Combined action of Escherichia coli glycogen synthase and branching enzyme in the so-called "unprimed" polyglucoside synthesis.

Mutants of Escherichia coli which are unable to synthesize glycogen were used to study the so-called “unprimed” synthesis of glycogen. The glycogen synthase has been partially purified from these mutants. During the purification, attempts were made to separate the activity which requires the addition of an exogenous primer (primed activity) from the activity which does not require a primer but is highly dependent on the presence of some salts such as citrate and EDTA (unprimed activity). No separation between these two activities could be achieved but the results obtained by chromatography on DEAE-Sephadex indicate that there is a single form of glycogen synthase which is responsible for both unprimed and primed activity. The evidence that a single protein was necessary to catalyze these two reactions was given by the findings that mutants defective in glycogen synthase activity were unable to catalyze glucosyl transfer without added primer. At low concentration, the glycogen synthase purified from a branching enzyme negative mutant catalyzed the unprimed reaction at a slow rate even in presence of salts. A protein activator of this reaction was found in mutants lacking glycogen synthase but not in mutants lacking branching enzyme. The hypothesis that this activator is the branching enzyme itself was supported by the observation that it co-purified with the branching enzyme from a E. coli strain defective in glycogen synthase activity. EDTA or Triton X-100 increased the stimulation of the unprimed synthesis by the branching enzyme. The apparent affinity of the glycogen synthase for glycogen was increased twofold in the presence of EDTA but the branching enzyme further increased the effect of EDTA. The combined action of the glycogen synthase and the branching enzyme on the endogenous glucan associated with the synthase may account for the unprimed activity observed in vitro.     

Differences in the Average Single Molecule Activities of E. coli β-Galactosidase: Effect of Source, Enzyme Molecule Age and Temperature of Induction

Using a capillary electrophoresis–based method, single enzyme molecule assays were performed on E. coli β-galactosidase from three different sets of samples. The first set consisted of lysates of induced cells from five different strains of the bacteria, as well as two different commercial preparations of the enzyme. These samples were found to have substantially different distributions of single molecule activities. For the second set of samples, β-galactosidase expression was induced for 1.5 hr, followed by further incubation where expression was repressed. Assays were performed on the lysates of the preinduction and on the lysates from aliquots taken set times postinduction. The recently induced enzyme had a 25% higher average single molecule activity than the basally expressed enzyme. This average activity returned to the basal value 3.5 hr postinduction and remained unchanged thereafter. Finally, β-galactosidase was induced at 26 and 42°C. The enzyme was assayed before and after partial thermal denaturation. The samples were found to be indistinguishable with respect to their average single molecule activities.     

Indoleacetic Acid Oxidase: A Dual Catalytic Enzyme?

The isolation of a unique enzyme capable of oxidizing indoleacetic acid, but devoid of peroxidase activity, has been reported for preparations from tobacco roots and commercial horseradish peroxidase. Experiments were made to verify these results using enzyme obtained from Betula leaves and commercial horseradish peroxidase. Both indoleacetic acid oxidase and guaiacol peroxidase activity appeared at 2.5 elution volumes from sulfoethyl-Sephadex. These results were obtained with both sources of enzyme. In no case was a separate peak of indoleacetic acid oxidase activity obtained at 5.4 elution volumes as reported for the tobacco enzyme using the same chromatographic system. Both types of activity, from both sources of enzyme, also eluted together during gel filtration. Successful column chromatography of Betula enzyme was dependent upon previous purification by membrane ultrafiltration. These results indicate indoleacetic acid oxidase activity and guaiacol peroxidase activity are dual catalytic functions of a single enzyme.     

