L-Phenylalanine ammonia-lyase from Phaseolus vulgaris: partial degradation of enzyme subunits in vitro and in vivo

L-Phenylalanine ammonia-lyase (EC 4.3.1.5) has been purified from suspension cultured cells of French bean (Phaseolus vulgaris L.) which had been exposed to polysaccharide elicitor preparations from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. After preliminary purification by ammonium sulphate fractionation and gel filtration, the enzyme was further purified by (a) ion-exchange chromatography followed by chromatofocussing, (b) chromatography on rabbit anti-(phenylalanine ammonia-lyase) IgG, or (c) affinity chromatography on L-aminooxy(p-hydroxyphenyl)propionic acid (or L-tyrosine) linked to epoxy-activated Sepharose 6B via the phenolic hydroxyl group. The purified enzyme preparations exhibited subunit M_r values of 77 000, 70 000 and 53 000, the relative proportions of these depending upon the enzyme source, length of time taken for purification, and inclusion of freeze-thaw steps. Four forms of the enzyme, differing in pI value, were resolved by chromatofocussing, although all forms from the same preparation consisted of similar proportions of the different subunit M_r forms. Peptide mapping and freeze-thaw studies indicate that the M_r 77 000 native phenylalanine ammonia-lyase subunit is inherently unstable in vitro and breaks down to yield the lower M_r partial degradation products. Such products could also be observed following in vitro translation of phenylalanine ammonia-lyase mRNA. Pulse-chase experiments indicated that the 77 000 → 70 000 → 53 000 subunit interconversion also occurs in vivo.     

Comparison of acetyl-CoA carboxylases from parsley cell cultures and wheat germ

The activity of acetyl-CoA carboxylase of suspension-cultured cells of parsley (Petroselinum hortense Hoffm.) is greatly stimulated by light soon after transferring cells to new culture medium. Parsley acetyl-CoA carboxylase has been purified from frozen cells by treatment of the crude protein extract with Dowex 1 × 2 and polyethyleneimine, precipitation with (NH₄)₂SO₄, chromatography on DEAE-cellulose and blue Sepharose CL-6B, and gel filtration on Sepharose 6B. A recovery of about 8% has been achieved with a 300-fold increase in specific activity. Wheat germ acetyl-CoA carboxylase has been purified 2180-fold by a similar procedure. The two carboxylases have the following characteristics: Molecular weights of 840,000 for the parsley carboxylase and 700,000 for the wheat germ carboxylase have been estimated from the elution volumes of a calibrated Sepharose 6B column. Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the carboxylases from parsley and wheat each are composed of one large subunit (M_r = 210,000 and 240,000, respectively) and possibly one smaller polypeptide component (M_r = 105,000 and 98,000, respectively). Avidin-binding experiments demonstrated that the 240,000 — M_r component of wheat germ carboxylase is the biotin-containing subunit of this enzyme. No isoenzymes of the parsley carboxylase could be demonstrated.     

Purification and characterization of the messenger ribonucleoprotein-associated casein kinase II of Artemia salina cryptobiotic gastrulae

The mRNP-associated protein kinase is purified to near homogeneity by ion-exchange chromatography on phosphocellulose and affinity chromatography on casein-Sepharose 4B and ATP-agarose. The cyclic nucleotide-independent enzyme phosphorylates casein using either ATP or GTP. The enzyme exists in two forms composed of subunits with M_r 36 500 (α) and 28 000 (β) and of subunits with M_r 36 500 (α), 33 000 (α′) and 28 000 (β). The undegraded enzyme has an M_r of 136 000 ± 7000. The enzyme is inhibited by heparin and hemin and stimulated by spermine. The mRNP-associated protein kinase may be classified as a casein kinase II. Main mRNP protein phosphate acceptors have M_r values of 112 000, 72 000, 65 000, 53 000, 38 000, 28 000, 23 500 and 21 000. Phosphorylation of the M_r 38 000 poly(A)-binding protein resulted in the generation of different acidic ionic species. From the observed inhibition of the translational activity after phosphorylation by the mRNP-associated protein kinase a function in the repression of mRNP is proposed.     

