A primer independent activity of rabbit muscle phosphorylase b.

Rabbit muscle phosphorylase b was found to be capable of forming protein bound alpha-1,4 glucosyl chains upon incubation of the enzyme with appropriate concentrations of glucose-1-phosphate with no primer addition (unprimed synthesis). This activity would only be present in a small fraction of the total muscle phosphorylase b activity, as judged from the high concentrations of enzyme which are required to demonstrate the occurrence of unprimed synthesis. Polyacrylamide gel electrophoresis shows the presence of a phosphorylase isoenzyme capable of accepting glucosyl moieties, giving rise to a glucosylated protein enzymatically active in the chain lengthening of its own glucan.     

Escherichia coli K-12 glycogen synthase: ability to use UDPglucose and ADP glucose as glucosyl donors in the absence of added primer.

A mechanism of initiation of glycogen biosynthesis in Escherichia coli has been previously postulated: In a first step, the glucosyl groups would be transferred into an acceptor protein from UDPglucose or ADPglucose by two glucosyl transferases, distinct from the glycogen synthase. In this work, the activity of transfer from UDPglucose into a methanol-insoluble fraction could not be found in the crude extracts of six independently isolated glycogen synthase-deficient mutants of E. coli K-12. Purified E. coli K-12 glycogen synthase was able to catalyze the unprimed reaction from ADPglucose and UDPglucose but at a very low rate; the rate with UDPglucose is 6–7% the rate observed with ADPglucose. With these two substrates, the unprimed reaction was strongly stimulated by the simultaneous presence of salts and branching enzyme. However the activity with UDPglucose increased rapidly at low concentrations of branching enzyme and was inhibited at physiological concentrations whereas the activity with ADPglucose reached a maximum only at these concentrations. Consequently, the relative activities found with ADPglucose and UDPglucose varied with the branching enzyme concentration. Transfer from UDPglucose was inhibited by low concentrations of ADPglucose and high concentrations of glycogen. These results suggest that the same enzyme, namely the glycogen synthase, catalyzes the unprimed transfer from ADPglucose and UDPglucose and that ADPglucose is probably the most important physiological donor in glycogen biosynthesis in E. coli.     

Inhibition of rabbit skeletal muscle phosphorylase phosphatase by spermine

Summary The effect of three naturally occurring polyamines (putrescine, spermidine, and spermine) on the activity of rabbit skeletal muscle phosphorylase phosphatase was investigated. Only spermine significantly inhibited the enzyme. The mode of inhibition (K_i value of 0.3mm) of the phosphatase by spermine appears to be different from that caused by divalent metal ions or by other organic cations, such as arginine and lysine esters, since it is noncompetitive with respect to the substrate, phosphorylasea.     

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.     

Differential scanning calorimetry study of glycogen phosphorylaseb-detergent interactions

The overall thermal denaturation of glycogen phosphorylaseb is irreversible and our results conform to the theoretical prediction of a reversible process followed by a slower irreversible process. The basic thermodynamic parameters of glycogen phosphorylaseb denaturation have been worked out and found to be: critical temperature 57.0±0.5°C, transition half-width 8±1°C, and calorimetric enthalpy change and Van't Hoff enthalpy change of the denaturation process 450±50 and 105±15 kcal/mol of enzyme monomer, respectively, at pH 7.4. These parameters have been found to be largely altered by the detergents octylglucoside, cholate, and deoxycholate at or below their critical micelle concentration, but not by Triton X-100 nor by lecithin liposomes. Organic solvents, such as dimethyl sulfoxide and methanol, and the presence of sarcoplasmic reticulum membranes produces an alteration of the denaturation thermogram of glycogen phosphorylaseb similar to that produced by the above-mentioned detergents. These results allow us to hypothesize that hydrophobic domains of glycogen phosphorylaseb are involved in its association to sarcoplasmic reticulum membranes in the sarcoplasmic reticulum/glycogenolytic complex of mammalian skeletal muscle.     

Characterization of Escherichia coli B glycogen synthase enzymatic reactions and products.