Purification and characterization of methylglyoxal reductase from Hansenula mrakii

Methylglyoxal reductase was purified from Hansenula mrakii IFO 0895 to a homogenous state on polyacrylamide gel electrophoresis. The enzyme consisted of a single polypeptide chain with a molecular weight of 34,000. The enzyme was specific to methylglyoxal (K_m = 1.92 mM) and NADPH (K_m = 40.8 μM). The activity of the enzyme was inhibited by p-chloromercuribenzoate and HgCl₂. NADP also inhibited the activity of the enzyme, and the K_i value was calculated to be 0.25 mM.     

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.     

Detergent compatible alkaline lipase produced by marine Bacillus smithii BTMS 11

Bacillus smithii BTMS 11, isolated from marine sediment, produced alkaline and thermostable lipase. The enzyme was purified to homogeneity by ammonium sulfate precipitation and ion exchange chromatography which resulted in 0.51 % final yield and a 4.33 fold of purification. The purified enzyme was found to have a specific activity of 360 IU/mg protein. SDS-PAGE analyses, under non-reducing and reducing conditions, yielded a single band of 45 kDa indicating the single polypeptide nature of the enzyme and zymogram analysis using methylumbelliferyl butyrate as substrate confirmed the lipolytic activity of the protein band. The enzyme was found to have 50 °C and pH 8.0 as optimum conditions for maximal activity. However, the enzyme was active over wide range of temperatures (30–80 °C) and pH (7.0–10.0). Effect of a number of metal salts, solvents, surfactants, and other typical enzyme inhibitors on lipase activity was studied to determine the novel characteristics of the enzyme. More than 90 % of the enzyme activity was observed even after 3 h of incubation in the presence of commercial detergents Surf, Sunlight, Ariel, Henko, Tide and Ujala indicating the detergent compatibility of B. smithii lipase. The enzyme was also found to be efficient in stain removal from cotton cloths. Further it was observed that the enzyme could catalyse ester synthesis between fatty acids of varying carbon chain lengths and methanol with high preference for medium to long chain fatty acids showing 70 % of esterification. Results of the study indicated scope for application of this marine bacterial lipase in various industries.     

Induction of acid phosphatase in cotton seedlings: Enzyme purification,subunit structure and kinetic properties

About a 16-fold rise in acid phosphatase (EC 3.1.3.2) activity was observed during the early stages of germination of cotton embryos. Administration of cyclobeximide to the germinating embryos significantly blocked the enhancement of acid phosphatase activity. This indicated that translational activity was essential for the induction of enzyme activity. Conclusive proof for the de novo synthesis of the enzyme was obtained by showing the incorporation of ³⁵S from ³⁵SO²⁻₄ into the cysteine residues of the purified acid phosphatase. The enzyme was purified (1046-fold) to electrophoretic homogeneity by ammonium sulphate fractionation, CM-Sephadex C-50 and affinity chromatography on concanavalin A-Agarose. PAGE gave two isozyme bands. The M, of the phosphatase was 200 k as determined by molecular sieving on Sephadex G-200. SDS-PAGE of acid phosphatase revealed a single band of M 55 k. Thus the native enzyme is a tetramer of four identical subunits. The K_m of the enzyme with p-nitrophenyl phosphate was 0.5 mM. Optimal enzyme activity was observed at pH 5.0, using p-nitrophenyl phosphate as substrate. The enzyme activity remained linear for 105 min at 37° and was proportional to the concentration of protein within the range 0.6–2.4 μg.     

Haemonchus contortus: enzymes. 3. Glutamate dehydrogenase

Adult male and female Haemonchus contortus were homogenized and subjected to differential centrifugation. The crude, high-speed, supernatant fraction contained more than 95% of the glutamate dehydrogenase activity. The enzyme was purified through use of DEAE-cellulose columns and sucrose density gradient centrifugation. The enzyme from both crude and purified preparations was detected as a single band of activity following starch or polyacrylamide-gel electrophoresis. The Haemonchus enzyme was compared with ovine and bovine liver glutamate dehydrogenases. The three enzymes were similar in molecular size, Michaelis constants, and pH optimums but differed in electrophoretic mobility in polyacrylamide-gels, activity with NADP as coenzyme, and effect of AMP and ADP on activity. Sheep anti-Haemonchus glutamate dehydrogenase serum inhibited Haemonchus glutamate dehydrogenase, but did not inhibit the ovine or bovine enzymes.     