Metabolism of purine nucleotides in the tomato plant

The enzymes 5′-nucleotidase (EC 3.1.3.5) and adenine phosphoribosyltransferase (EC 2.4.2.7) from the roots and leaves of tomato (Lycopersicon esculentum) have been purified and characterized. Two forms (root 1 and root 2) of 5′-nucleotidase from tomato roots were separated by chromatography on DEAE-cellulose. These were further purified by affinity chromatography on Blue Sepharose CL-6B. The enzyme from leaves appeared in only one form (leaf) when purified by similar methods. Root 2 and leaf enzymes were very similar in all respects including M_r (ca 68 000) whilst root 1 appeared distinct with a M_r close to 18 000. Tomato 5′-nucleotidase catalysed hydrolysis of isopentenylAMP and its action on AMP was inhibited in the presence of nucleoside monophosphates including isopentenylAMP. Adenine phosphoribosyltransferase existed in one form in roots and leaves and these differed from one another in several respects, e.g. pH optimum, M_r. Both enzymes catalysed phosphoribosylation of benzyladenine and the conversion of adenine to AMP was inhibited by the presence of cytokinin bases. The enzymes from the two sources differed in their patterns of inhibition by cytokinin bases.     

Proteins in the nervous system — structure and function : Progress in clinical and biological research; volume 79

We have purified a unique enzyme, α-amino-?-caprolactam racemase 945-fold from an extract of Achromobacter obae by Octyl—Sepharose CL-4B and Thiopropyl—Sepharose 6B and some other chromatographies. The purified enzyme was found homogeneous by sodium dodecyl sulfate—polyacrylamide gel electrophoresis and analytical ultracentrifugation. The enzyme has a monomeric structure with M_r ～ 50 000 and a sedimentation coefficient (s_20,w) of 4.28 S. The enzyme contains pyridoxal 5'-phosphate as a coenzyme. The pH optimum for the enzyme activity is ～9.0. D- and L-α-amino-?-caprolactams are the only substrates. The K_m values for the D- and L-isomers are, 8 and 6 mM, respectively.     

Allantoinase from nodules of pigeonpea (Cajanus cajan)

Allantoinase was purified about 10-fold from nitrogen fixing root nodules of pigeonpea (Cajanus cajan) using (NH₄)₂S0₄ fractionation and chromatography on Sephadex G-100. The purified preparation showed a specific activity of 1.73 nkat/mg protein, M_r of 125 000, pH optimum between 7.5 and 7.7 and K_m of 13.3 mM. The enzyme was heat stable up to 70dg and metal ions, except Hg²⁺, had no effect on the enzyme activity. The enzyme was inhibited significantly by reducing agents. Amino acids, ammonium, nitrate, potential precursors of allantoin and a number of other intermediate metabolites of ureide biosynthetic pathway had no effect on enzyme activity. It is suggested that allantoinase is unlikely to regulate the production of ureides in the nodule tissue.     

Purification and Partial Characterization of Potato (Solanum tuberosum) Invertase and Its Endogenous Proteinaceous Inhibitor

Invertase plays an important role in the hydrolysis of sucrose in higher plants, especially in the storage organs. In potato (Solanum tuberosum) tubers, and in some other plant tissues, the enzyme seems to be controlled by interaction with an endogenous proteinaceous inhibitor. An acid invertase from potato tubers (variety russet) was purified 1560-fold to electrophoretic homogeneity by consecutive use of concanvalin A-Sepharose 4B affinity chromatography, DEAE-Sephadex A-50-120 chromatography, Sephadex G-150 chromatography, and DEAE-Sephadex A-50-120 chromatography. The enzyme contained 10.9% carbohydrate, had an apparent molecular weight of 60,000 by gel filtration, and was composed of two identical molecular weight subunits (M_r 30,000). The enzyme had a K_m for sucrose of 16 millimolar at pH 4.70 and was most stable and had maximum activity around pH 5. The endogenous inhibitor was purified 610-fold to homogeneity by consecutive treatment at pH 1 to 1.5 at 37°C for 1 hour, (NH₄)₂SO₄ fractionation, Sephadex G-100 chromatography, DEAE-Sephadex G-50-120 chromatography, and hydroxylapatite chromatography. The inhibitor appears to be a single polypeptide (M_r 17,000) without glyco groups. The purified inhibitor was stable over the pH range of 2 to 7 when incubated at 37°C for 1 hour.     

