERIZATION OF THE AMYLASES AND R-ENZYME OF SPINACH LEAVES

The properties of two amylase activities which differ in their substrate specificity and subcellular location as well as a chloroplast-associated R-enzyme (debranching activity) are reported. An extrachloroplastic amylase is resolved by gel filtration chromatography into two activities of 80,000 and 40,000 daltons. Both extrachloroplastic activities hydrolyze amylopectin and shellfish glycogen and only slowly hydrolyze rabbit liver glycogen, β-limit amylopectin, and amylose. In contrast, the major chloroplastic amylase attacks all of these glucans at comparable rates. Glucan hydrolysis by both the extrachloroplastic and chloroplastic amylase generates not only maltose but appreciable amounts of other oligosaccharides, whereas maltotetraose hydrolysis produces glucose, maltose, and maltotriose. The action patterns displayed by the amylase activities indicate that both are endoamylases, although they lack the typical Ca²⁺ requirement or heat stability of seed endosperm α-amylases. Dithiothreitol, glutathione (oxidized or reduced), ascorbate, dehydroascorbate, and dithiothreitol plus thioredoxin have no effect on either the chloroplastic or extrachloroplastic amylase activities.     

Localization of alpha-Amylase in the Apoplast of Pea (Pisum sativum L.) Stems

Most of the activity of an α-amylase present in crude pea (Pisum sativum L. cv Laxton's Progress No. 9) leaf preparations cannot be found in isolated pea leaf protoplasts. The same extrachloroplastic α-amylase is present in pea stems, representing approximately 6% of total stem amylolytic activity and virtually all of the α-amylase activity. By a simple infiltration-extraction procedure, the majority (87%) of this α-amylase activity was recovered from the pea stem apoplast without significantly disrupting the symplastic component of the tissue. Only 3% of the β-amylase activity and less than 2% of other cellular marker enzymes were removed during infiltration-extraction.     

Starch degradation in the cotyledons of germinating lentils

Starch, total amylolytic and phosphorylase activities were determined in lentil cotyledons during the first days of germination. Several independent criteria show that the amylolytic activity is due mainly to an amylase of the α type. Starch is degraded slowly in the first days; during this time, α- and β-amylase activity are very low, while phosphorylase increases and reach a peak on the 3rd day. On the 4th day, there is a more rapid depletion of starch which coincides with an increase in α-amylase activity. By polyacrylamide gel electrophoresis of the crude starch-degrading enzyme, five bands were obtained: one phosphorylase, three α-amylases, and one β-amylase. Based on their heat lability or heat stability, two sets of α-amylase seem to exist in lentil cotyledons.     

Respiration and protein synthesis in Escherichia coli membrane-envelope fragments. VI. Solubilization and characterization of the electron transport chain

Membranes obtained from Escherichia coli have been solubilized with deoxycholate. The solubilized dehydrogenases and cytochromes are not sedimented at 105,000 g. These components readily penetrate the "included space" of Sepharose 4B (Pharmacia Fine Chemicals Inc., Uppsala, Sweden) and polyacrylamide gels and have been fractionated on the basis of molecular size. Solubilization destroys nicotinamide adenine dinucleotide, reduced form (NADH) oxidase and D-lactate oxidase activities, but leaves an appreciable part of the original succinoxidase activity intact. Evidence for a succinate dehydrogenase-cytochrome b ₁ complex is given. Menadione added to the solubilized preparation does not elicit NADH oxidase activity nor stimulate the existing succinoxidase activity, but does provoke an active D-lactate oxidase activity. This D-lactate oxidase activity, however, does not use cytochromes and is not sensitive to cyanide.     

Glutamate dehydrogenase in developing endosperm, chloroplasts, and roots of castor bean

All the glutamate dehydrogenase activity in developing castor bean endosperm is shown to be located in the mitochondria. The enzyme can not be detected in the plastids, and this is probably not due to the inactivation of an unstable enzyme, since a stable enzyme can be isolated from castor bean leaf chloroplasts. The endosperm mitochondrial glutamate dehydrogenase consists of a series of differently charged forms which stain on polyacrylamide gel electrophoresis with both NAD⁺ and NADP⁺. The chloroplast and root enzymes differ from the endosperm enzyme on polyacrylamide gel electrophoresis. The amination reaction of all the enzymes is affected by high salt concentrations. For the endosperm enzyme, the ratio of activity with NADH to that with NADPH is 6.3 at 250 millimolar NH₄Cl and 1.5 at 12.5 millimolar NH₄Cl. K_m values for NH₄⁺ and NAD(P)H are reduced at low salt concentrations. The low K_m values for the nucleotides may favor a role for glutamate dehydrogenase in ammonia assimilation in some situations.     

