Light modulation of maize leaf phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (PEPC) was extracted from maize (Zea mays L. cv Golden Cross Bantam T51) leaves harvested in the dark or light and was partially purified by (NH₄)₂SO₄ fractionation and gel filtration to yield preparations that were 80% homogeneous. Malate sensitivity, PEPC activity, and PEPC protein (measured immunochemically) were monitored during purification. As reported previously, PEPC from dark leaves was more sensitive to malate inhibition compared to enzyme extracted from light leaves. Extraction and purification in the presence of malate stabilized the characteristics of the two forms. During gel filtration on Sephacryl S-300, all of the PEPC activity and PEPC protein emerged in a single high molecular weight peak, indicating that no inactive dissociated forms (dimers, monomers) were present. However, there was a slight difference between the light and dark enzymes in elution volume during gel filtration. In addition, specific activity (units at pH 7/milligram PEPC protein) decreased through the peak for both enzyme samples; because the dark enzyme emerged at a slightly higher elution volume, it contained enzyme with a relatively lower specific activity. The variation in specific activity of the dark enzyme corresponded with changes in malate sensitivity. Immunoblotting of samples with different specific activity and malate sensitivity, obtained from gel filtration, revealed only a single polypeptide with a relative molecular mass of 100,000. When the enzyme was extracted and purified in the absence of malate, characteristic differences of the light and dark enzymes were lost, the enzymes eluted at the same volume during gel filtration, and specific activity was constant through the peak. We conclude that maize leaf PEPC exists in situ as a tetramer of a single polypeptide and that subtle conformation changes can affect both enzymic activity and sensitivity to malate inhibition.     

Kinetic Ramifications of the Association-Dissociation Behavior of NAD Malic Enzyme : A Possible Regulatory Mechanism

NAD malic enzyme can exist in dimer, tetramer, or octamer form. Freshly prepared enzyme from Solanum tuberosum var. Chieftan exists predominantly as the octamer and during storage is progressively converted into lower molecular weight forms. High ionic strength favors dimer formation, whereas high concentrations of malate or citrate favor tetramer formation. The tetramer is the most active form, having a low K_m for malate and a high V_max. The dimer, with its high K_m and low V_max, is the least active form. Malate may regulate NAD malic enzyme by controlling its state of oligomerization.     

Alkaline phosphatase from the excretory system of the grasshopper,Poekilocerus bufonius

Alkaline phosphatase from the excretory system of the grasshopper, Poekilocerus bufonius was purified with ammonium sulphate fractionation and chromatography on Bio-Gel A-0.5 m. The specific activity of the enzyme is 152 units/mg of protein. The enzyme is a tetramer and the M_r value of the subunit is 72,000 ± 2500 as shown by gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme has a pH optimum of 9.6 and an apparent K_m value of 0.28 × 10⁻³ M. The activity of the enzyme reached a maximum at 75°C and the enzyme showed stability at 65°C. The enzyme was inhibited by Ca²⁺, Na⁺ and Fe³⁺ and was stimulated by Zn²⁺, Mn²⁺ and Mg²⁺.     

Evidence for dimer/tetramer equilibrium in Trypanosoma brucei 6-phosphogluconate dehydrogenase

6-Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway (PPP), is essential for biosyntheses and oxidative stress defence. It also has the function of depleting 6PG, whose accumulation induces cell senescence. 6PGDH is a proposed drug target for African trypanosomiasis caused by Trypanosoma brucei and for other microbial infections and cancer. Gel filtration, density gradient sedimentation, cross-linking and dynamic light scattering were used to assay the oligomerization state of T. brucei 6PGDH in the absence and presence of several ligands. The enzyme displays a dimer–tetramer equilibrium and NADPH (but not NADP) reduces the rate of approach to equilibrium, while 6PG is able to antagonize the NADPH effect. The different behaviour of the two forms of coenzyme appears to be related to the differences in ΔC_p, with NADP binding ΔC_p closer to what is expected of crystallographic structures, while NADPH ΔC_p is three times larger. The estimated dimer–tetramer association constant is 1.5 · 10⁶ M^? 1, and the specific activity of the tetramer is about 3 fold higher than the specific activity of the dimer. Thus, cellular conditions promoting tetramer formation could allow an efficient clearing of 6PG. Experiments carried out on sheep liver 6PGDH indicate that tetramerization is a specificity of the parasite enzyme.     