Properties of γ-glutamyl arylamidase activity of the heavy subunit of γ-glutamyl arylamidase from Bacillus sp. strain No. 12

γ-Glutamyl arylamidase of Bacillus sp. strain No. 12, composed of two heavy (M_r 56 000) and two light (M_r 46 000) subunits, was dissociated and inactivated by mild SDS treatment. The activity was restored in the isolated heavy subunit but not in the light subunit when SDS was removed by dialysis. The restored activity of the heavy subunit was similar to that of the native enzyme with regard to substrate specificity and inhibition and activation by α- and γ-glutamyl compounds, free amino acids, peptides, enzyme inhibitors, and anti-native enzyme antibody.     

Purification and characterization of a nicotinamide adenine dinucleotide-dependent secondary alcohol dehydrogenase from Candida boidinii

From the yest Candida biodinili grown on glucose a new secondary alcohol dehydrogenase was purified 426-fold by heat treatment, column chromatography on DEAE-Sephacel, affinity chromatography on Blue Sepharose Cl-6b, and gel filtration on Sephacryl S-300. The purified enzyme was homogeneous as judged by analytical polyacrylamide gel electrophoresis. The molecular weight was found to be 150 000 by sedimentation equilibirum as well as by flitration. The enzyme appears to be composed of four identical subunits (M_r = 38000) as determined by SDS-gel electrophoresis. The enzyme catalyzes the oxidation of isopropanol to acetone in the presence of NAD⁺ as an electron acceptor. The K_m values were found to be 0.099 mM for isopropanoi and 0.14 mM for NDA⁺. Besides isopropanol also other secondary alcohols like butan-2-ol, pentan-2-ol, pentan-3-ol, hexan-2-ol, cyclobutanol, cyclopentanol, and cyclohexanol served as a substrate and were oxidazed to the correponding ketones. Isopropanol seems to be the best substrate for this enzyme which we therefore call isopropanol dehydrogenase. Primary alcohols are not oxidized by the enzyme. The optimum pH for enzymatic activity in the oxidation reaction was found to be 9.0, the optimal temperature is 45°C. The isolectric point of the isopropanol dehydrogenase was found to be pH 4.9. The enzyme is inactivated by mercaptide-forming reagents and chelating agents, 2-mercaptoethanol is an inhibitor. Zinc ions appear necessary for enzyme productuion.     

An enzyme mediating the conversion of zeatin to dihydrozeatin in phaseolus embryos

A reductase catalyzing the conversion of zeatin to dihydrozeatin was detected in soluble fractions of immature Phaseolus vulgaris embryos. The enzyme was partially purified by ammonium sulfate fractionation and affinity, gel filtration, and anion exchange chromatography. NADPH was the only cofactor required for enzyme activity, and the pH optimum was 7.5 to 8.0. The enzyme did not recognize compounds closely related to zeatin, such as ribosylzeatin, cls-zeatin, O-xylosylzeatin, N⁶-(Δ²-isopentenyl)adenine, or N⁶-(Δ²-isopentenyl)adenosine. No conversion of dihydrozeatin to zeatin by the enzyme was observed. Two forms of the reductase could be separated by either gel filtration or anion exchange high performance liquid chromatography. The high molecular weight isozyme (M_r 55,000 ± 5,000) eluted as the second peak from the anion exchange column, while the low molecular weight isozyme (M_r 25,000± 5000) was less negatively charged. The results suggest that side chain reduction occurs at the free base level. In addition, Phaseolus embryos are useful for the detection of zeatin-specific metabolic enzymes.     

The purification and partial amino acid sequence of a polypeptide from the glutelin fraction of rice grains; homology to pea legumin

The glutelin fraction was extracted from grain meals of rice (Oryzea sativa) with 50 mM Tris-HCl buffer (pH 8.8) containing 6 M urea and 10 mM 2-mercaptoethanol. Polypeptides of glutelin were separated and purified by ion-exchange chromatography under denaturing conditions. Analysis by two-dimensional gel electrophoresis showed that 2 major polypeptides of the rice glutelin fraction, M_r 36 000 and 22 000, were linked in disulphide bonded pairs containing one M_r 36 000 and one M_r 22 000 subunit. A partial amino acid sequence of the purified M_r 22 000 glutelin subunit showed it to be homologous to the β-subunit of pea legumin, a storage protein which also contains disulphide-linked subunit pairs (M_r 38 000 and M_r 22 000). It is therefore proposed that the major component of rice glutelin is a legumin-like protein.     