Previous reports implicate UDPglucose as an active glucosyl donor for the unprimed reaction and “glucoprotein” formation in glycogen biosynthesis in Escherichia coli. Results presented here indicate that UDPglucose and GDPglucose are glucosyl donors in the primed and unprimed reactions catalyzed by purified E. coli B glycogen synthase at less than 5% the rate observed when ADPglucose is the donor. The unprimed reaction is stimulated by 0.25 m citrate and a high molecular weight product is formed similar to that produced when ADPglucose is the glucosyl donor. Physiological amounts of branching enzyme and high concentrations of glycogen inhibit transfer from UDPglucose and GDPglucose. In addition, transfer from UDPglucose is inhibited by ADPglucose. These results strongly suggest that ADPglucose is the physiological donor in both the primed and unprimed reactions. Furthermore, these and previously reported results suggest that one enzyme is involved in the catalysis of the primed, unprimed, and TCA-insoluble product formation reactions. Antiserum prepared against purified E. coli B glycogen synthase inactivates transfer of glucose from either ADPglucose or UDPglucose in the above reactions catalyzed by E. coli B crude extracts. Purified E. coli B glycogen synthase preparations contain significant amounts of α-glucan primer. Evidence shows that this glucan is not covalently attached to the enzyme. Results presented show that formation of material insoluble in TCA and previously considered to be due to “glucoprotein” formation, is in fact due to the generation of long chain length glucan molecules intrinsically acid insoluble. The data suggest that previous results purported to be de novo synthesis of glycogen are due to glucan associated with the glycogen synthase and not to formation of a “glucoprotein” intermediate which then acts as primer for further oligosaccharide synthesis.     

Glucose-6-phosphate dehydrogenase in cell free extracts of Zymomonas mobilis

Summary Glucose-6-phosphate dehydrogenase activity in cell free extracts o Zymomonas mobilis showed marked differences when compared with the corresponding enzyme of Escherichia coli. It exhibited 3 times higher activity and the reaction rate over 10 min gave linearity only up to a cell free protein concentration of 0.15 mg protein. This different behaviour was not a function of environmental growth conditions of the culture nor of the nine different assay methods employed. A constant relationship existed between the specific G-6-P dehydrogenase protein and the total protein concentration in the cell free extract. The enzyme was stable for at least 5 h at 4°C in Tris-NaCl-MgCl₂-buffer.An investigation of the properties of G-6-P dehydrogenase from Z. mobilis revealed a pH optimum of 8.7 with a rapid decline towards the acidic and a small decrease towards the alkaline side. The K _m values were 5×10^-4 m for glucose-6-phosphate and 3.6×10^-5 m NADP⁺. The addition of 1×10^-2 m MgCl₂ produced optimal activity but higher concentrations inhibited the enzyme reaction.These results were discussed with those from other sources and found to be unique for Zymomonas mobilis.Meinem hochverehrten Lehrer Herrn Professor A. Rippel zum 80. Geburtstage.     

Isocitrate dehydrogenase of Tetrahymena pyriformis

Summary We have studied the isocitrate dehydrogenase ofTetrahymena pyriformis. This enzyme is able to utilize both NAD and NADP, but kinetic studies suggest that the enzymatic activity with NAD is not of physiological significance.Some of the factors that might regulate the NADP-dependent isocitrate dehydrogenase were also studied. This enzyme has an absolute requirement for divalent cations; Mg²⁺ and Mn²⁺ will serve as cofactors but the latter is more effective than the former.It is known that this enzyme is subject to a concerted inhibition by oxaloacetate and glyoxylate. Either glyoxylate or oxaloacetate alone also are capable of inhibiting the enzyme although higher concentrations are required. We have found concerted inhibition also for the NAD-dependent isocitrate dehydrogenase from rat liver and yeast. The activity of theTetrahymena pyriformis enzyme is inhibited by NADPH. This inhibition is competitive with NADP. The Ki and Km values are, respectively, 23µ m and 18µ m.     

Purification,characterization and regulation of a monomeric l-phenylalanine dehydrogenase from the facultative methylotroph Nocardia sp. 239