Biochemical Studies on Ricin Part X Effect of the Two Constituent Polypeptide Chains of Ricin D toward Mouse Sarcoma Ascite Tumor Cells

A maltotetraose-forming amylase from Pseudomonas stutzeri was highly purified by adsorption on starch granules and by chromatographies on Sephadex G-100 and DEAE-cellulose. The purified enzyme showed a single band in polyacrylamide gel electrophoreses with or without sodium dodecylsulfate. The optimum pH for enzyme action on starch was 6.0-6.5, and the optimum temperature was 45°C. The purified enzyme attacked starch from the non-reducing end to produce α-anomer oligosaccharides. This indicated that the enzyme was an exo-α-amylase which had not hitherto been found. The enzyme activity was markedly inhibited by the addition of Cu²⁺, Hg²⁺, N-bromosuccinimide and 2,3-butanedione. The molecular weight of the enzyme determined by the method of Weber and Osborn was about 5.7 × 10⁴. The isoelectric point of the enzyme was estimated to be 5.3 by polyacrylamide gel electrofocusing. The Km and k₀ values of this enzyme for starch, glycogen, short chain amylose and some maltooligosaccharides were calculated from Lineweaver-Burk plots.     

Purification and Properties of 2-Carboxy-d-Arabinitol 1-Phosphatase

Carboxyarabinitol 1-phosphatase (2-carboxy-d-arabinitol 1-phosphate phosphohydrolase), a chloroplast enzyme that metabolizes the naturally occurring inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase, was isolated from tobacco (Nicotiana tabacum) leaves. The enzyme was purified more than 3500-fold using a protocol that included ammonium sulfate fractionation and gel filtration, ion-exchange, and hydrophobic interaction chromatography. Analysis of the final preparation by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed the presence of a single polypeptide with a molecular mass of 53 kilodaltons. The enzyme exhibited an apparent K_m (carboxyarabinitol 1-phosphate) of 33 micromolar and a pH optimum of 7.5. Enzyme activity did not require divalent cations and was unaffected by the metal chelators EDTA and cysteine. Carboxyarabinitol 1-phosphatase activity was inhibited by zinc, copper and molybdate and stimulated by sulfate. Chloroplast metabolites that affected activity included inorganic phosphate and ATP, which were inhibitory, and ribulose-1,5-bisphosphate, fructose-1,6-bisphosphate and NADPH which stimulated activity 2.5-fold. Activation of carboxyarabinitol 1-phosphatase activity by these positive effectors, together with the previously reported requirement for dithiothreitol, explain the light/dark modulation of carboxyarabinitol 1-phosphatase activity in vivo.     

Amylases in Pea Tissues with Reduced Chloroplast Density and/or Function

Pea (Pisum sativum L.) tissues with reduced chloroplast density (e.g. petals and stems) or function (i.e. senescent leaves and leaves darkened for prolonged periods) were surveyed to determine whether tissues with genetically or environmentally reduced chloroplast density and/or function also have significantly different amylolytic enzyme activities and/or isoform patterns than leaf tissues with totally competent chloroplasts. Native PAGE followed by electrophoretically blotting through a starch or β-limit dextrin containing gel and KI/I₂ staining revealed that the primary amylases in leaves, stems, petals, and roots were the primarily vacuolar β-amylase (EC 3.2.1.2) and the primarily apoplastic α-amylase (EC 3.2.1.1). Among tissues of light grown pea plants, petals contained the highest levels of total amylolytic (primarily β-amylase) activity and considerably higher ratios of β- to α-amylase. In aerial tissues there was an inverse relationship between chlorophyll and starch concentration, and β-amylase activity. In sections of petals and stems there was a pronounced inverse relationship between chlorophyll concentration and the activity of α-amylase. Senescing leaves of pea, as determined by age, and protein and chlorophyll content, contained 3.8-fold (fresh weight basis) and 32-fold (protein basis) higher α-amylase activity than fully mature leaves. Leaves maintained in darkness for 12 days displayed a 14-fold (fresh weight basis) increase in α-amylase activity over those grown under continuous light. In senescence and prolonged darkness studies, the α-amylase that was greatly increased in activity was the primarily apoplastic α-amylase. These studies indicate that there is a pronounced inverse relationship between chloroplast function and levels of apoplastic α-amylase activity and in some cases an inverse relationship between chloroplast density and/or function and vacuolar β-amylase activity.     