A comparison of the membrane-bound and extracellular cyclic AMP phosphodiesterases of Dictyostelium discoideum

The Dictyostelium discoideum membrane-bound and extracellular cyclic nucleotide phosphodiesterases (EC 3.1.4.17) shear several properties including the ability to react with a specific glycoprotein inhibitor and small inhibitory molecules. We have partialy purified the membrane-bound enzyme and compared its properties to those of the extracellular form. The kinetic properties of the two forms were similar except that, while associated with membrane particles, the membrane-bound form exhibited non-linear kinetics when assayed ove a broad substrate range. The isoelectric point of the membrane-bound phosphodiesterase was identical to that of the extracellular enzyme when isoelectrofocusing was done in the presence of 6 M urea. The molecular weights of membrane-bound and extracellular enzyme, determined by gel filtration, were the same following isoelectrofocusing in the presence of 6 M urea. When precipitated with an antiserum prepared against purified extracellular phosphodiesterase, the partially purified membrane-bound enzyme preparation was shown to contain a M_r 50 000 polypeptide comigrating with the extracellular enzyme during SDS polyacrylamide gel electrophoresis. When the iodinated extracellular enzyme and the iodinated M_r 50 000 polypeptide from membrane-bound enzyme were subjected to partial proteolytic digestion, similar profiles were obtained indicating extensive regions of homology.     

Purification and properties of α-amino--caprolactam racemase from Achromobacter obae

We have purified a unique enzyme, α-amino--caprolactam racemase 945-fold from an extract of Achromobacter obae by Octyl—Sepharose CL-4B and Thiopropyl—Sepharose 6B and some other chromatographies. The purified enzyme was found homogeneous by sodium dodecyl sulfate—polyacrylamide gel electrophoresis and analytical ultracentrifugation. The enzyme has a monomeric structure with M_r 50 000 and a sedimentation coefficient (s_20,w) of 4.28 S. The enzyme contains pyridoxal 5'-phosphate as a coenzyme. The pH optimum for the enzyme activity is 9.0. D- and L-α-amino--caprolactams are the only substrates. The K_m values for the D- and L-isomers are, 8 and 6 mM, respectively.     

Purification and properties of a high affinity guanosine 3′: 5′-cyclic monophosphate-binding phosphatase from silver beet leaves

A cyclic nucleotide-binding phosphatase was purified from silver beet leaves by a procedure involving chromatography on CM-Sepharose CL-6B, DEAE-Sephacel, casein-Sepharose 4B, concanavalin A-agarose and Ultrogel AcA44. The enzyme is eluted from concanavalin A-agarose by 0.5 M α-methylglucoside at high ionic strength. The enzyme is monomeric, having a subunit molecular weight (M_r) of 28 000; the native M_r is 31 000 as determined from gel filtration. The enzyme catalyzes the hydrolysis of a range of phosphomonoesters including various nucleotides and O-phosphotyrosine but not O-phosphoserine or O-phosphothreonine. The leaf phosphatase is competitively inhited by guanosine 3′ : 5′-cyclic monphosphate (cGMP) and adenosine 3′ : 5′-cyclic monophosphate (cAMP) (K_i-values: 0.4 μM and 3.3 μM, respectively). The leaf phosphotase has the highest affinity for cGMP yet reported for a plant protein.     

A rapid four column purification of 2-deoxy-D-glucoside-2-sulphamate sulphohydrolase from human liver

2-Deoxy-D-glucoside-2-sulphamate sulphohydrolase was extracted from human liver and purified 40 000-fold by a simple four column procedure. The purification was followed using a specific substrate isolated from an acid hydrolysate of heparin, $\text{O-(α-2-}\text{sulphamino}\text{-2-}\text{deoxy-}\text{D}\text{-glucopyranosyl}\text{)-(1→3)-}\text{L}\text{-[6,}{}^3\text{H]}\text{idonic}$ acid. Only one form of the enzyme was seen on either ion exchange chromatography or isoelectric focussing, with a pI of 6.8. The apparent M_r of the haloenzyme as determined by gel filtration was 190 000 ± 20 000. Two other larger M_r protein peaks observed on gel filtration appear to be an inactive dimer of the 190 000 dalton peak and a larger aggregate near the exclusion limit of the column. On polyacrylamide disc gel electrophoresis in sodium dodecyl sulphate, with or without prior reduction, each protein peak from the gel filtration column electrophoreced as a single major band with an apparent M_r corresponding to 55 000 ± 6000.     