Purification and some properties of polyphenol oxidase from the yam tubers, Dioscorea bulbifera

In a comparison of the polyphenol oxidase activity of various species of yam tubers the greatest enzyme activity was found in D. bulbifera. The enzyme was purified from acetone powder extracts of this plant. Ammonium sulphate fractionation, followed by ion exchange chromatography and gel filtration gave 22-fold purification. The final product gave a single band on polyacrylamide disc gel electrophoresis. The purified enzyme showed activity towards catechol, pyrogallol and dl-β-3,4-dihydroxyphenylalanine (dl-DOPA) and had a MW 115000 ± 2000. It was characterized by response to various inhibitors. β-Mercaptoethanol, dithioerythritol, l-cysteine, sodium metabisulphite and KCN inhibited strongly.     

Purification and properties of spinach leaf debranching enzyme

Starch debranching enzyme was purified from intact spinach (Spinacia oleracea L. cv Vital) chloroplasts and from a spinach leaf extract using affinity chromatography on Sepharose 6B-bound cycloheptaamylose (Schardinger β-dextrin). The enzyme from both sources was homogeneous upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Spinach leaf debranching enzyme appears to consist of a single polypeptide chain, since the molecular weight of the native protein (110,000 daltons) was not changed by treatment with sodium dodecyl sulfate. Only one spinach leaf debranching enzyme band could be detected after electrophoresis of a leaf extract on amylopectin-containing polyacrylamide gel, the retardation factor of which coincided with that of the single band seen with the chloroplast enzyme. The purified enzyme exhibited strong pullulanase activity, the specific activity being 69 units per milligram protein with pullulan and 22 units per milligram protein with amylopectin. Cycloheptaamylose is a potent competitive inhibitor of spinach leaf debranching enzyme. The pH optimum of the enzyme was found to be 5.5. The purified enzyme is rather unstable at both 20° and 0°C. Part of the activity lost under storage or at a suboptimal pH could immediately be restored by the addition of thiols. The reactivatable protein, being of the same molecular weight as the native enzyme, exhibited a somewhat altered electrophoretic mobility resulting in one or two minor bands on a zymogram.     

Carboxypeptidase activity in the insulin secretory granule

Carboxypeptidase activity was studied in subcellular fractions from a transplantable rat insulinoma and found to be localised principally in the insulin secretory granule. The activity, which was specific for peptide substrates with C-terminal basic amino acids, appeared to be a single enzyme with M_r 54 000. This enzyme differed with respect to size and pH optimum from other basic amino acid-specific carboxypeptidases, such as carboxypeptidases B and N, and may be a secretory granule-specific enzyme involved in propolypeptide processing.     

Structure and function characterization of the phytoene desaturase related to the lutein biosynthesis in Chlorella protothecoides CS-41

Phytoene desaturase is the key enzyme involved in the biosynthesis pathway of lutein. The unicellular microalga, Chlorella protothecoides CS-41, had been selected for the heterotrophic production of high concentrations of lutein. In this study, a cDNA copy of the pds gene from C. protothecoides was obtained using the rapid amplification of cDNA ends (RACE) technique. Phylogenetic analysis of the deduced amino acid sequence revealed that the phytoene desaturases derived from the algal family. Expression of the pds gene in Escherichia coli produced a single protein of 61 kDa. The PDS activity of the expressed protein was confirmed by the production of ζ-carotene as the result from the action of the enzyme’s desaturation activity, which was identified by high-performance liquid chromatography and heterologous complementation analysis. Using random and site-directed mutagenesis, a single amino acid mutation (N144D) was identified and confirmed. This mutant encodes an inactive enzyme, which implies that amino acid 144 is crutial to the activity of the PDS enzyme. Therefore, by gene cloning and expression in prokaryotic cells, the gene for ζ-carotene production or as part of the biosynthetic pathway of lutein had been characterized from Chlorella protothecoides CS-41.     

