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Lists the research papers to be published in the succeeding issues of the "European Journal of Biochemistry." "The structure of DNA junctions and their interaction with enzymes," by D.R. Duckett and colleagues; "CD4 gene transcription is transiently repressed during differentiation of myeloid cells to macrophage-like cells," by F.X. Pimentel-Muiñoz and colleagues; "Malonate decarboxylase of Malonomas rubra: a novel type of biotin-containing acetyl-enzyme," by H. Hilbi and colleagues.

Lists papers to be published in the "European Journal of Biochemistry." Chemical modification of phenol hydroxylase; Fructose metabolism; Ester composition of carnitine; Structure of sidechain lipopolysaccharide from Erwinia amylovora T.

Lists articles about biochemistry. "Modulation of the interaction between the two halves of troponin C by the other troponin subunits," by C.-L.A. Wang and J. Gergely; "Sequence determinants of cytosolic N-terminal protein processing," by C. Flinta, B. Persson, H. Jornvall and G. von Heijne; "Effect of benzoate on the metabolism of fructose 2,6-biphosphate in yeast," by J. Francois, E. van Schaftingen and H.-G. Hers; "Isolation and properties of porcine cholesterol acyltransferase," G. Knipping; Others.

This article presents information related to forthcoming research papers on biochemistry published in the October 1991 issue of "European Journal of Biochemistry." Few of the listed papers are: "Atrial Natriuretic Peptide (ANP) is a High Affinity Substrate for Rat Insulin Degrading Enzyme,"; "Synthesis of Dinucleoside Polyphosphates Catalyzed by Firefly Luciferase,"; "The Mechanism of Receptor Mediated Endocytosis,"; Restriction Enzymes Permit Quantitative Determination of Defined Chromatin Structures Within the Chromosome."

This article presents information about some forthcoming research papers related to the "European Journal of Biotechnology." Some of those papers are: "Mutation-Induced Instability of Antibiotic-Resistant Mammalian Ribosomes," by P.J. Wejksnora and J.R. Warner; "Phosphoribosylpyrophosphate Synthetase of Escherichia coli. Identification of a Mutant Enzyme," by B. Hove-Jensen, P. Nygaard; "The Fl-Gene Product of Bacteriophage Lambda. Purification and Properties," by S. Benchimol and A. Becker.

Lists papers to be published in the "European Journal of Biochemistry" after November 1986. "Intracellular pathway followed by invertase endocytosed by rat liver," by M. Jadot, et al; "Biochemical analysis of synoviocytes from normal and arthritic rats — Evidence for an activated state associated with adjuvant polyarthritis," by C. Bernabeu, et al; "Purification and properties of guanylate kinase from bovine retina and rod outer segments," by S. W. Hall and H. Kühn.

The presence of bound D-glucuronic acid in the endotoxin of Bordetella pertussis was demonstrated. The branched chain trisaccharide named in the title was isolated after hydrolysis of the endotoxin with 3 M HCl for 2 h at 100°C. Its structure was established by chemical and enzymic degradation. [ABSTRACT FROM AUTHOR]

The O antigen polysaccharide of Salmonella montevideo was isolated from a core-defective mutant by the phenol/water procedure, and was suspected to contain phosphomonoester and cyclic phosphodiester at its reducing end in anology to the O hapten from Salmonella typhimurium (Kent and Osborn, 1968). Therefore, it was chromatographed on a DEAE-cellulose column. Whereas one part eluted with water the other part of the polysaccharide could only be eluted with buffer. Both fractions were further purified on Sephadex G100 and contained mannose, glucose, N-acetylglucosamine and phosphate in a molar ratio of 4:1:1: < 0.1. In order to specifically label the reducing end phosphate was removed enzymatically, or the presumed cyclic diester was cleaved by mild hydrolysis, and the fractions were reduced with sodium boro[³H]hydride. Both fractions yield mainly [³H]glycerol after hydrolysis and paper chromatography. In addition, [³H]mannitol and [³H]monohydroxyacetone could be identified by paper chromatography and were concluded to be the result of phosphate migration and β-elimination reactions taking place during the isolation procedure and the various treatments prior to sodium boro[³H]-hydride reduction. These findings in addition to periodate oxidation studies indicated that the O antigen polysaccharide of Salmonella montevideo had glyceraldehyde phosphate at its reducing end. From the incorporation of ³H into the polysaccharide the O antigen was calculated to consist of about 19 repeating units of 6 sugar residues each. [ABSTRACT FROM AUTHOR]

The presence of sulfated uronic acid residues in dermatan sulfate has been known for some time. In view of the copolymeric nature of pig skin dermatan sulfate, it was of interest to see whether the extra sulfate groups were attached to iduronic acid or glucuronic acid residues. Hyaluronidase-degraded dermatan sulfate was fractionated on Dowex-1 to yield fractions of varying sulfate content. It was observed that the degree of sulfation was inversely proportional to the glucuronic acid content. Oversulfated dermatan sulfate was subsequently treated with chondroitinase-AC to yield a completely glucuronic acid-free polymeric product still bearing all the extra sulfate groups. More direct evidence for the presence of sulfated iduronic acid moieties was obtained by Smith degradation of oversulfated, chondroitinase-AC treated dermatan sulfate. A fast-moving component isolated by preparative electrophoresis had the molar ratio sulfate: hexosamine 1.8:1 and contained exclusively L-iduronic acid. [ABSTRACT FROM AUTHOR]

The pectin methyl esterase from soybean cell walls has been isolated and purified to homogeneity. It is a protein with a relative molecular mass close to 33000. The enzyme is maximally active at a pH close to 8 and its pH dependence may be explained by a classical Dixon model, where the two interconvertible enzyme ionization states coexist. The outflux of protons from cell walls, upon raising the ionic strength, may be taken as an indirect estimate of the fixed charge density. If the cell-wall fragments are pre-incubated at pH values between 5 and 9, the outflux of protons rises with the pH of pre-incubation. This implies, as postulated from the theory developed in the preceding paper, that alkaline pH favours the activity of pectin methyl esterase and that this enzyme effectively generates the fixed negative charges of the cell wall. Therefore the pectin methyl esterase reaction builds up the Donnan potential, Δψ, at the cell surface. The cell-wall charge density, estimated from the proton outflux, as well as from the titration of methyl groups on the cell wall, reaches a maximum between the third and the fourth day of growth. While the cell-wall volume increases and reaches a plateau, the fixed charge density increases at first and then declines. This is understandable if one assumes that the building up of a high charge density is a co-operative phenomenon and that the local pH inside the wall rises during cell growth. When both the cell-wall volume and the charge density increase together, this suggests that the local pH inside the wall lies within the critical pH range associated with the steep response of the system. When the cell-wall volume increases together with a decrease of the fixed charge density, the local pH should have dropped below this critical pH range. Under these conditions the pectin methyl esterase remains inactive, or poorly active. As the number of fixed negative charges increases, calcium becomes tightly bound to cell walls. This binding is so tight that the net charge density is minimum when the calcium concentration is maximum. The experimental results, presented above, offer experimental support to two important ideas discussed in the preceding paper, namely that pectin methyl esterase reaction builds up the Donnan potential at the cell surface, and that this response may be co-operative with respect to pH. [ABSTRACT FROM AUTHOR]