Multiple forms of hydroxycinnamate : CoA ligase in etiolated pea seedlings

A survey of a range of plant tissues showed that the hydroxycinnamate CoA ligase in crude extracts of pea shoots had a high relative activity towards sinapic and other methoxycinnamic acids, together with high activity with p-coumaric acid. The pea enzyme has been resolved by chromatography on DEAE-cellulose into two peaks which differ in their substrate specificity. The form which elutes at relatively low salt concentrations has a ratio activity towards p-coumaric and sinapic acids of about 1.8:1 while the form eluting at higher salt concentrations, although showing very high activity with p-coumaric acid, is inactive towards sinapic acid. The pattern of elution of these forms following gel filtration on Ultragel AcA 34 and Sephadex G100 suggests that these two isoenzymes which differ in ionic properties and substrate specificity can exist in two or three molecular weight forms and there is evidence that these forms are under certain circumstances interconvertible.     

Polymers of Cu/Zn Superoxide Dismutase Produced by Cross-Linking with Glutaraldehyde

Soluble polymers of bovine Cu/Zn superoxide dismutase (EC 1.15.1.1) have been prepared using the homobifunctional cross-linking reagent, glutaraldehyde. A form of the enzyme, a tetramer. with a molecular weight of 64, 000 has been purified by gel filtration. The functional properties of the tctrarner have been investigated. Reconstitution with copper and zinc was required for full activity. After metal reconstitution, the specific activity of the tetramer was shown to be close to 90% that of the native dimerism enzyme.

The serum half-life of the tetramer in rats was found to be increased by a factor of six when compared with native superoxide dismutase. The tissue distribution of the two forms was also found to be direrent with the tetrarner accumulating predominantly in the liver.     

Purification and properties of betaine aldehyde dehydrogenase from Xanthomonas translucens

Betaine aldehyde dehydrogenase from Xanthomonas translucens was purified to apparent homogeneity by ammonium sulfate fractionation, followed by ion-exchange, butyl-Toyopearl and gel filtration chromatography. The amino acid composition and the N-terminal sequence of 35 amino acid residues were determined. The enzyme was found to be a tetramer with identical 50 kDa subunits. Both NAD and NADP could be used as a cofactor for the enzyme and K_m values for NAD and NADP were 70 μM and 50 μM, respectively. The enzyme was highly specific for betaine aldehyde and the K_m value for betaine aldehyde was 0.19 mM.     

Multiple forms of α-galactosidase in mature leaves of Cucurbita pepo

α-D-Galactosidase has been purified from mature leaves of Cucurbita pepo using pH and ammonium sulphate fractionation, Sephadex gel filtration and DEAE Sephadex gel chromatography. Gel filtration produced one peak of α-galactosidase activity from which three distinct enzyme forms were resolved on DEAE Sephadex and designated LI, LII and LIII. Purirications obtained were ca 75, 120 and 30 fold for LI, Lll and LIII respectively. Ll was slightly contaminated with β-galactosidase and LII with β-fructosidase activity. All forms hydrolysed the α-galactosyl linkages of raffinose and stachyose. Differences between each form were found in their pH optima, reactivity toward metal ions, thermal stability and K_m values using either p-nitrophenyl-α-D-galactoside (NPG) or raffinose as substrates. All forms were inhibited by NPG at high concentrations and by α-D-galactose. It is proposed that α-galactosidases may be components of a lysosomal system in plant cells.     

Liver protein phosphatases: studies of the presumptive native forms of phosphorylase phosphatase activity in liver extracts and their dissociation to a catalytic subunit of Mr 35,000.

Several aspects of the properties of phosphorylase phosphatase in crude rat liver extracts were investigated. Treatment of tissue extracts with either trypsin, ethanol, or urea was found to dissociate phosphorylase phosphatase activity to a form of M_r 35,000. The M_r 35,000 enzyme form was derived from three native enzyme forms. The major cytosolic form of phosphorylase phosphatase had a molecular weight of 260,000 as determined by gel filtration and was dissociated to a M_r 35,000 form by treatment with either ethanol or urea. Treatment of the M_r 260,000 form with trypsin led to its conversion to M_r 225,000 and a M_r 35,000 form. A minor cytosolic form of M_r 200,000 was also present. This minor activity was latent until activated by trypsin treatment and was converted to a M_r 35,000 form by such treatment. The third form was found to chromatograph as a form of molecular weight greater than 500,000 on gel filtration and, like the M_r 200,000 form, was only detected after activation with trypsin. Subsequent to this treatment, it too behaved as a M_r 35,000 enzyme. Although a single major enzyme form was present in the cytosol, multiple molecular weight forms could be generated in crude extracts simply by the use of vigorous mechanical homogenization procedures. This suggested that artifactual forms of enzyme may readily be produced, possibly by proteolytic cleavage of the native enzyme.     

