Purification to homogeneity of pyrroline-5-carboxylate reductase of barley

An enzyme has been purified to homogeneity from barley seedlings which has `proline dehydrogenase' and the pyrroline-5-carboxylic acid reductase activities. The purification achieved is 39,000-fold as calculated from the proline dehydrogenase activity. The subunit molecular weight of the protein is 30 kilodaltons. The native enzyme has molecular weights up to 480 kilodaltons, depending on the buffer environment. From the pH profiles, the specific activities and thermodynamic considerations, it is concluded that the plant proline dehydrogenase functions in vivo as a pyrroline-5-carboxylate reductase.     

Barley aleurone layers secrete a nuclease in response to gibberellic Acid : purification and partial characterization of the associated ribonuclease, deoxyribonuclease, and 3'-nucleotidase activities

Incubation of barley (Hordeum vulgare L. cv Himalaya) half-seeds with gibberellic acid enhances the secretion of ribonuclease and deoxyribonuclease from aleurone tissue (MJ Chrispeels, JE Varner 1967 Plant Physiol 42: 398-406; L Taiz, JE Starks 1977 Plant Physiol 60: 182-189). These activities were over 50-fold greater in medium of half-seeds incubated with gibberellic acid than in control medium. Ribonuclease and deoxyribonuclease activities initially appeared in the medium 24 to 48 hours after hormone induction and increased for up to 96 hours. Both activities had a pH optimum of 6.0 and a temperature optimum of 55°C. When the medium from gibberellic acid-treated half-seeds was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the major ribonuclease and deoxyribonuclease activity bands comigrated. The two enzyme activities remained associated throughout a 2,700-fold purification employing ammonium sulfate fractionation, Heparin-Agarose affinity chromatography, and Reactive Blue 2-Agarose affinity chromatography. Also accompanying the ribonuclease and deoxyribonuclease activities throughout purification was the ability to hydrolyze the 3′-phosphoester linkage of 3′-AMP. The purified protein was composed of a single polypeptide with an apparent molecular weight of 36 kilodaltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It is concluded that in response to gibberellic acid, barley aleurone tissue secretes a nuclease having ribonuclease, deoxyribonuclease, and 3′-nucleotidase activities.     

Molecular cloning, overexpression, and characterization of autophosphorylation in calcium-dependent protein kinase 1 (CDPK1) from Cicer arietinum

In plants, calcium-dependent protein kinases (CDPKs) are key intermediates in calcium-mediated signaling that couple changes in Ca²⁺ levels to a specific response. In the present study, we report the high-level soluble expression of calcium-dependent protein kinase1 from Cicer arietinum (CaCDPK1) in Escherichia coli. The expression of soluble CaCDPK1 was temperature dependent with a yield of 3–4 mg/l of bacterial culture. CaCDPK1 expressed as histidine-tag fusion protein was purified using Ni–NTA affinity chromatography till homogeneity. The recombinant CaCDPK1 protein exhibited both calcium-dependent autophosphorylation and substrate phosphorylation activities with a V _max and K _m value of 13.2 nmol/min/mg and 34.3 μM, respectively, for histone III-S as substrate. Maximum autophosphorylation was seen only in the presence of calcium. Optimum temperature for autophosphorylation was found to be 37 °C. The recombinant protein showed optimum pH range of 6–9. The role of autophosphorylation in substrate phosphorylation was investigated using histone III-S as exogenous substrate. Our results show that autophosphorylation happens before substrate phosphorylation and it happens via intra-molecular mechanism as the activity linearly depends on enzyme concentrations. Autophosphorylation enhances the kinase activity and reduces the lag phase of activation, and CaCDPK1 can utilize both ATP and GTP as phosphodonor but ATP is preferred than GTP.     

Solubilization of the receptor for N-1-naphthylphthalamic Acid

A receptor protein for the auxin transport inhibitor, N-1-naphthylphthalamic acid (NPA), has been solubilized from corn coleoptile membranes using Triton X-100. [³H]NPA binding activity of the receptor was compared in soluble and membrane-bound states. Both activities are abolished by treatment with trypsin. Differences between the two are observed in pH optima and rates of heat inactivation.     