After CNBr cleavage of p-hydroxybenzoate hydroxylase from Pseudornonas fluorescens, five peptides and free homoserine were isolated (see preceding paper in this journal). The amino acid sequences of the three smallest peptides, viz. CB3, CB4 and CB5, were determined by automated Edman degradation and analysis of enzymatic subdigests. These peptides form a continuous stretch of 110 residues from the N terminus: [This equation can not be represent in ASCII.TXT]. [ABSTRACT FROM AUTHOR]

The effect of N-substitution with various acyl groups at glucosamine residues on lysozyme- catalyzed hydrolysis of cell wall peplidoglycan and its oligosaccharides was studied. The lysozyme-resistant cell wall peptidoglycan from Bacillus cereus, N-unsubstituted at most of its glucosamine residues, was N-acylated and used as substrate for lysozyme. The N-acetylated, N-formylated, N-propionylated and N-butyrylated preparations of the peptidoglycan were hydrolyzed by lysozyme with relative maximum rates of 1.00, 0.05, 0.38 and 0.10 respectively, and with apparent Km values, expressed in disaccharide unit concentration, of 0.1, 0.6, 2.4 and 10 mM respectively, whereas the N-succinylated preparation was unsusceptible to this enzyme. Peptidoglycan oligosaccharides, GlcN-MurAc-GlcNAc-MurAc, GlcNAc-MurAc-G1cN-MurAc- GlcNAc-MurAc and GlcN-MurAc-GIcN-MurAc-GlcNAc-MurAc, were also N-acylated to be examined as substrates for lysozyme. The N-acelylated, N-formylated, N-propionylated and N- butyrylated preparations from the tetrasaccharide were hydrolyzed at 0.5 mM with relative rates of 1.00, 0.04, 0.1 and 0.01 respectively, but the N-succinylated tetrasaccharide was unaffected by lysozyme. The N-formylated, N-propionylated, N-butyrylated and N-succinylated preparations from the former hexasaccharide were hydrolyzed with relative rates of 1.5, 22, 1.6 and 0.8 respectively, as compared to the value of about 2000 for the N-acetylated hexasaccharide. The N-acylated preparations from the latter hexasaccharide, except the N-succinylated one, were hydrolyzed at rates similar to those of hydrolysis of the corresponding preparations from the former hexasaccharide. The above results seem to be accounted for by the predominant importance of the alkyl moieties of the N-acyl substituents on the glucosamine residues located in subsite C in the enzyme-substrate complexes. [ABSTRACT FROM AUTHOR]

RNA ligase catalyzed the joining of pC-C-Ap with C-A-A in the synthesis of C-A-A-C-C-Ap, which has the sequence of the Escherichia coli tRNAfMet 3′-end. pC-C-A was also shown to be joined to C-A-A without any undesired self-polymerization. Joining of pC-C-A to various synthetic ribotriplets, such as C-C-A, A-A-A, C-C-C, U-U-U, U-A-G, C-C-G AND U-U-C, was performed as well as joining to the partially substituted trimers with a photolabile o-nitrobenzyl group, C-Anbzl-A and C-C-Anbzl. The yields were C-A-A-C-C-A (69 %), C-C-A-C-C-A (38%), A-A-A-C-C-A (66%), C-C-C-C-C-A (71%), U-U-U-C-C-A (50%), U-A-G-C-C-A (23%), C-C-G-C-C-A (43%) AND U-U-C-C-C-A (46%). C-Anbzl-A was a slightly poorer acceptor than C-A-A and C-C-Anbzl did not serve as an acceptor. Recognition of acceptor molecules by RNA ligase is discussed in terms of affinity of oligonucleotides for the enzyme. [ABSTRACT FROM AUTHOR]

Procedures are described for the assay of collagen galactosyltransferase and collagen glucosyltransferase activities. The methods are based on the transfer of [14C]galactose or [14C]glucose from the corresponding radioactive UDP-glycoside to hydroxylysyl or galactosylhydroxylysyl residues in calf-skin gelatin substrate, and on the specific assay of the products of the enzymic reactions. The assay of the products includes precipitation of the protein, and removal of most of the radioactivity present in the free UDP-glycoside by several washings of the precipitate. The [14C]galactosylhydroxylysine or [14C]glucosylgalactosylhydroxylysine is then liberated by alkaline hydrolysis, and purified further by a modified Dowex chromatography and by paper electrophoresis. The procedure is entirely specific when judged by amino acid analysis, and it is possible to assay a series of 36 samples in 2 days. The sensitivity of the method permits assays in extracts from several tissues, including tissues with low transferase activities, such as liver. Preliminary characterization of the transferases from chick embryo cartilage indicated that the galactosyltransferase did not catalyze the transfer of galactose to free hydroxylysine. Dialyzable peptides were found to act as substrates for both transferases. The Km for the gelatinized collagen substrate was 150 g/1 in the reaction with the galactosyltransferase, and 14 g/1 in that with the glucosyltransferase. The Km for UDP-galactose or UDP-glucose in the reaction with the corresponding transferase was about 30 μM. Both transferases required a metal co-factor, and manganese was found to be far more effective than any other metal tested. The optimal manganese concentration for both transferases was 10 mM. No stimulation of the glucosyltransferase activity was found with folate, which has recently been reported to stimulate collagen glucosyltransferase from rat kidney. [ABSTRACT FROM AUTHOR]