Purification and characterization of acetate kinase from veillonella alcalescens ATCC 17748.

Acetate kinase was isolated in highly purified form from Veillonella alcalescens through a combination of ammonium sulfate fractionation, DEAE-Sephadex column chromatography, and gel filtration. It had a specific activity about 60-fold that of crude extracts. Purity of the enzyme preparation was estimated to be 90% as judged by polyacrylamide gel electrophoresis. The molecular weights of the enzyme as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by gel filtration were 43,000 and 66,000, respectively. Succinate was unnecessary for the activity of this enzyme. This result is markedly different from that reported previously (Bowman, C. M. et al., 1976, J. Biol. Chem. 251, 3117–3121). This may, however, be due to the difference in bacterial strains used. The enzyme reaction with propionate was equal to about two-thirds that of its reaction with acetate. Apparent K_m values for ATP, acetate, ADP, and acetylphosphate were about 2, 30,0.3, and 1.2 mm, respectively. Phosphate donors, ATP, and acetylphosphate exhibited cooperativity while phosphate acceptors, ADP, acetate, and propionate did not. The enzyme had a broad pH optimum from 7.2 to 10, and required magnesium ions, whose optimal molar ratio to ATP was 1:1. The activity was inhibited by several SH-inhibitors, but not stimulated by free SH groups.     

Purification and properties of two forms of hepatic phenylalanine:pyruvate transaminase from glucagon treated rats.

Two forms of phenylalanine:pyruvate transaminase (EC 2.6.1. aminotransferases, the exact EC number has not been assigned) termed A and B were obtained from the liver supernatant fraction of glucagon-treated rats by DEAE-Sephadex A-50 column chromatography. Each of the two forms was further purified by hydroxylapatite, Sephadex G-100 chromatography, and preparative gel electrophoresis. Both the A and B forms have been purified to homogeneity as judged by analytical and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Moreover, histidine was found to be a competitive inhibitor of phenylalanine with both purified proteins. These findings conclusively support the view that phenylalanine:pyruvate transaminase and histidine:pyruvate transaminase reactions are catalyzed by the same protein. The overall purification was 710-fold for the A form and 1200-fold for the B form. The apparent molecular weight for both A and B are 74,000 ±6000 as determined by gel filtration. Sodium dodecyl sulfate gel electrophoresis revealed that the A form has two identical subunits of molecular weight 42,000, whereas the B form has two nonidentical subunits of molecular weight 42,000 and 44,000. The amino acid composition for the A and B forms of the enzyme are different. The major differences are in glycine, alanine and leucine. The isoelectric point for A was 7.8 and for B was 7.3. However, the A and B forms of the enzyme are of immunological identity. The substrate specificity determined for both the A and B form was phenylalanine >asparagine >alanine >leucine >histidine. The K_m for phenylalanine was 7.70 mm for the A form, 6.00 mm for the B form. For histidine, the K_m was 13.70 mm for the A form, 12.50 mm for the B form.     

Structure and function of amylases. II. Multiple forms of bacillus subtilis -amylase

The subunit structure of Bacillus subtilis α-amylase has been studied by gel filtration and by SDS-gel electrophoresis. The crystalline enzyme was found to be a 96,000 dalton zinc tetramer. Incubation of the 96,000 species at pH 5.5 or with EDTA produced a 48,000 zinc-free dimer; incubation with 100 mm sodium chloride produced a 72,000 zinc trimer; incubation at pH 8.5 produced a 48,000 zinc dimer and a 24,000 zinc-free monomer. Incubation of the 48,000 zinc dimer with EDTA produced a 24,000 monomer. After standing, the 48,000 zinc dimer formed insoluble aggregates that could be dissolved by treatment with EDTA. The aggregates had molecular weights between 125,000 and 400,000. The 72,000 zinc trimer also aggregated to form a single 144,000 species. All of the forms were enzymatically active, although with widely differing specific activities. Schematic diagrams for the structures of the multiple forms and their interconversions are presented.     