Properties and Ontogenic Development of Membrane-Bound Histidine Decarboxylase from Rat Brain

Abstract: Histidine decarboxylase (HD) activity was determined in high-speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates. Twenty-eight percent of the HD activity was associated with membranes, and the remaining was soluble. Several properties of the soluble and membrane-bound HD were compared. No significant differences in the values of K _m for histidine and pyridoxal 5'-phosphate were observed. The solubilization of membrane-bound HD with Triton X-100 resulted in an increase of 60% over the nonsolubilized activity with no changes in the K _m for substrate and cofactor. The proportion of free pyridoxal 5'-phosphate-independent activity was identical in both fractions. The soluble and membrane-bound forms of the enzyme differ slightly in their pH-activity profiles, although both enzymes showed an optimum pH near 6.5. The HD activities present in soluble and membrane fractions were determined at different postnatal ages. The soluble activity increased until day 90, whereas the membrane-bound activity became stabilized from day 20.     

NADH-Ferricyanide Reductase of Leaf Plasma Membranes : Partial Purification and Immunological Relation to Potato Tuber Microsomal NADH-Ferricyanide Reductase and Spinach Leaf NADH-Nitrate Reductase

Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b₅ reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber microsomal NADH-ferricyanide reductase (NADH-cytochrome b₅ reductase).     

Quinoprotein D-glucose dehydrogenases inAcinetobacter calcoaceticus LMD 79. 41: Purification and characterization of the membrane-bound enzyme distinct from the soluble enzyme

Acinetobacter calcoaceticus is known to contain soluble and membrane-bound quinoprotein D-glucose dehydrogenases while other oxidative bacteria such asPseudomonas orGluconobacter contain only membrane-bound enzyme. The two different forms were believed to be the same enzyme or interconvertible. Present results show that the two different forms of glucose dehydrogenase are distinct from each other in their enzymatic and immunological properties as well as in their molecular size.The soluble and membrane-bound glucose dehydrogenases were separated after French press-disruption by repeated ultracentrifugation, and then purified to nearly homogeneous state. The soluble enzyme was a polypeptide of 55 Kdaltons, while the membrane-bound enzyme was a polypeptide of 83 Kdaltons which is mainly monomeric in detergent solution. Both enzymes showed different enzymatic properties including substrate specificity, optimum pH, kinetics for glucose, and reactivity for ubiquinone-homologues. Furthermore, the two enzymes could be distinguished immunochemically: the membrane-bound enzyme is cross-reactive with an antibody raised against membrane-bound enzyme purified fromPseudomonas but not with antibody elicited against the soluble enzyme, while the soluble enzyme is not cross-reactive with the antibody of membrane-bound enzyme.Data also suggest that the membrane-bound enzyme functions by linking to the respiratory chain via ubiquinone though the function of the soluble enzyme remains unclear.     

Purification and Characterization of Soluble (Cytosolic) and Bound (Cell Wall) Isoforms of Invertases in Barley (Hordeum vulgare) Elongating Stem Tissue

Three different isoforms of invertases have been detected in the developing internodes of barley (Hordeum vulgare). Based on substrate specificities, the isoforms have been identified to be invertases (β-fructosidases EC 3.2.1.26). The soluble (cytosolic) invertase isoform can be purified to apparent homogeneity by diethylaminoethyl cellulose, Concanavalin-A Sepharose, organomercurial Sepharose, and Sephacryl S-300 chromatography. A bound (cell wall) invertase isoform can be released by 1 molar salt and purified further by the same procedures as above except omitting the organo-mercurial Sepharose affinity chromatography step. A third isoform of invertase, which is apparently tightly associated with the cell wall, cannot be isolated yet. The soluble and bound invertase isoforms were purified by factors of 60- and 7-fold, respectively. The native enzymes have an apparent molecular weight of 120 kilodaltons as estimated by gel filtration. They have been identified to be dimers under denaturing and nondenaturing conditions. The soluble enzyme has a pH optimum of 5.5, K_m of 12 millimolar, and a V_max of 80 micromole per minute per milligram of protein compared with cell wall isozyme which has a pH optimum of 4.5, K_m of millimolar, and a V_max of 9 micromole per minute per milligram of protein.     

A simple linear relationship and definition of a unit for proteinase activity

A mathematical transformation involving the $\text{3}\text{2}$ power of the number of milligrams of soluble nitrogen released from the substrate makes possible the expression of proteolytic activity in terms of the quantity of enzyme required to cause a given change in the substrate. The relationship is linear for several different enzymes including papain, patent flour, malted barley, malted wheat flour, and several fungal preparations acting on either Bacto-hemoglobin or gluten substrate.One unit of proteinase activity is defined as that activity contained by a quantity of enzyme-active material which gives an increase in soluble nitrogen in a 10-ml. aliquot from the filtrate corresponding to the intersection of the straight line with the transformed value representing 1 ml. of 0.0714 N alkali.This method of expressing proteolytic activity greatly simplifies the expression of proteolytic activity and has been found convenient for analytical purposes as well as for comparing activities of different enzymes acting on the same substrate.     