A Lipoxygenase from Leaves of Tomato (Lycopersicon esculentum Mill.) Is Induced in Response to Plant Pathogenic Pseudomonads

Lipoxygenase (LOX) mRNA, enzyme protein, and enzyme activity were found to be induced in leaves of tomato (Lycopersicon esculentum Mill. cv Moneymaker) on inoculation with plant pathogenic bacteria. The rate of enzyme activity with linoleic or linolenic acid as substrate was approximately 10 times greater than that with arachidonic acid. Optimum activity was at pH 7.0. In the incompatible interaction, which was associated with a hypersensitive reaction (HR), a single band with relative molecular weight approximately 100,000 was revealed by probing western blots of enzyme extracts with antiserum raised against a pea lipoxygenase. Changes in the intensity of this band reflected the changes observed in LOX enzyme activity after bacterial inoculations. In the hypersensitive reaction, i.e. after inoculation with Pseudomonas syringae pv syringae, LOX mRNA was induced by 3 hours and enzyme activity began to increase between 6 and 12 hours and had reached maximum levels by 24 to 48 hours. In tomato leaves inoculated with P. syringae pv tomato (compatible interaction), LOX mRNA was induced later and enzyme activity changed only marginally in the first 24 hours, then increased steadily up to 72 hours, reaching the levels seen in the HR.     

Marked increase in ascorbate oxidase protein in pumpkin callus by adding copper

Ascorbate oxidase from pumpkin (Cucurbita sp.) was purified from a commercially available preparation. A single polypeptide band with M_r 64,000 was detected after sodium dodecylsulfate-polyacrylamide gel electrophoresis of the purified enzyme. In double immunodiffusion tests, antiserum against the purified preparation formed a single precipitin line with the crude extract from pumpkin fruit tissue or the callus as well as with the purified preparation. Immunological blotting method showed that mol wt of ascorbate oxidase subunit in pumpkin callus was the same as that of the purified preparation. Analysis with the single radial immunodiffusion method showed that the increase in ascorbate oxidase activity during the growth of pumpkin callus correlated with an increase in the enzyme protein. Furthermore, enzyme protein in the callus grown in the presence of 10 micromolar CuSO₄ for 2 weeks was about eight times that grown in the presence of 0.1 micromolar CuSO₄. The synthesis of ascorbate oxidase in pumpkin callus may be induced by copper, a prosthetic metal of the enzyme, or copper may help stabilize the enzyme against proteolytic breakdown.     

An unusual allantoinase from Dolichos biflorus

An unusual allantoinase from Dolichos biflorus has been purified 62-fold. The purified enzyme has an unusual pH activity profile with a shoulder at pH 4 and a peak at pH 7.5. This is due to a single enzyme which does not need metal ions for activation. In the fully reduced state the enzyme exhibits a single sharp peak at 7.5; when it is not in the sulfhydryl form (in the fully oxidized SS form?) the enzyme shows a single pH optimum at pH 4. K_m values for (±)-allantoin were 5.5 mM at pH 4 and 1.43 mM at pH 7.5. Allantoinase activity has been demonstrated in the resting seed, and increased linearly with time during the first 5 days of seedling growth.     