In Nocardia sp. 239 d-phenylalanine is converted into l-phenylalanine by an inducible amino acid racemase. The further catabolism of this amino acid involves an NAD-dependent l-phenylalanine dehydrogenase. This enzyme was detected only in cells grown on l- or d-phenylalanine and in batch cultures highest activities were obtained at relatively low amino acid concentrations in the medium. The presence of additional carbon- or nitrogen sources invariably resulted in decreased enzyme levels. From experiments with phenylalanine-limited continuous cultures it appeared that the rate of synthesis of the enzyme increased with increasing growth rates. The regulation of phenylalanine dehydrogenase synthesis was studied in more detail during growth of the organism on mixtures of methanol and l-phenylalanine. Highest rates of l-phenylalanine dehydrogenase production were observed with increasing ratios of l-phenylalanine/methanol in the feed of chemostat cultures. Characteristic properties of the enzyme were investigated following its (partial) purification from l- and d-phenylalanine-grown cells. This resulted in the isolation of enzymes with identical properties. The native enzyme had a molecular weight of 42 000 and consisted of a single subunit; it showed activity with l-phenylalanine, phenylpyruvate, 4-hydroxyphenyl-pyruvate, indole-3-pyruvate and -ketoisocaproate, but not with imidazolepyruvate, d-phenylalanine and other l-amino acids tested. Maximum activities with phenylpyruvate (310 mol min^-1 mg^-1 of purified protein) were observed at pH 10 and 53°C. Sorbitol and glycerol stabilized the enzyme.Abbreviations RuMP ribulose monophosphate - HPS hexulose-6-phosphate synthase - HPT hexulose-6-phosphate isomerase - FPLC fast protein liquid chromatography     

Factors enhancing l-valine production by the growth-limited l-isoleucine auxotrophic strain Corynebacterium glutamicum ΔilvA ΔpanB ilvNM13 (pECKAilvBNC)

Cell growth limitation is known to be an important condition that enhances l-valine synthesis in Corynebacterium glutamicum recombinant strains with l-isoleucine auxotrophy. To identify whether it is the limited availability of l-isoleucine itself or the l-isoleucine limitation-induced rel-dependent ppGpp-mediated stringent response that is essential for the enhancement of l-valine synthesis in growth-limited C. glutamicum cells, we deleted the rel gene, thereby constructing a relaxed (rel ⁻ ) C. glutamicum ΔilvA ΔpanB Δrel ilvNM13 (pECKAilvBNC) strain. Variations in enzyme activity and l-valine synthesis in rel ⁺ and rel ⁻ strains under conditions of l-isoleucine excess and limitation were investigated. A sharp increase in acetohydroxy acid synthase (AHAS) activity, a slight increase in acetohydroxyacid isomeroreductase (AHAIR) activity, and a dramatic increase in l-valine synthesis were observed in both rel ⁺ and rel ⁻ cells exposed to l-isoleucine limitation. Although the positive effect of induction of the stringent response on AHAS and AHAIR upregulation in cells was not confirmed, we found the stringent response to be beneficial for maintaining increased AHAS, dihydroxyacid dehydratase, and transaminase B activity and l-valine synthesis in cells during the stationary growth phase.     

Cloning and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an l-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da. The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni–NTA chromatography. The molecular mass of the purified enzyme was estimated to be ~53 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme required a divalent metal ion, either Mn²⁺or Co²⁺, for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50°C, respectively, with a k _cat of 12,455 min⁻¹ and a k _cat/K _m of 34 min⁻¹ mM⁻¹ for l-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for l-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of l-ribulose. This work describes one of the most catalytically efficient L-AIs characterized thus far.     

Purification of tobacco O-methyltransferases by affinity chromatography and estimation of the rate of synthesis of the enzymes during hypersensitive reaction to virus infection

The three tobacco (Nicotiana tabacum L.) S-adenosyl-L-methionine: o-diphenol-O-methyltransferases (OMTs; EC 2.1.1.6) were purified to homogeneity by affinity chromatography on adenosine-agarose. Amounts and catalytic actities of the enzymes were measured in tobacco leaves during the hypersensitive reaction to tobacco mosaic virus. The drastic increase in activity of each enzyme upon infection was shown to arise from the accumulation of enzymatic protein with constant specific enzymatic activity. Rates of OMT synthesis were determined from pulse-labeling experiments with L-[¹⁴C]leucine injected into the leaves. The specific radioactivities of the homogenous enzymes were compared in healthy and tobacco mosaic virus-infected tobacco. The results demonstrated that increase in OMT amounts is a consequence of de novo synthesis of the enzymes.Abbreviations DEAE diethylaminoethyl - OMT O-methyltransferase - SAM S-adenosyl-L-methionine - TMV tobacco mosaic virus     

Catalytic properties and substrate specificity of 3-hexulose phosphate synthase fromMethylomonas M15

Summary 3-Hexulose phosphate synthase was purified in 94% yield from Methylomonas M15. The enzyme did not form a Schiff-base intermediate with d-ribulose 5-phosphate that could be reduced by NaBH₄. However, the enzyme required Mg²⁺ or Mn²⁺ ions for activity and was inactivated in the presence of EDTA. The latter is a property of class II aldolases. The enzyme accepted a wide range of other aldehydes in addition to its natural substrate formaldehyde, while d-ribulose 5-phosphate could not be replaced. This makes it an attractive tool for the synthesis of higher sugar phosphates. Offprint requests to: M.-R. Kula     