The fate of the hydrogen atoms at C-3 and C-4 of uridine diphospho-D-glucuronic acid (UDPGlcUA) in the course of its conversion to uridine diphospho-apiose (UDP-api) was investigated. The specifically labeled UDP-GlcUA substrates were prepared enzymatically from D-[3-³H]glucose and D-[4-³H]glucose by the combined action of hexokinase, phosphoglucomutase, UDPGpyrophosphorylase, and UDPG dehydrogenase. UDP-[U-14C, 3-³H]GlcUA, UDP-[U14C, 4-³H]GlcUA, and UDP-[U14C, 3,4-³H2]GlcUA were separately converted to UDP-Api by an enzyme preparation from Lemna minor. In order to obtain an apiose (3-C-hydroxymethyl-D-erythrofuranose) derivative which was reasonably stable and which could be produced without passing through the aldehydo form, the apiosyl radical of UDP-Api was enzymatically transferred to 7-glucosylapigenin by an enzyme preparation from parsley. The product of this reaction, apiin (7-O-[β-D-apiofuranosyl-(1→2)-β-D-glucosyl-5,7,4'-trihydroxyflavone), was subjected to mild periodate oxidation to convert the branch hydroxymethyl group of the apiosyl residue to formaldehyde. Approximately 14-16% of each of the substrates was converted to UDP-Api. The transfer of the apiosyl moiety to 7-glucosylapigenin was essentially quantitative in all cases. The isolated apiin was shown to have retained 53.5% of the tritium from the substrate labeled at C-3, 99% from the substrate labeled at C-4, and 73% from the substrate labeled at C-3,4. All of the tritium and one-fifth of the 14C of apiin produced from the variously labeled substrate was accounted for in the formaldehyde-dimedone derivative. These results establish the occurrence of a hydride shift from C-4 of UDP-GlcUA to C-3' of apiose in the biosynthesis of UDP-Api from UDPGlcUA. [ABSTRACT FROM AUTHOR]

The enzyme terminal deoxynucleotidyl transferase catalyzes the addition of one or two riboadenylie acid residues to deoxyoligonucleotides. This reaction allows the specific labeling of deoxyoligonucleotides at the 3′-ends if [α-32P]ATP is used as substrate. In order to eliminate the product of the addition of two riboadenylie acid residues the total reaction mixtures after incubation with terminal transferase are treated with alkali and subsequently with phosphatase. Deoxyoligonucleotidyl [32P]pAr is then obtained as the sole reaction product. Digestion with spleen phosphodiesterase yields the deoxynucleotide-3′-[32P]monophosphates corresponding to the 3′-ends of the deoxyoligonucleotides. The riboadenosyl residues at the 3′-termini of the monoaddition products also can be eliminated by treatment with periodate and cyclohexylamine whereupon deoxyoligonucleotides specifically labeled at the 3′-termini with [32P]phosphomonoester groups are obtained. The sensitivity of the method and the faithfulness in the identification of the 3′-terminal deoxynucleoside residues could be tested using as little as 0.1 A250 units (5 μg) of oligodeoxynucleotides of specific base sequences. [ABSTRACT FROM AUTHOR]

The availability of a high-resolution structure of the Thermobifida fusca endocellulase Cel6A catalytic domain makes this enzyme ideal for structure-based efforts to engineer cellulases with high activity on native cellulose. In order to determine the role of conserved, noncatalytic residues in cellulose hydrolysis, 14 mutations of six conserved residues in or near the Cel6A active-site cleft were studied for their effects on catalytic activity, substrate specificity, processivity and ligand-binding affinity. Eleven mutations were generated by site-directed mutagenesis using PCR, while three were from previous studies. All the CD spectra of the mutant enzymes were indistinguishable from that of Cel6A indicating that the mutations did not dramatically change protein conformation. Seven mutations in four residues (H159, R237, K259 and E263) increased activity on carboxymethyl cellulose (CM-cellulose), with K259H (in glucosyl subsite -2) creating the highest activity (370%). Interestingly, the other mutations in these residues reduced CM-cellulose activity. Only the K259H enzyme retained more activity on acid-swollen cellulose than on filter paper, suggesting that this mutation affected the rate-limiting step in crystalline cellulose hydrolysis. All the mutations lowered activity on cellotriose and cellotetraose, but two mutations, both in subsite +1 (H159S and N190A), had higher kcat/Km values (6.6-fold and 5.0-fold, respectively) than Cel6A on 2,4-dinitrophenyl-β-d-cellobioside. Measurement of enzyme : ligand dissociation constants for three methylumbelliferyl oligosaccharides and cellotriose showed that all mutant enzymes bound these ligands either to the same extent as or more weakly than Cel6A. These results show that conserved noncatalytic residues can profoundly affect Cel6A activity and specificity. [ABSTRACT FROM AUTHOR]

As shown in the preceding paper [Ito, M., Ikegami, Y., Tai, T. & Yamagata, T. (1993) Eur. J. Biochem. 218, 637–643], endoglycoceramidase (EGCase: EC,3,3,1,123) was found to hydrolyze the cell-surface glycosphingolipids (GSLs) of erythrocytes without any damage to other cell membrane components. This paper represent the kinetics of EGCase action toward cell-surface GSLs of erythrocytes. Without activator or detergents, cell-surface GSLs were found to be hydrolyzed by EGCase II very slowly at pH 7.0. The initial reaction velocity of EGCase II under this condition was 0.038 pmol⋅min ¹ ⋅ mU ¹ for horse erythrocyte cell-surface GM3 and 0.032 pmol ⋅ min ¹ ⋅ mU ⋅ for guinea pig erythrocyte cell-surface Gg3Cer. The addition of activator protein (60 μM), which stimulates EGCase II in the absence of detergents, increased the initial reaction velocity of the enzyme 616-fold for cell-surface GM3 and 468-fold for Gg3Cer, while no increased hemolysis was observed with the addition of activator. However, even in the presence of the activator, the cell-surface GSLs were very resistant to hydrolysis by EGCase II compared to GSL vesicles (or micelles) under same conditions. In contrast to the activator, Triton X-100 (0.4%, mass/vol.) not only stimulated the enzyme activity but also solubilized erythrocyte GSLs into detergent micelles, inducing further increment of the enzyme activity but also solubilized erythrocyte GSLs into detergent micelles, inducing further increment of the enzyme reaction velocity. The apparent Km and Vmax values of EGCase II were calculated from the Lineweaver­Burk plot as 47 μM and 35 pmol min ¹ mU ¹ for horse erythrocyte cell-surface GM3 and 44 μM and 27 pmol ⋅ min ¹ ⋅ mU ¹ for guinea pig erythrocyte cell-surface Gg3Cer, at pH 7.0 in the presence of activator at a concentration of 60 μM. [ABSTRACT FROM AUTHOR]