Purification and some properties of two polyphenol oxidases from bartlett pears

Two polyphenol oxidases (enzymes A and B) from Bartlett pear (Pyrus communis) peelings were purified to electrophoretic homogeneity according to polyacrylamide gel by a combination of Sephadex gel filtration, diethylaminoethyl cellulose chromatography and hydroxyl apatite chromatography. While the two enzymes differ electrophoretically at pH 9.3, chromatographically on hydroxyl apatite, and in the effect of ionic strength on activity, they are similar with respect to chromatography on diethylaminoethyl cellulose, substrate specificity, pH activity relations, inhibition by p-coumaric and benzoic acids, and heat stability. The two enzymes are o-diphenol oxidases with no detectable monophenolase or laccase activities. Pyrocatechol, 4-methyl catechol, chlorogenic acid, and d-catechin are good substrates of the enzymes with K_m values in the range of 2 to 20 mm. Dependences of activity on oxygen and chlorogenic acid concentrations indicate a sequential mechanism for binding of these substrates to enzyme B. V_max and K_m values for oxygen and chlorogenic acid were 103 μmoles O₂ uptake per minute per milligram of enzyme, 0.11 mm and 7.2 mm, respectively, for enzyme B at pH 4.0. Both enzymes had maximum activity at pH 4.0 on chlorogenic acid. K_m values for chlorogenic acid were independent of pH from 3 to 7; the V_max values for both enzymes gave bell-shaped curves as a function of pH. p-Coumaric acid is a simple, linear noncompetitive inhibitor with respect to chlorogenic acid at pH 6.2 with K_i values of 0.38 and 0.50 mm for enzymes A and B, respectively. Benzoic acid is a linear competitive inhibitor with respect to chlorogenic acid at pH 4.0 with K_i values of 0.04 and 0.11 mm for enzymes A and B, respectively.     

Resolution and reconstitution of succinate-cytochrome c reductase. Preparations and properties of high purity succinate dehydrogenase and ubiquinol—cytochrome c reductase

An improved method was developed to sequentially fractionate succinate-cytochrome c reductase into three reconstitutive active enzyme systems with good yield: pure succinate dehydrogenase, ubiquinone-binding protein fraction and a highly purified ubiquinol-cytochrome c reductase (cytochrome b-c₁ III complex).An extensively dialyzed succinate-cytochrome c reductase was first separated into a succinate dehydrogenase fraction and the cytochrome b-c₁ complex by alkali treatment. The resulting succinate dehydrogenase fraction was further purified to homogeneity by the treatment of butanol, calcium phosphate gel adsorption and ammonium sulfate fractionation under anaerobic condition in the presence of succinate and dithiothreitol. The cytochrome b-c₁ complex was separated into cytochrome b-c₁ III complex and ubiquinone-binding protein fractions by careful ammonium acetate fractionation in the presence of deoxycholate.The purified succinate dehydrogenase contained only two polypeptides with molecular weights of 70 000 and 27 000 as revealed by the sodium dodecyl sulfate polyacrylamide gel electrophoretic pattern. The enzyme has the reconstitutive activity and a low K_m ferricyanide reductase activity of 85 μmol succinate oxidized per min per mg protein at 38°C.Chemical composition analysis of cytochrome b-c₁ III complex showed that the preparation was completely free of contamination of succinate dehydrogenase and ubiquinone-binding protein and was 30% more pure than the available preparation.When these three components were mixed in a proper ratio, a thenoyl-trifluoroacetone- and antimycin A-sensitive succinate-cytochrome c reductase was reconstituted.