THE DISTRIBUTION OF CALCIUM-DEPENDENT PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHODIESTERASE IN RAT BRAIN

Abstract— The properties of Ca2⁺-dependent phosphatidylinositol-phosphodiesterase in membrane fractions and supernatants prepared from rat brain have been examined with the aim of providing firm evidence for the existence of a membrane-bound activity distinct from the soluble enzyme found in the cytosol (EC 3.1.4.10). The soluble enzyme is either stimulated or inhibited at pH 7.0 by deoxycholate depending on the ratio of detergent to substrate. The effects of deoxycholate are pH dependent and result in a shift of the enzyme optimum to a higher pH if the enzyme is assayed in the presence of deoxycholate. The soluble enzyme cannot hydrolgse membrane-bound phosphatidylinositol (in ₃₂P-labelled rat liver microsomes) unless deoxycholate is present. The pH optimum is 6.7 for this detergent-dependent hydrolysis and this is probably dependent on the ionization of deoxycholic acid. The lactate dehydrogenase (EC 1.1.1.27) content of rat brain membrane fractions has been measured to estimate the contamination of these fractions by supernatant phosphatidylinositol-phosphodiesterase. No evidence has been found for phosphatidylinositol-phosphodiesterase activities that cannot be explained by such contamination. It is concluded that all the properties of calcium-dependent phospha-tidylinositol-phosphodicsterase in rat brain can be explained by the existence of only the solublc cyto-plasmic enzyme: no evidence confirming a distinct membrane-bound activity has been obtained.     

A soluble acid invertase is directed to the vacuole by a signal anchor mechanism

Enzyme activities in the vacuole have an important impact on the net concentration of sucrose. In sugarcane (Saccharum hybrid), immunolabelling demonstrated that a soluble acid invertase (β-fructofuranosidase; EC 3.2.1.26) is present in the vacuole of storage parenchyma cells during sucrose accumulation. Examination of sequences from sugarcane, barley and rice showed that the N-terminus of the invertase sequence contains a signal anchor and a tyrosine motif, characteristic of single-pass membrane proteins destined for lysosomal compartments. The N-terminal peptide from the barley invertase was shown to be capable of directing the green fluorescent protein to the vacuole in sugarcane cells. The results suggest that soluble acid invertase is sorted to the vacuole in a membrane-bound form.     

Subcellular distribution of soluble and membrane-bound Leu-, Arg- and Asp-beta-naphthylamide-hydrolysing activities in rat brain

Subcellular distribution of soluble and membrane-bound Leu-, Arg- and Asp-beta-naphthylamide hydrolysing activities (arylamidase activity) was studied from left and right rat brains, each including hemisphere, cerebellum and brain stem. Both soluble Leu- and Arg-beta-naphthylamide hydrolysing activities showed the highest levels in the synaptosomal fraction. However, the microsomal fraction presented the highest levels when membrane-bound activity was assayed. When we used Asp-beta-naphthylamide as substrate, there were no differences among fractions in the membrane-bound activity, and the highest soluble activity was present at the mitochondrial level. Two different patterns in the subcellular distribution of enzymatic activity were observed: One of them was the result of the use of Leu- or Arg-beta-naphthylamide as substrate and the other when Asp-beta-naphthylamide was employed. No differences between left and right brains in soluble or membrane-bound activities were found.     

Comparison of physico-chemical properties of two forms of dopamine-beta-hydroxylase from chromaffin granules

The purification procedures of both soluble and membrane-bound forms of dopamine-beta-hydroxylase have been developed. The membrane-bound form was solubilized by a detergent. Both preparations have been obtained in electrophoretically homogeneous form. The yield of soluble and membrane-bound enzyme forms by a described procedure was 22 mg and 15 mg, correspondingly, from 100 g of chromaffin granules paste. A comparative analysis of the main physico-chemical properties of the two enzyme forms has shown their identity. The effects of pH, ionic strength, oxidants and reducers on the EPR spectra of the two forms of dopamine-beta-hydroxylase have been investigated. A comparison of the EPR spectra of the two forms of the enzyme suggests that the copper environment of soluble and membrane-bound dopamine-beta-hydroxylases is practically identical.     