This paper describes a rapid and inexpensive method for homogeneous enzyme preparation from SDS/polyacrylamide gels with subsequent renaturation. The method was optimized for an enzyme of pyrimidine metabolism, thymidine-5′-triphosphatase (dTTPase), present in human serum in small amounts. After gel electrophoresis, the enzyme was eluted from gel pieces in an elution chamber based on a tube gel electrophoresis system. Renaturation conditions were optimized in preliminary tests. The best results were obtained with an initial acetone precipitation to remove sodium dodecyl sulfate. The precipitate was then dissolved in 8 M guanidine hydrochloride and diluted 50-fold for renaturation. Adding 1.5 mg/ml lauryl maltoside to the renaturation buffer, followed by subsequent dialysis of the renaturating samples, improved the renaturation yield up to 95%. This method was used to purify dTTPase to homogeneity from a partially purified sample, and to determine the molecular mass of the subunits. The procedure can also be applied to other enzymes and could give rise to a general strategy for enzyme purification. [ABSTRACT FROM AUTHOR]

The kinetic parameters of 20β-hydroxysteroid dehydrogenase were determined in aqueous solutions and in reversed micellar media composed with either an anionic, a cationic or a nonionic surfactant, at low and at high ionic strength. The velocity data were analysed in two ways: first by extrapolation to infinite concentrations of both substrates to determine ‘apparent’ Michaelis constants and V values, and secondly by comparison to reaction rates calculated using the model presented (see first of this series of papers in this issue of the journal). Data analysis according to the first method reveals some differences in the kinetic parameters in reversed micelles as compared to those in aqueous solution, though the kinetic parameters of the enzyme seem not to be much affected by enclosure in reversed micelles. It is shown that the changes that do occur are not caused by a shift of the intramicellar pH or by electrostatic interactions between the enzyme and the surfactant head groups. Interpretation of the data using the second method assumes that the enzyme is not affected by the enclosure in reversed micelles, and that deviations with respect to the aqueous parameters are caused by exchange phenomena between distinct aqueous droplets in the organic phase and by a high effective intramicellar substrate concentration. This model is able to predict reaction rates that agree rather well with experimentally determined rates and explains why the enzyme mechanism in reversed micelles is, at all progesterone concentrations used, the same as observed at high progesterone concentrations in aqueous solution. Furthermore it clarifies the occurrence of substrate inhibition in sodium-di(ethylhexyl)sulphosuccinate-reversed micelles and the observed low activity in Triton-reversed micelles, as arising from the high partition coefficient of progesterone and the slow rate of diffusion of progesterone into the reversed micelles. From these results, and those reported for enoate reductase (see preceding paper in this issue of the journal) it can be concluded that the theory presented before (see first of this series of papers in this issue of the journal) offers a good explanation for the observed kinetic behaviour in reversed micelles, and emphasizes the importance of exchange processes between micelles. [ABSTRACT FROM AUTHOR]

An 11450-base DNA fragment containing the gene for the extracellular active-site serine DD-peptidase of Streptornyces R61 was cloned in Streptornyces lividans using the high-copy-number plasmid pi J702 as vector. Amplified expression of the excreted enzyme was observed. Producing clones were identified with the help of a specific antiserum directed against the pure DD-peptidase. The coding sequence of the gene was then located by hybridization with a specific nucleotide probe and sub-fragments were obtained from which the nucleotide sequence of the structural gene and the putative promoter and terminator regions were determined. The sequence suggests that the gene codes for a 406-amino-acid protein precursor. When compared with the excreted, mature DDpeptidase, this precursor possesses a cleavable 31-amino-acid N-terminal extension which has the characteristics of a signal peptide, and a cleavable 26-amino-acid C-terminal extension. On the basis of the data of Joris et al. (following paper in this journal), the open reading frame coding for the synthesis of the DD-peptidase was established. Comparison of the primary structure of the Streptomyces R61 DD-peptidase with those of several active-site serine β-lactamases and penicillin-binding proteins of Escherichia coli shows homology in those sequences that comprise the active-site serine residue. When the comparison is broadened to the complete amino acid sequences, significant homology is observed only for the pair Streptomyces R61 DD-peptidase/Escherichia coil ampC β-lactamase (class C). Since the Streptomyces R61 DD-peptidase and β-lactamases of class A have very similar three-dimensional structures [Kelly et al. (1986) Science (Wash. DC) 231, 1429-1431; Samraoui et al. (1986) Nature (Lond.) 320, 378-380], it is concluded that these tertiary features are probably also shared by the β-1actamases of class C, i.e. that the Streptomyces R61 DD-peptidase and the β-lactamases of classes A and C are related in an evolutionary sense. [ABSTRACT FROM AUTHOR]

Soybean cell walls display a process of autolysis which results in the release of reducing sugars from the walls. Loosening and autolysis of cell wall are involved in the cell-wall growth process, for autolysis is maximum during both cell extension and cell-wall synthesis. Autolysis goes to completion within about 50 h and is an enzymatic process that results from the activity of cell wall exo- and endo-glycosyltransferases. The optimum pH of autolysis is about 5. Increasing the ionic strength of the bulk phase where cell-wall fragments are suspended, results in a shift of the pH profile towards low pH. This is consistent with the view that at 'low' ionic strength, the local pH in the cell wall is lower than in the bulk phase. One of the main ideas of the model proposed in a preceding paper, is that pectin methyl esterase reaction, by building up a high fixed charge density, results in proton attraction in the wall. Low pH must then activate the wall loosening enzymes involved in autolysis and cell growth. This view may be directly confirmed experimentally. The pH of a cell-wall suspension, initially equal to 5, was brought to 8 for 20 min, then back to S. Under these conditions, the rate of cell-wall autolysis was enhanced with respect to the rate of autolysis obtained with cell-wall fragments kept at pH 5. The pH response of the multienzyme plant cell-wall system basically relies on opposite pH sensitivities of the two types of enzymes involved in the growth process. Pectin methyl esterase, which generates the cell-wall Donnan potential, is inhibited by protons, whereas the wall-loosening enzymes involved in cell growth are activated by protons. [ABSTRACT FROM AUTHOR]