Regulation of the NAD Malic Enzyme from Crassula

Using size exclusion chromatography, the nicotinamide adenine dinucleotide malic enzyme purified to near homogeneity from leaves of Crassula argentea was found to exist in at least three aggregational states (dimer, tetramer, and octamer). These forms differ in their apparent kinetic characteristics in initial rate assays, but all display similar characteristics at the steady state. The presence of 50 millimolar malate during chromatography causes a shift in favor of the smaller forms with the tetramer predominating. The native enzyme, when diluted 1/1000 and incubated 18 hours in buffer of high ionic strength, changes its steady state kinetic parameters to ones which indicate a low activity and low affinity for malate. When 50 millimolar malate or 50 micromolar coenzyme A are present the loss of activity and increase in K_m is reduced. When both malate and coenzyme A are present the effects in minimizing the change in kinetic characteristics are additive.     

Ferredoxin-dependent Nitrite Reductase from Spinach Leaves

A ferredoxin-dependent nitrite reductase from Spinacea oleracea was purified approximately 180-fold, with a specific activity of 285 units/mg protein. This purified enzyme also had methyl viologen-dependent nitrite reductase activity, with a specific activity of 164 units/mg protein. After disc electrophoresis with polyacrylamide gel, the purified enzyme showed one major and one minor protein band.

The molecular weight of the enzyme was estimated to be 86,000 from Ultrogel filtration. This purified enzyme in oxidized form had absorption peaks at 278, 390, 573 and 690 nm. The absorbance ratios, A₃₉₀: A₂₇₈ and A₆₇₃: A₃₉₀ were 0.61 and 0.37, respectively.

By applying the purified enzyme to DEAE-Sephadex A–50 column chromatography, the ferredoxin-dependent nitrite reductase activity was selectively decreased. However, the methyl viologen-dependent nitrite reductase activity was increased, with a specific activity of 391 units/mg protein. This modified enzyme was homogeneous by disc electrophoresis with polyacrylamide gel.     

Occurrence and Regulatory Properties of Uridine Diphosphatase in Fully Expanded Leaves of Soybean (Glycine max Merr.) and Other Species

High activities (100-200 micromoles UDP hydrolyzed per milligram chlorophyll per hour) of uridine-5′ diphosphatase (UDPase) have been identified in extracts of fully expanded soybean (Glycine max Merr.) leaves. In desalted crude extracts, UDPase activity was strongly inhibited by low concentrations of Mg:ATP (I₅₀ = 0.3 millimolar). Two forms of the enzyme were resolved by gel filtration on Sephadex G-150. The higher molecular weight form (UDPase I, about 199 kilodaltons by gel filtration) retained ATP sensitivity (I₅₀ = 0.3 millimolar), whereas the major, lower molecular weight form (UDPase II, about 58 kilodaltons) was markedly less sensitive to ATP inhibition (I₅₀ = 2.7-3.0 millimolar). Subsequent purification of UDPase I by ion-exchange chromatography on DEAE cellulose produced a lower molecular weight enzyme (about 74 kilodaltons by gel filtration) that had reduced ATP sensitivity similar to UDPase II. Ion-exchange chromatography of UDPase II did not alter molecular weight or ATP sensitivity. UDPase II, after the DEAE-cellulose step, was specific for nucleoside diphosphates. Maximum reaction velocity decreased in the following sequence; UDP > GDP > CDP. ADP was not a substrate for the enzyme. The reaction catalyzed was hydrolysis of the terminal-P of UDP to form UMP. The enzyme was stimulated by Mg²⁺ and the pH optimum was centered between pH 6.5 and 7.0. In a survey of various species, soybean cultivars had highest activities of apparent UDPase and other species ranged in apparent activity from 0 to 30 micromoles hydrolyzed per milligram chlorophyll per hour.     

Inhibition of the O2−-generating NADPH oxidase of guinea-pig polymorphonuclear leukocytes by rabbit antibody to homologous liver NADPH-cytochrome c (P-450) reductase

Rabbit antibody highly specific for guinea-pig liver NADPH-cytochrome c (P-450) reductase was found to inhibit dose-dependently the O₂^?-generating activity of the membrane fraction isolated from phorbol-myristate acetate-stimulated, homologous polymorphonuclear leukocytes. In addition, the antibody also could inhibit the NADPH-cytochrome c (Nitroblue tetrazolium) reductase from the membrane fractions and phagosomes of leukocytes by polyacrylamide gel electrophoresis or gel filtration on a Sephacryl S-300 column in the presence of 0.2% Triton X-100. These results demonstrate that the NADPH-cytochrome c reductase in the membrane fractions of leukocytes is antigenically cross-reactive with homologous liver NADPH-cytochrome c reductase, and also suggest that the enzyme of leukocytes participates in the respiratory burst.     