Immunological comparison of the starch branching enzymes from potato tubers and maize kernels

Starch branching enzyme was purified from potato (Solanum tuberosum L.) tubers as a single species of 79 kilodaltons and specific antibodies were prepared against both the native enzyme and against the gel-purified, denatured enzyme. The activity of potato branching enzyme could only be neutralized by antinative potato branching enzyme, whereas both types of antibodies reacted with denatured potato branching enzyme. Starch branching enzymes were also isolated from maize (Zea mays L.) kernels. All of the denatured forms of the maize enzyme reacted with antidenatured potato branching enzyme, whereas recognition by antinative potato branching enzyme was limited to maize branching enzymes I and IIb. Antibodies directed against the denatured potato enzyme were unable to neutralize the activity of any of the maize branching enzymes. Antinative potato branching enzyme fully inhibited the activity of maize branching enzyme I; the neutralized maize enzyme was identified as a 82 kilodalton protein. It is concluded that potato branching enzyme (M_r = 79,000) shares a high degree of similarity with maize branching enzyme I (M_r = 82,000), in the native as well as the denatured form. Cross-reactivity between potato branching enzyme and the other forms of maize branching enzyme was observed only after denaturation, which suggests mutual sequence similarities between these species.     

A novel glucosyltransferase from Catharanthus roseus cell suspensions

The potential applications of glycosyltransferases in glycoconjugate synthesis have attracted considerable interest from the biotechnology community in recent years. In this work, we present a novel glucosyltransferase from Catharanthus roseus cell cultures. The enzyme was purified to one spot in SDS-polyacrylamide gel electrophoresis, and its molecular weight was about 51 kDa. The optimum temperature was 35 °C, and the optimum pH was 7.6. Sodium ion has weak effect on enzyme activity, whereas divalent ions inhibit enzyme activity strongly. The K_m values were 0.112, 0.077, 0.064 and 1.0 mM for scopoletin, 5,7-dihydroxyflavone, 5,7-dihydroxyflavanone and UDPG, respectively. Substrate screening with the purified enzyme was performed against a range of phenolic compounds using UDPG as sugar donor. The enzyme showed activity towards a number of coumarins including umbelliferone, scopoletin, isoscopoletin and esculetin, and flavonoids including a flavone, a flavanone and chalcones. No activity was detected with compounds characterized by a single aromatic ring, i.e. simple and acidic phenols. The substrate specificity and the regioselectivity suggest enzyme structural features that are different from those of other glucosyltransferases.     

D-Ribulose-1,5-bisphosphate carboxylase/oxygenase from chemolithotrophically grown Rhizobium japonicum

Ribulose-1,5-bisphosphate carboxylase/oxygenase has been purified from chemolithotrophically grown Rhizobium japonicum SR and ribulose-5-phosphate kinase activity has also been detected in extracts of such cells. Electrophoretically homogeneous ribulosebisphosphate carboxylase/oxygenase purified in the presence of PMSF showed two types of large subunits of 55 000 and 53 000 daltons and small subunits of 14 200 daltons. The heterogeneity of large subunits was not observed when the enzyme was prepared in the presence of PMSF and DIFP. Ribulose-1,5-bisphosphate carboxylase from R. japonicum was inhibited by antibodies to this enzyme and a single precipitin band from the antibody-enzyme interaction was observed on double diffusion plates. Antibodies to R. japonicum enzyme did not cross-react on immunodiffusion plates with the ribulosebisphosphate carboxylase/oxygenases from wheat, spinach, soybean and tobacco.     

Regulation and function of glutamate synthase in Neurospora crassa

In Neurospora crassa two enzymes can provide glutamate: the NADPH dependent GDH and the NADH dependent GOGAT. An elevated GOGAT activity was found in Neurospora wild-type under ammonium limitation in contrast to a 4-fold lower activity on excess of a$\text{m}$ monium. Glutamate and glutamine repress this enzyme. On excess of ammonium the GDH-NADPH deficient mutant am-1 grows poorly with an elevated GOGAT activity. A GOGAT less mutant was found. It presented a lag-phase to grow on ammonium. It is concluded that N. crassa glutamate synthase provides glutamate from low am-monium concentrations. The enzyme was purified to homogeneity and shown to be composed of a single type of monomer with a molecular weight above 200,000.     

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.