The complete ‘centre-of-subunit structure’ of the multisubunit enzyme DNA-dependent RNA polymerase was determined by triangulation of the subunit positions using the intersubunit distances calculated from scattering difference measurements and from the corresponding radii of gyration R. In addition to the centre-to-centre distances d between the core subunits α 2, β and βʹ presented in the preceding paper, the values of between initiation factor σ and α (8.4 ± 1.6 nm), β (4.4 ± 2.2 nm) and βʹ (10.7 ± 1.5 nm) were derived from R of σ (4.1 ± 0.3 nm) in situ and of the pairs α2 – σ (6.1 ± 0.4 nm), β – σ (5.6 ± 0.3 nm) and βʹ – σ (7.5 ± 0.4 nm) within the holoenzyme (α2β βʹσ). The structural parameters of the subunits within their molecular complex are accessible for neutron small-angle scattering measurements using labelling of the different subunits (deuteration), total reconstitution of isotopic hybrids, scattering length density matching of‘hydrogenated’ molecular parts and extended exposure times because of weak scattering effects. The overall shape of σ bound to core enzyme (α2ββʹ) proved to be identical (within experimental resolution) with σ in the isolated state measured recently by X-ray small-angle scattering. The refined shape of isolated σ was reduced to an ellipsoid which was orientated with respect to the core structure (α2 — β — βʹ) in a ’space-filling’ way around the position of the σ centre obtained by triangulation. The complete subunit arrangement of holoenzyme is shown in a three-dimensional model. [ABSTRACT FROM AUTHOR]

The DNA-stimulated 75 000-Mr ATPase described in the preceding paper is shown to be a further catalytic DNA unwinding principle (DNA unwinding enzyme II) made in Escherichia coli cells (the first being the 180 000-Mr ATPase of the cells: DNA unwinding enzyme I). Unwinding depends, strictly, on the supply of ATP. It occurs only under conditions permitting ATP dephosphorylation and it proceeds as long as enzyme molecules are permitted to enter the enzyme · DNA complex. The enzyme binds specifically to single-stranded DNA yielding a complex of only limited stability. These results are interpreted in terms of a distributive mode of action of the enzyme. It is argued that chain separation starts near a single-stranded DNA region and that, forced by continued adsorption of enzyme molecules to the DNA, it develops along the duplex. This mechanism is different from that deduced previously for DNA unwinding enzyme I. Complicated results were obtained using ATPase prepared from rep3 mutant cells. [ABSTRACT FROM AUTHOR]

The kinetic and regulatory properties of enzymes involved in the biosynthesis of UDP-D-galactose, UDP-N-acetylglucosamine, GDP-α-D-mannose and GDP-β-L-fucose from D-glucose 6-phosphate in various reticulo-endothelial tissues was studied. The tissues examined include bovine liver, thyroid, spleen, salivary gland, lung, intestine and mesenteric, pulmonary, portal and sub-maxillary lymph nodes. The maximum rates of specific enzymes in these pathways which were slow enough to be rate-limiting in the formation of glycoproteins in these tissues was determined. UDP-D-galactose 4-epimerase was consistently the rate-limiting reaction in the conversion of D-glucose 6-phosphate to UDP-D-galactose in all of the tissues examined. The series of reactions leading to the formation of GDP-α-D-mannose and GDP-β-L-fucose were limited by the activity of GDP-α-D-mannose pyrophosphorylase and GDP-α-D-mannose oxidoreductase, respectively. The formation of UDP-N-acetylglucosamine was limited by the rate of the amination reaction which converts D-fructose 6-phosphate to D-glucosamine 6-phosphate in the presence of glutamine. Several of these rate-limiting enzymes were partially purified from mesenteric lymph node extracts, and their regulatory properties were examined. GDP-α-D-mannose was found to be a competitive inhibitor of GDP-α-D-mannose pyrophosphorylase. The apparent Km for GTP was 0.06 mM and the Ki for GDP-α-D-mannose was 0.03 mM. The concentrations of GTP and GDP-α-D-mannose in lymph node extracts were determined to be 0.095 and 0.012 μmol per g, respectively. UDP-N-acetylglucosamine and UDP-D-glucose inhibited D-fructose 6-phosphate amidotransferase in a manner competitive with D-fructose 6-phosphate. The Km for fructose 6-phosphate was 0.3 mM, while the Ki for UDP-D-glucose and UDP-N-acetylglucosamine were determined to be 0.4 mM and 0.045 mM, respectively. The concentrations of these metabolites in lymph node tissue were; UDP-D-glucose, 0.42; UDP-N-acetylglucosamine 0.095; and D-fructose 6-phosphate, 0.073 μmol per g wet weight of tissue. The results obtained in these studies show that specific rate-limiting enzymes in the pathways for the biosynthesis of nucleoside diphosphate sugars in reticulo-endothelial tissues may be subject to cumulative feedback inhibition by the nucleoside diphosphate sugars which are the final products of these systems and the initial precursors of the oligosaccharide units of glycoproteins in these tissues. [ABSTRACT FROM AUTHOR]

There is now experimental evidence that association of different enzymes as a multi-enzyme complex may result in an alteration of the catalytic properties of the enzymes present in this complex. This effect is not related to the channeling of reaction intermediates between different active sites. It appears as a consequence of an information transfer that occurs within the multi-enzyme complex. A theory, based on statistical thermodynamics, has been developed which provides an understanding, on a physical basis, for how isologous as well as heterologous interactions between identical, or different, enzymes of the complex may modulate the catalytic properties of an oligomeric enzyme of that complex. The theory predicts three possible types of effects: an alteration, through heterologous interactions, of an already existing co-operativity of the oligomeric enzyme within the complex; a co-operativity, generated by heterologous interactions in the complex that could not occur if the oligomeric enzyme were isolated from the rest of the complex; a Michaelis-Menten character of the oligomeric enzyme within the complex, but with altered values of Vm and Km relative to what would have been observed with the naked enzyme. All these effects appear as a consequence of a transfer of information between different enzymes of the same multi-protein complex. The following paper in this journal shows how one can demonstrate and characterize experimentally these effects in a multi-enzyme complex containing ribulose bisphosphate carboxylase-oxygenase. [ABSTRACT FROM AUTHOR]