Proteolytic activity in Plagiodera versicolora Laicharting (Coleoptera: Chrysomelidae): Characterization of digestive proteases and effect of host plants

Proteolytic profiles in the midgut of Plagiodera versicolora were studied using biochemical approaches, and the effects of host plants on possible changes in their activity were determined. Morphology of the alimentary canal revealed several areas of sections, namely bucca, pharynx, esophagus, crop, midgut, ileum, rectum and anus. A pH of 6 and 11 was found to be optimal for soluble and membrane-bound fractions, by using azocasein 2% as a substrate. Determination of specific proteases demonstrates the presence of trypsin-like, chymotrypsin-like, elastase, cathepsin B, cathepsin L and cathepsin D, as well as two exopeptidases. Regarding site of activity for each specific protease, it was found that the major activity of cathepsin B and cathepsin L was in the soluble fraction, chymotrypsin, cathepsin D and two exopeptidases in membrane-bound fraction. Additionally, trypsin-like and elastase activities had no significant differences between fractions. The presence of the above mentioned specific proteases was verified using the specific inhibitors PMSF, TLCK, TPCK, cystatin, phenanthroline and DTT. Feeding of the beetle on four host plants: including Salix aegyptica, S. alba, Populus alba and P. caspica, from the 1st larval instar to adult, revealed the highest trypsin-, chymotrypsin-like and elastase activities in the individuals fed on S. aegyptica and S. alba, respectively. Regarding cathepsins B and L, the highest activities were observed on S. alba and S. aegypticum but cathepsin D was higher in S. Alba and P. alba. Feeding on S. alba and S. aegypticum showed the highest activities of amino- and carboxy-peptidases, respectively.     

In vitro synthesis of the large subunit of ribulose diphosphate carboxylase on 70 S ribosomes

Polyribosomes isolated from greening barley leaves were active in directing protein synthesis, using soluble components isolated from Escherichia coli. A peptide of 55,000 molecular weight was a major product of translation activity. This peptide was precipitated by antibody to ribulose 1,5-diphosphate carboxylase (RuDPCase) and comigrated with the large subunit of RuDPCase on sodium dodecyl sulfate-polyacrylamide gels. Cyanogen bromide peptides of the peptide of 55,000 molecular weight also corresponded to the peptides prepared from authentic RuDPCase large subunit. The peptides synthesized were shown by sucrose density gradient sedimentation to be largely associated with 70 S ribosomes.     

Isolation and Partial Characterization of a Factor from Barley Aleurone that Modifies alpha-Amylase in Vitro

Posttranslational modifications that give rise to multiple forms of α-amylase (EC 3.2.1.1) in barley (Hordeum vulgare L. cv Himalaya) were studied. When analyzed by denaturing polyacrylamide gel electrophoresis, barley α-amylase has a molecular mass of 43 to 44 kilodaltons, but isoelectric focusing resolves the enzyme into a large number of isoforms. To precisely identify these isoforms, we propose a system of classification based on their isoelectric points (pl). α-Amylases with pls of approximately 5, previously referred to as low pl or Amy1 isoforms, have been designated HAMY1, and α-amylases with pls of approximately 6, referred to as high pl or Amy2, are designated HAMY2. Individual isoforms of HAMY1 and HAMY2 are identified by their pls. For example, the most acidic α-amylase synthesized and secreted by barley aleurone layers is designated HAMY1(4.56). Some of the diversity in the pls of barley α-amylases arises from posttranslational modifications of the enzyme. We report the isolation of a factor from barley aleurone layers and incubation media that can modify HAMY1 isoforms in vitro. This factor has a molecular mass between 30 and 50 kilodaltons, and it can catalyze the conversion of HAMY1(4.90) and HAMY1(4.64) to isoforms 4.72 and 4.56, respectively. The in vitro conversion of HAMY1 isoforms by the factor is favored by pH values of approximately 5 and is inhibited at approximately pH 7. The level of this factor in aleurone layers and incubation media is not affected by treatment of the tissue with gibberellic acid. The amylase-modifying activity from barley will also modify α-amylases isolated from human saliva and porcine pancreas. An activity that can modify HAMY1 isoforms in vitro has also been isolated from Onozuka R10 cellulase. Because the activity isolated from barley lowers the pl of α-amylase from barley, human saliva, and porcine pancreas, we speculate that it is a deamidase.     

In Vitro Biosynthesis of Vicia faba Polyphenoloxidase

Poly A⁺ mRNA was isolated from Vicia faba leaves and translated in vitro using a rabbit reticulocyte translation system. From analysis of the total translation products, the major proteins synthesized in vitro were 32 kilodaltons and 20 kilodaltons. When antibodies to Vicia faba polyphenoloxidase were added, a specific immunoprecipitable protein was observed. This protein's molecular weight was shown to be similar to that of the isolated enzyme (45 kilodaltons). The isolated enzyme successfully competed with the in vitro synthesized product for antipolyphenoloxidase. In addition, the in vitro synthesized product was not immunoprecipitated with antitomato peroxidase and comigrated with isolated and/or iodinated enzyme in sodium dodecylsulfate-polyacrylamide gel electrophoresis. Using in vitro translation and specific immunoprecipitation, a primary translation product corresponding to Vicia faba polyphenoloxidase was identified as a 45 kilodaltons protein.     