Purification of cassava linamarase

Linamarase was purified from parenchymal tissue of cassava by extraction with acetate buffer, fractional precipitation with ammonium sulphate, followed by column chromatography on DEAE-cellulose and Sepharose-6-B gel filtration. The specific activity is increased 350 fold with 35% recovery. The K_ms for linamarin and p-nitro-phenyl β-D-glucoside are 1.45 × 10^?3 M and 0.46 × 10^?3 M, respectively. The pH optimum in 50 mM NaPi is pH 6 and the specific activity is 26.5 nkat/mg. The enzyme can be prepared from cassava peel using the same procedure and has similar properties.     

The molecular heterogeneity of purified human liver lysosomal alpha-glucosidase (acid alpha-glucosidase).

An α-glucosidase active at acid pH and presumably lysosomal in origin has been purified from human liver removed at autopsy. The enzyme has both α-1,4-glucosidase and α-1,6-glucosidase activities. The K_m of maltose for the enzyme is 8.9 mm at the optimal pH of 4.0. The K_m of glycogen at the optimal pH of 4.5 is 2.5% (9.62 mm outerchain end groups). Isomaltose has a K_m of 33 mm when α-1,6-glucosidase activity is tested at pH 4.2. The enzyme exists in several active charge isomer forms which have pI values between 4.4 and 4.7. These forms do not differ in their specific activities. Electrophoresis in polyacrylamide gels under denaturing conditions indicates that the protein is composed of two subunits whose approximate molecular weights are 88,000 and 76,000. An estimated molecular weight of 110,000 was obtained by nondenaturing polyacrylamide gel electrophoresis. When the protein was chromatographed on Bio-Gel P-200 it was separated into two partially resolved active peaks which did not differ in their charge isomer constitution or in subunit molecular weights. One peak gave a strongly positive reaction for carbohydrate by the periodic acid-Schiff method and the other did not. Both had the same specific activity. The enzyme was antigenic in rabbits, and the antibodies so obtained could totally inhibit the hydrolytic action of the enzyme on glycogen but were markedly less effective in inhibiting activity toward isomaltose and especially toward maltose. Using these antibodies it was found that liver and skeletal muscle samples from patients with the “infantile” form or with the “adult” form of Type II glycogen storage disease, all of whom lack the lysosomal α-glucosidase, do not have altered, enzymatically inactive proteins which are immunologically cross-reactive with antibodies for the α-glucosidase of normal human liver.     

Purification and properties of neutral β-galactosidases from human liver

Two neutral β-galactosidase isozymes were purified from human liver. The initial step of purification was removal of the acidic β-galactosidases by adsorption on concanavalin A-Sepharose 4B conjugate. Subsequent purification steps included ammonium sulfate precipitation, diethylaminoethyl cellulose column chromatography, Sephadex G-100 gel filtration, and preparative polyacrylamide-gel isoelectric focusing. The final step of purification was affinity chromatography of the separated isoelectric forms on ?-aminocaproyl-β-d-galactosylamine-Sepharose 4B conjugate. The purified β-galactosidase isozymes had activity toward both β-d-galactoside and β-d-glucoside derivatives of 4-methylumbelliferone and p-nitrophenol with a pH optimum around 6.2. These enzyme forms were also found to possess lactosylceramidase II activity with a pH optimum in the range of 5.4 to 5.6, but not lactosylceramidase I activity and no activity toward galactosylceramide or GM₁-ganglioside. The molecular weight was found to be in the range of 37,500–39,500 for the two neutral isozymes and they had similar K_m and V values; the more acidic form (designated β-galactosidase N₁) was more heat stable than the other form (designated β-galactosidase N₂). Antibodies evoked against the N₁ and N₂ β-galactosidases gave identical precipitin lines retaining enzymatic activity. No cross-reactivity was observed between the neutral and the acidic isozymes when examined with the respective antisera.     

The lectin nature of α-galactosidases from Vicia faba seeds

α-Galactosidase from Vicia faba seeds has been resolved into three molecular forms, I, II¹ and II², respectively. Enzyme I is a tetramer (M_r 160 000) consisting of identical sub-units (M_r 44000 ± 2000). All three forms display lectin activity with glucose/mannose specificity. Enzyme I has been further studied with respect to its lectin specificity and various factors affecting this property. The results indicate that the catalytic and the lectin sites reside in the same protein molecule. The results presented are unique in that the enzyme activity is specific for galactose and its lectin activity is specific for glucose/mannose.