De novo Synthesis of Polyglucans by a Phosphorylase Isoenzyme in Algae

The α2 phosphorylase isoenzyme of Oscillatoria princeps, Rhodymenia pertusa and Chlorella pyrcnoidosa has been found to be capable of the synthesis of amylose-like polyglucans without the necessity for addition of primers. This enzyme differs in this respect from the a1 isoenzyme of blue-green and green algae. The a2 enzyme appears to be a glyco-protein and synthesis of linear polyglucans appears to occur by the apposition of glucosyl residues from glucose-1-phos-phate directly to the glycosyl moiety of its molecule.     

Interactions of lanthanum with purified and intact cell acetylcholinesterase of electrophorus electricus

Summary The effects of lanthanum on the activity of purified preparations of acetylcholinesterase (AChE) from the electric organ ofE. electricus and on the activity of AChE in intact electro-plaques from the same species were studied. 0.1mm LaCl₃ produced an initial inhibition of purified AChE which was followed by a delayed activation of the enzyme. Upon pretreatment of purified enzyme with LaCl₃, initial activity was markedly increased. LaCl₃ exerted a marked, concentration-dependent inhibition of intact cell AChE.La³⁺ and Ca²⁺ appear to interact competitively. In the presence of both 10mm CaCl₂ and 0.1mm LaCl₃, the initial activity of purified AChE was increased at lower ACh concentrations and inhibited at ACh concentrations greater than 3 × 10^–4 m. Inhibition of intact cell enzyme by 0.1mm LaCl₃ was relieved by increasing the CaCl₂ concentration to 10mm at ACh concentrations less than 2 × 10^–4 m.The data were analyzed assuming Michaelis-Menten kinetics and interpreted with reference to the differential binding of divalent and trivalent cations to regulatory anionic sites which are separate and distinct from the anionic site of the active center of the enzyme.     

Purification and characterization of a novel l-2-amino-Δ2-thiazoline-4-carboxylic acid hydrolase from Pseudomonas sp. strain ON-4a expressed in E. coli

l-2-Amino-Δ²-thiazoline-4-carboxylic acid hydrolase (ATC hydrolase) was purified and characterized from the crude extract of Escherichia coli, in which the gene for ATC hydrolase of Pseudomonas sp. strain ON-4a was expressed. The results of SDS–polyacrylamide gel electrophoresis and gel filtration on Sephacryl S-200 suggested that the ATC hydrolase was a tetrameric enzyme consisted of identical 25-kDa subunits. The optimum pH and temperature of the enzyme activity were pH 7.0 and 30–35°C, respectively. The enzyme did not require divalent cations for the expression of the activity, and Cu²⁺ and Mn²⁺ ions strongly inhibited the enzyme activity. An inhibition experiment by diethylpyrocarbonic acid, 2-hydroxy-5-nitrobenzyl bromide, and N-bromosuccinimide suggested that tryptophan, cysteine, or/and histidine residues may be involved in the catalytic site of this enzyme. The enzyme was strictly specific for the l-form of d,l-ATC and exhibited high activity for the hydrolysis of l-ATC with the values of K _m (0.35 mM) and V _max (69.0 U/mg protein). This enzyme could not cleave the ring structure of derivatives of thiazole, thiazoline, and thiazolidine tested, except for d,l- and l-ATC. These results show that the ATC hydrolase is a novel enzyme cleaving the carbon–sulfur bond in a ring structure of l-ATC to produce N-carbamoyl-l-cysteine.     

Biochemical and genetic factors in the heat inactivation of murine β-glucuronidase

The enzymes coded for by two alleles at the glucuronidase structural locus (Gus) were compared in their response to pH, buffering anion, buffer molarity, ionic strength, and temperature. The heat-labile Gus^h gene product responded in a qualitatively similar but quantitatively reduced manner compared to the relatively heat-stable Gus ^b gene product. In all buffers tested, the enzyme was most heat stable at pH 5.0. Ranking of the various buffer anions tested, according to increasing heat stabilization, was water acetate phosphate < citrate. Varying the molarity of the buffers from 0.01 to 0.6 m at pH 5.0 revealed further differences among the buffers. Increasing ionic strength exerted a destabilizing force on the protein. The half-life of the enzyme decreased by as much as a hundredfold between 71 and 75 C. The Gus^h/Gus^h genotype also results in decreased activity levels in all tissues, reportedly because of decreased synthesis. The heat inactivation curves of Gus^b/Gus^h heterozygotes were incompatible with any theoretical curve based on the assumption that the Gus^b and Gus^h chromosomes in the heterozygote behave in a manner similar to that seen in the homozygotes.This research was supported by a Basil O'Connor Starter Research Grant from the National Foundation—March of Dimes (R. J. M.) and by a grant from The Jane Coffin Childs Memorial Fund for Medical Research (K. H.).Fellow of The Jane Coffin Childs Memorial Fund for Medical Research.     