Localization, purification, and characterization of shikimate oxidoreductase-dehydroquinate hydrolyase from stroma of spinach chloroplasts

The stroma of chloroplasts is probably the sole site of the shikimate pathway enzymes shikimate oxidoreductase/dehydroquinate hydrolyase (SORase/DHQase) in spinach leaves. (a) The chromatographic behavior of the bifunctional protein SORase/DHQase on several separation materials with extracts from stroma compared with leaf extracts showed only one peak of enzymic activity originating from the stroma. (b) Polyacrylamide gel electrophoresis (PAGE) of these extracts followed by specific staining resulted in the same pattern without a band of extraplastidic enzyme. (c) In protoplast fractionation experiments it was shown that SORase/DHQase was present only in the soluble chloroplast protein fraction.

An improved purification procedure for SORase/DHQase from stroma of chloroplasts, yield 40%, 1600 times as pure, gave essentially one protein band on sodium dodecyl sulfate-PAGE. Our results demonstrate that both enzyme functions are carried out by a single polypeptide. Nondenaturing PAGE exhibited a pattern of four bands with SORase/DHQase showing that they differ in charge but not in their molecular weight. Molecular weight was determined to be 67 kilodaltons (gel filtration) and 59 kilodaltons (PAGE) for all four forms. It was proven they were not due to artifacts. The four forms show similar kinetic properties, their K_m and pH optima differing only very slightly. Response to some metabolites is reported.

     

Comparison of the Soluble and Membrane-Bound Forms of the Puromycin-Sensitive Enkephalin-Degrading Aminopeptidases from Rat

Enkephalin degradation in brain has been shown to be catalyzed, in part, by a membrane-bound puromycin-sensitive aminopeptidase. A cytosolic puromycin-sensitive aminopeptidase with similar properties also has been described. The relationship between the soluble and membrane forms of the rat brain enzyme is investigated here. Both of these aminopeptidase forms were purified from rat brain and an antiserum was generated to the soluble enzyme. Each of the aminopeptidases is composed of a single polypeptide of molecular mass 100 kilodaltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography. The antisoluble aminopeptidase antiserum reacts with both enzyme forms on immunoblots and inhibits both with nearly identical inhibition curves. The isoelectric points (pI = 5.0) of both forms were shown to be identical. N-terminal sequencing yielded a common sequence (P-E-K-R-P-F-E-R-L-P-T-E-V-S-P-I-N-Y) for both enzyme forms, and peptide mapping yielded 26 peptides that also appeared identical between the two enzyme forms. Studies on the nature of the association of the membrane enzyme form with the cell membrane suggest that this enzyme form does not represent the soluble form trapped during the enzyme preparation. It is suggested that the membrane form of the puromycin-sensitive aminopeptidase is identical to the soluble enzyme and that it associates with the membrane by interactions with other integral membrane proteins.     

A Proteinase from Germinating Barley : I. Purification and Some Physical Properties of a 30 kD Cysteine Endoproteinase from Green Malt

A proteinase was purified from germinated barley (green malt from Hordeum vulgare L. cv Morex) by acidic extraction, ammonium sulfate fractionation and successive chromatographies on CM-cellulose, hemoglobin sepharose, Sephadex G-75 and organomercurial agarose columns. The overall purification and final recovery were 290-fold and 7.5%, respectively. The purified enzyme was homogeneous on analytical gel electrophoresis, yielding a single protein associated with protease activity. An apparent molecular weight of about 20 kilodaltons was estimated for the native enzyme from gel filtration. SDS-gel electrophoresis revealed a single polypeptide of about 30 kilodaltons. The optimum pH for the hydrolysis of hemoglobin was around 3.8. The enzyme was strongly inhibited by leupeptin but was insensitive to phenylmethylsulfonyl fluoride, indicating that it was a cysteine proteinase. It hydrolyzed several large proteins from various origins. The ability of the enzyme to digest barley storage proteins in vitro was examined using SDS-gel electrophoresis. The hydrolysis patterns obtained showed that the enzyme rapidly hydrolyzed the large hordein polypeptides into relatively small fragments. The results of this study suggest that this 30 kilodalton enzyme is one of the predominant cysteine proteinases secreted into the starchy endosperm during barley germination and that it plays a major role in the mobilization of storage proteins.