Regulation of Protein Synthesis in Plant Embryo by Protein Phosphorylation : I. Purification and Characterization of a cAMP-Independent Protein Kinase and Its Endogenous Substrate

A cyclic AMP-independent protein kinase, which strongly inhibits in vitro protein synthesis, was purified to homogeneity from barley embryo by affinity and ion exchange chromatography. The M_r of the purified enzyme is 95,000 with two nonidentical subunits of M_r 58,000 and 39,000. The enzyme activity is not stimulated by cAMP, cGMP, or calmodulin. The endogenous phosphate acceptor of this kinase is a protein of M_r 52,000, was isolated by purified protein kinase immobilized Sepharose column. Using antibodies raised against this protein kinase, the levels of the enzyme during embryogenesis and germination are determined. An inverse relationship has been observed between protein kinase level and rate of protein synthesis.     

Stress Responses in Alfalfa (Medicago sativa L.): II. Purification, Characterization, and Induction of Phenylalanine Ammonia-Lyase Isoforms from Elicitor-Treated Cell Suspension Cultures

l-Phenylalanine ammonia-lyase has been purified from elicitor-treated alfalfa (Medicago sativa L.) cell suspension cultures using two protocols based on different sequences of chromatofocusing and hydrophobic interaction chromatography. Three distinct forms of the intact enzyme were separated on the basis of affinity for Octyl-Sepharose, with isoelectric points in the range pH 5.1 to 5.4. The native enzyme was a tetramer of M_r 311,000; the intact subunit M_r was about 79,000, although polypeptides of M_r 71,000, 67,000 and 56,000, probably arising from degradation of the intact subunit, were observed in all preparations. Two-dimensional gel analysis revealed the presence of several subunit isoforms of differing isoelectric points. The purified isoforms of the native enzyme had different K_m values for l-phenylalanine in the range 40 to 110 micromolar, although mixtures of the forms in crude preparations exhibited apparent negative rate cooperativity. The enzyme activity was induced approximately 16-fold within 6 hours of exposure of alfalfa cells to a fungal elicitor or yeast extract. Analysis by hydrophobic interaction chromatography revealed different proportions of the different active enzyme isoforms, depending upon either time after elicitation or the elicitor used. The elicitor-induced increase in enzyme activity was associated with increased translatable phenylalanine ammonia-lyase mRNA activity in the polysomal fraction.     

Hydrogenase from the unicellular cyanobacterium,Microcystis aeruginosa

A hydrogenase was isolated from a unicellular and non-nitrogen-fixing cyanobacterium, Microcystis aeruginosa strain NIES 44. The enzyme was easily solubilized and was capable of evolving hydrogen gas in the presence of reduced methyl viologen and benzyl viologen. The enzyme was stimulated by divalent ions and showed a pH optimum around 6.8. The M_r of the enzyme, estimated by gel filtration, was 50 000.     

Subunit structure of γ-conglycinin in soybean seeds

Dissociated subunits of purified γ-conglycinin were isolated on a DEAE-Sephadex A-50 column. A single band was seen on two kinds of gel electrophoresis and isoleucine was shown as the only N-terminal amino acid. The isolated subunit reacted with antisera to the native γ-conglycinin. The M_r of the subunit was 51 000–51 500 estimated by urea-acetic acid and SDS-urea gel electrophoresis. A value of 50 000 was obtained by gel filtration with guanidine-hydrochloric acid on Sepharose CL-6B. The γ-conglycinin molecule was found to be made up of three subunits. This was determined by cross-linking the subunits and then submitting them to gel electrophoresis. Differences and similarities of subunit structure among γ-conglycinin, β-conglycinin and glycinin are discussed.     

Purification and subunit structure of phenylalanyl-tRNA synthetase from hen liver mitochondria

Hen liver mitochondrial phenylalanyl-tRNA synthetase is purified to homogeneity by a series of steps including salting-out chromatography, salting-out affinity chromatography in the presence of tRNA^Phe, dissociation of the enzyme-tRNA complex on DEAE-cellulose, chromatography on DEAE-Sepharose CL-6B and Sepharose 6B. The enzyme appears to be a tetrameric enzyme with a molecular weight of 255 000, as determined by gel filtration, with a subunit structure of α₂β₂ (α = 57 000, β = 66 000), as determined by sodium dodecyl sulfate gel electrophoresis.