6-Phosphogluconate dehydrogenase in cell free extracts of Escherichia coli K-12

Summary Investigations into the properties of 6-PG dehydrogenase in cell free extracts of Escherichia coli revealed a pH optimum at pH 9.5 with a sharp decline on both sides of the optimum. The addition of 1.0×10^-2 m MgCl₂ produced maximal activity, whereas higher concentrations caused inhibition. The K _m values were 2.5×10^-4 m for 6-phosphogluconate and 2.5×10^-5 m for NADP⁺ as substrate. The enzyme was extremely stable for at least 5 hours if stored at 4°C in Tris–NaCl–MgCl₂ buffer at pH 7.5. 6-PG dehydrogenase activity was shown to be proportional to cell free extract concentration over the range 0–0.3 mg protein. An assay method based on the new optimal conditions has been established and has been shown to be 33% more sensitive than a number of commonly used methods.Meinem hochverehrten Lehrer Herrn Professor A. Rippel zum 80. Geburtstage.     

Purification and characterization of naringinase from a newly isolated strain of <Emphasis Type="Italic">Aspergillus niger</Emphasis> 1344 for the transformation of flavonoids

Summary An extracellular naringinase (an enzyme complex consisting of α-L-rhamnosidase and β-D-glucosidase activity, EC 3.2.1.40) that hydrolyses naringin (a trihydroxy flavonoid) for the production of rhamnose and glucose was purified from the culture filtrate of Aspergillus niger 1344. The enzyme was purified 38-fold by ammonium sulphate precipitation, ion exchange and gel filtration chromatography with an overall recovery of 19% with a specific activity of 867 units per mg of protein. The molecular mass of the purified enzyme was estimated to be about 168 kDa by gel filtration chromatography on a Sephadex G-200 column and the molecular mass of the subunits was estimated to be 85 kDa by sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 4.0 and temperature of 50 °C, respectively. The naringinase was stable at 37 °C for 72 h, whereas at 40 °C the enzyme showed 50% inactivation after 96 h of incubation. Hg²⁺, SDS, p-chloromercuribenzoate, Cu²⁺ and Mn²⁺ completely inhibited the enzyme activity at a concentration of 2.5–10 mM, whereas, Ca²⁺, Co²⁺ and Mg²⁺ showed very little inactivation even at high concentrations (10–100 mM). The enzyme activity was strongly inhibited by rhamnose, the end product of naringin hydrolysis. The enzyme activity was accelerated by Mg²⁺ and remained stable for one year after storage at −20 °C. The purified enzyme preparation successfully hydrolysed naringin and rutin, but not hesperidin.     

A novel N-carbamoyl-L-amino acid amidohydrolase of Pseudomonas sp. strain ON-4a: purification and characterization of N-carbamoyl-L-cysteine amidohydrolase expressed in Escherichia coli

N-carbamoyl-l-cysteine amidohydrolase (NCC amidohydrolase) was purified and characterized from the crude extract of Escherichia coli in which the gene for NCC amidohydrolase of Pseudomonas sp. strain ON-4a was expressed. The enzyme was purified 58-fold to homogeneity with a yield of 16.1% by three steps of column chromatography. The results of gel filtration on Sephacryl S-300 and SDS-polyacrylamide gel electrophoresis suggested that the enzyme was a tetramer protein of identical 45-kDa subunits. The optimum pH and temperature of the enzyme activity were pH 9.0 and 50°C, respectively. The enzyme required Mn²⁺ ion for activity expression and was inhibited by EDTA, Hg²⁺ and sulfhydryl reagents. The enzyme was strictly specific for the l-form of N-carbamoyl-amino acids as substrates and exhibited high activity in the hydrolysis of N-carbamoyl-l-cysteine as substrate. These results suggested that the NCC amidohydrolase is a novel l-carbamoylase, different from the known l-carbamoylases.