PC-1 is an ecto-enzyme possessing alkaline phosphodiesterase I and nucleotide pyrophosphatase activities. In this paper, we demonstrate the expression, biochemical characterization and biosynthesis of human PC-1. Previously, there has been uncertainty concerning which of two methionine residues is the initiator. It is now shown that expression of PC-1 is much greater if the first methionine residue is present, and that the sequence between the two methionine residues is translated in both human and mouse, in both transfected cells and cells naturally expressing PC-1. The first methionine residue is therefore the initiator. Human PC-1 is capable of autophosphorylation, and conditions are described in which PC-1 is the only labelled phosphoprotein on the plasma membranes of intact cells, allowing the demonstration that the mature membrane form of human PC-1 is approximately 10 kDa larger than that of the mouse form. Pulse-chase biosynthetic studies and treatment with two different endoglycosidases show that most of this difference is due to N-linked oligosaccharides. The polypeptide backbone of human PC-1 is 20 amino acids longer than that of the mouse PC-1, with most of the difference in polypeptide chain length being in the cytoplasmic domain. The revised cytoplasmic domain of human PC-1 has 76 amino acids, while the mouse cytoplasmic domain has 58 amino acids. Optimal alignment of mouse and human cytoplasmic domains reveals areas of sequence conservation in which the third bases vary. It is suggested that these regions of conservation may point to functionally important sequences in the cytoplasmic domain. [ABSTRACT FROM AUTHOR]

The mechanism of formation of the formyl group of chlorophyll b has long been obscure but, in this paper, the origin of tile 7-formyl-group oxygen of chlorophyll b in higher plants was determined by greening etiolaled maize leaves, excised from dark-grown plants, by illumination under while light in the presence of either H2 18O or 18O2 and examining the newly synthesized chlorophylls by mass spectroscopy. To minimize the possible loss of 18O label from the 7-formyl substituent by reversible formation of chlolophyll b-7¹-gem-diol (hydrate) with unlabelled water in the cell, the formyl group was reduced to a hydroxymethyl group during extraction with methanol containing NaBH4: chlorophyll a remained unchanged during this rapid reductive extraction process, Mass spectra of chlorophyll a and 17-hydroxymethyll-chlorophyll b extracted from leaves greened in the presence of either H, 18O or 18O. revealed that so was incorporated only from molecular oxygen but into both chlorophylls: the mass spectra were consistent with molecular oxygen providing an oxygen atom not only for incorporation into the 7-formyl group of chlorophyll b but also for the well-documented incorporation into the 13¹-oxo group of both chlorophylls a and b [see Walker. C. J., Mansfield, K. E., Smith, K. M. & Casteffranco. P. A. (1989) Biochem. J. 257, 599–602]. The incorporation of isotope led to as much as 77% enrichment of the 13¹-oxo group of chlorophyll a: assuming identical incorporation into the 13¹ oxygen of chlorophyll b, then enrichment of the 7-formyl oxygen was as much as 93%. Isotope dilution by re-incorporation of photosyn-thetically produced oxygen from unlabelled water was negligible as shown by a greening experiment in the presence of 3-(3.4-dichlorophenyl)-l.1-dimethylurea. The high enrichment using 18O, and the absence of labelling by unequivocally demonstrates that molecular oxygen is the sole precursor of the 7-formyl oxygen of chlorophyll b in higher plants and strongly suggests a single pathway for the formation of the chlorophyll b formyl group involving the participation of an oxygenase-type enzyme. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that Corynebacterium cyclohexanicum degrades cyclohex-anecarboxylic acid, a bacteriocide, through a pathway including the aromatization of 4-oxocyclohex-anecarboxylic acid to 4-hydroxybenzoic acid [Kaneda, T. (1974) Biochem. Biophys. Res. Commun. 58, 140–144]. Aromatization has now been shown to be catalysed by two desaturase enzymes. Under the action of desaturase I, 4-oxocyclohexanecarboxylic acid is converted to (+)-4-oxocyclohex-2-enecarboxylic acid which is then aromatized by desaturase II to 4-hydroxybenzoic acid. The latter reaction is presumed to occur via the unstable intermediate, 4-oxocyclohex-2,5-dienecarboxylic acid, which is spontaneously isomerized to 4-hydroxybenzoic acid. Desaturase I has been purified in an electrophoretically homogeneous form. It is monomeric with a molecular mass of 67 kDa and contains one tryptophan, one histidine and two cysteine residues per enzyme molecule. The enzyme produces an equivalent amount of 4-oxocyclohex-2-enecarboxylic acid and hydrogen peroxide from 4-oxocyclohexanecarboxylic acid. The properties of desaturase I have been studied in detail. Desaturase II is unstable and has been partially purified. Its characterization is therefore limited. However, the molecular mass of desaturase II was estimated to be 43 kDa by gel filtration chromatography. The characterization of both desaturase enzymes is described in this paper. The possible environmental importance of microbial aromatization in the biodegradation of compounds with the cyclohexane structure is discussed. [ABSTRACT FROM AUTHOR]

We extend the analysis of unbranched chains (preceding paper) to large parameter changes in branched systems using linear kinetic assumptions. More complex relationships between flux control coefficients and deviation indices are established. In particular, the deviation index in such systems depends on more than one control coefficient as well as on the magnitude of the enzyme change. Non-additivity of the indices is the general rule. Combined changes of groups of enzymes, whether co-ordinate or not, have also been formulated. Control coefficients can be estimated from a small number of independent large-change experiments. Alternatively, the amplification factors can be calculated given the knowledge of the control coefficients. A 'case study' using published data is presented. The movement of intermediate metabolites as a consequence of large parameter changes can be dealt with in a similar manner. Experimental methods for showing the admissibility of assuming the simplifying assumptions used are summarised. Some simulation studies show possible limits of the application of the approach and some aspects of the general, non-linear, case are discussed. It is concluded that, although metabolic systems are in principle non-linear, many behave, in practice, as quasi-linear systems. The relationships established between deviation indices and control coefficients therefore provide a practical way of predicting the effects of large-scale changes in parameters for many metabolic systems. [ABSTRACT FROM AUTHOR]

Metabolic control analysis [Kacser and Burns (1973) Symp. Soc. Exp. Biol. 27, 65-104; Heinrich and Rapoport (1974) Eur. J. Biochem. 42, 89-95] leads to a description of the systemic properties of a metabolic system (expressed as control coefficients) in terms of the local kinetic properties of the individual enzyme-catalyzed reactions (expressed as elasticity coefficients). This paper describes a non-algebraic diagrammatic method which generates the mathematical expressions for flux or concentration-control coefficients in terms of elasticity coefficients. According to a set of simple rules, 'flux-control patterns' or 'concentration-control patterns' are drawn on a metabolic diagram. Each control pattern represents a product of elasticity coefficients that occurs as a term in the expression for a control coefficient. The rules also generate the correct sign that precedes each term. The control patterns are then used to build the expressions for control coefficients. The procedure was developed in such a way that each control pattern can be understood in terms of a 'chain of local effects' which shows how a perturbation in the activity of an enzyme is propagated through the metabolic pathway. [ABSTRACT FROM AUTHOR]

Recently, we reported that the synthesis of human lysozyme in yeast is inhibited when artificially designed non-native signal sequences are used [Yamamoto et al. (1987) Biochem. Biophys. Res. Commun. 149, 431- 436; (1989) Biochemistry 28, 2728-2732]. Pulse/chase experiments described in the present paper have now revealed that precursor lysozymes are actually synthesized with these signal sequences, but that they are spontaneously degraded, suggesting that the enzyme is destabilized by the signal sequences. The data further show a good correlation between the secretory capability of the artificial signal sequences and the efficiency of the processing of precursor lysozyme. [ABSTRACT FROM AUTHOR]

The cAMP-dependent phosphorylation of α-crystallin was investigated. The major products of in vitro phosphorylation of total bovine lens homogenate are the αA1 and αB1 polypeptides, but in addition a minor labeled spot is present which might correspond with a double phosphorylated αB chain. It is demonstrated that the A1 and B1 subunits of α-crystallin from bovine eye lenses are solely the result of phosphorylation of the primary gene products αA2 and αB2, respectively, as judged from the stoichiometry of the phosphate content of these polypeptides. Both the in vitro and in vivo phosphorylation sites of the A chain of bovine α-crystallin were determined and found to be the same. After in vitro incubation the majority of the 32P label was found in the tryptic peptides T17a and T16-17a, the latter being the result of incomplete tryptic cleavage between T16 and T17a. The in vivo phosphorylation site is also located in T17a, as could be concluded from the retention times on reversed-phase HPLC of T16-17a and T17a from αA1 as compared to those from αA1 and from the differences in their mobilities on high-voltage paper electrophoresis at pH 6.5. Furthermore, both T17a and T16-17a and αA1 contain approximately 1 mol phosphate/mol peptide. Thermolytic digestion of T16-17a of both αA2 and αA1, followed by separation on RP-HPLC, demonstrated that Ser-122 is the phosphorylation site of the A chain of bovine lens α-crystallin. The replacement of this phosphorylation site or the lack of basic amino acids at the N-terminal side of Ser-122 in some vertebrate species apparently results in the absence of phosphorylation of α-crystallin A both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

A β-glucosyltransferase, extracted and purified from the cell walls of isolated soybean cells, displays hysteretic behaviour. The enzyme is monomeric and has a negative co-operative between pH 5.5 and 7.5. Below and above these pH values, the enzyme follows or approaches, classical Michaelis-Menten kinetics. The free enzyme and the enzyme-glucose complex exhibit, upon pH jumps, conformational transitions which may be followed by monitoring the fluorescence of enzyme-bound toluidinylnaphthalene sulfonate. Taken together these results are consistent with the model of pH-induced co-operativity described in the preceding paper in this journal. This special type of co-operativity relies on a change of the pK value of a strategic ionizable group located outside the active site in a region (or a domain) of the protein which undergoes the conformational transition. The result that at ‘low’ and ‘high’ pH values, the enzyme follows or approaches Michaelis-Menten kinetics is explained by assuming that the conformational changes do not affect the active site. [ABSTRACT FROM AUTHOR]

The particulate enzyme from Bacillus subtilis AHU 1031 catalyzed the synthesis of a polysaccharide and glycolipids from UDP — N-acetylmannosaminuronic acid (UDP-ManNAcUA), UDP — N-acetylglucosamine (UDP-GlcNAc), and UDP-glucose (UDP-Glc). The polysaccharide synthesis required UDP-ManNAcUA and UDP-GlcNAc, proceeded optimally at pH 8.5 and in the presence of 5 mM MgCl2 and 2.5 mM dithiothreitol, and was stimulated by the addition of UDP-Glc. The molar ratio of ManNAcUA, GlcNAc, and Glc incorporated into polysaccharide was calculated to be 1:1:1.8 from chemical analysis involving reduction with water soluble carbodiimide; its relative molecular mass was estimated to be 12000. The analysis of Smith degradation products revealed that the polysaccharide backbone is composed of repeating trisaccharide units comprising ManNAcUA, GlcNAc, and Glc. Based on the data regarding the time course of the incorporation of glucose into the polysaccharide, extra glucose seems to be attached to the polysaccharide backbone as lateral branches. The saccharide moieties of the glycolipids were identified as GlcNAc, ManNAcUA-GlcNAc, and Glc-ManNAcUA-GlcNAc from several analytical criteria. The addition of antibiotic 24010, a tunicamycin-like antibiotic, at 10 μg/ml resulted in almost complete inhibition of the synthesis of glycolipids and polysaccharide. It is therefore concluded that the glycolipids function as intermediates in polysaccharide formation. Incubation of the ManNAcUA-GlcNAc-linked lipid. (labeled in the ManNAcUA moiety) with the particulate enzyme and UDP-Glc resulted incorporation of radioactivity into a trisaccharide-linked lipid and a polysaccharide. These results suggest that the particulate enzyme utilizes the trisaccharide moiety of the Glc-ManNAcUA-GlcNAc-linked lipid for the elongation of the main polysaccharide chain presumed to be cell wall acidic polysaccharide of this strain. [ABSTRACT FROM AUTHOR]

Purified extracellular endo-1,4-β-xylanase (EC 3.2.1.8) of the yeast Cryptococcus albidus was found to catalyze not only the known l,4-β-transfer, but an alternative transglycosylation reaction leading to the formation of 1,3-β-glycosidic linkages. From a mixture of products of β-xylanase degradation of phenyl β-D-xylopyranoside three xylooligosaccharide fractions, differring chromatographically from the 1,4-β-linked products, were isolated by preparative paper chromatography. Their structure was elucidated by mass spectrometry, 13C-NMR spectroscopy and enzymic hydrolysis by β-xylanase and β-xylosidase. The isomeric xylotriose was identified as 3-O-β-D-xylopyranosyl-4-O-β-D-xylopyranosyl-D-xylose. The fraction of isomeric tetrasaccharides was found to be represented mainly by 4-O-β-D-xylopyranosyl-3-O-β-D-xylopyranosyl-4-O-β-D-xylopyranosyl-D-xylose. The xylooligosaccharides containing one 1,3-β-linkage were also produced on the enzyme treatment of 1,4-β-xylotriose and 1,4-β-xylan. When treated with the enzyme responsible for their synthesis, the isomeric xylooligosaccharides were hydrolyzed at the 1,3-β-linkage, despite the fact the enzyme does not attack 1,3-β-xylan. The results are interpreted in the relation to the characterized four-subsite substrate-binding site of the enzyme. [ABSTRACT FROM AUTHOR]

Oxidative phosphorylation operates at optimal efficiency if and only if the condition of conductance matching L33/L11 = √1-q⊃ is fulfilled. In this relation L11 is the phenomenological conductance of phosphorylation, L33 the phenomenological conductance of the load, i.e. the irreversible ATP-utilizing processes in the cell, and q the degree of coupling of oxidative phosphorylation driven by respiration. Since during short time intervals L11 and q are constant whereas L33 fluctuates in the cell, oxidative phosphorylation would only rarely operate at optimal efficiency due to violation of conductance matching. This paper demonstrates that the reversible ATP-utilizing reaction catalyzed by adenylate kinase can effectively compensate deviations from conductance matching in the presence of a fluctuating L33 and hence allows oxidative phosphorylation to operate at optimal efficiency in the cell. Since the adenylate kinase reaction was found to buffer a thermodynamic potential, i.e. the phosphate potential, this finding was generalized to the concept of thermodynamic buffering. The thermodynamic buffering ability of the adenylate kinase reaction was demonstrated by experiments with incubated rat-liver mitochondria. Considerations of changes introduced in the entropy production by the adenylate kinase reaction allowed to establish the theoretical framework for thermodynamic buffering. The ability of thermodynamic buffering to compensate deviations from conductance matching in the presence of fluctuating loads was demonstrated by computer simulations. The possibility of other reversible ATP-utilizing reactions, like the ones catalyzed by creatine kinase and arginine kinase, to contribute to thermodynamic buffering is discussed. Finally, the comparison of the theoretically calculated steady-state cytosolic adenine nucleotide concentrations with experimental data from perfused livers demonstrated that in livers from fed rats conductance matching is fulfilled on a time average and that the degree of coupling corresponded to qpec = 0.97 permitting the most economic maintenance of a maximal output power of oxidative phosphorylation. For the case of livers from starved rats this analysis suggested that the degree of coupling corresponded to qfec = 0.95, permitting the most economic maintenance of a maximal net rate of ATP synthesis at optimal efficiency of oxidative phosphorylation. [ABSTRACT FROM AUTHOR]

Functional interrelationships between the acyl transferases of yeast fatty acid synthetase were investigated. In binding assays with synthetase modified by 5,5′-dithiobis(2-nitrobenzoic acid), 4–5 malonyl transferase entities per multienzyme complex molecule could be titrated. In the presence of palmitoyl-CoA these malonyl transferases were found inaccessible to malonyl-CoA, whereas the acetyl transferases were reactive towards acetyl-CoA. Between four and five palmitoyl transferase entities per synthetase equivalent were found reactive towards palmitoyl-CoA, the palmitoyl binding being inhibited by malonyl-CoA. Following palmitoyl binding the acetyl transferases were found reactive towards acetyl-CoA. Substrate model assays were consistent with these data. It is concluded that malonyl and palmitoyl transferases are closely coupled enzyme components of the multienzyme complex which are fairly independent of the acetyl transferase entities. The molecular basis for the observed coupling will be given in the following paper. [ABSTRACT FROM AUTHOR]

The enzymatic hydrolysis of thyroliberin (< Glu-His-Pro-NH2) by serum enzymes can be preferentially inhibited by 1 mM diisopropylfluorophosphate. Under such conditions, pyroglutamic acid and His-Pro-NH2 are formed as the only enzymatic split products. As a secondary metabolite, histidylproline diketopiperazine is formed non-enzymatically by intramolecular condensation of the dipeptide amide. Due to their differences in charge, the enzymatically formed split products can easily be separated from thyroliberin by ion-exchange chromatography on cellulose-phosphate paper and thus, by using radioactively labelled thyroliberin as tracer, highly sensitive tests could be developed for the quantitative determination of the thyroliberin-degrading enzymatic activity. By gel filtration, the thyroliberin-degrading serum enzyme could be partially purified and separated from two enzymes which catalyze the hydrolysis of pyroglutamyl β-naphthylamide, the substrate of the known pyroglutamate aminopeptidases. For the serum tested, the extremely low activities of the pyroglutamate aminopeptidases did not contribute to the observed rapid degradation of thyroliberin. On the other hand, pyroglutamyl β-naphthylamide was not effectively hydrolyzed by the thyroliberin-degrading serum enzyme. Furthermore, in contrast to the pyroglutamate aminopeptidases, which are activated by EDTA and dithiothreitol, the thyroliberin-degrading serum enzyme was inhibited by these agents. This enzyme, having an approximate molecular weight of 260000, is also effectively inhibited by other chelating agents, by heavy metal ions and by p-chloromercuribenzoate but not by 2-iodoacetamide and N-ethylmaleimide or by serine protease inhibitors. When tested in vitro, the activity of the thyroliberin-degrading serum enzyme was not affected by thyroid hormones, steroids or adenohypophyseal hormones. These results demonstrate that the degradation of thyroliberin at the pyroglutamyl-histidine bond is catalyzed by a serum enzyme which exhibits unique enzyme-chemical characteristics distinctly different from those of the known pyroglutamate aminopeptidases catalyzing the same reaction. [ABSTRACT FROM AUTHOR]

When yeast fatty acid synthetase is treated with iodoacetamide at 0 °C, three carbamoyimethyl residues are incorporated into the protein under concomitant complete loss of the enzymatic activity [Oesterhelt, D., Bauer, H., Kresze, G.-B., Steber, L. and Lynen, F. (1977) Eur. J. Biochem. 79, 173-180]. Here we report that, in this reaction, only cysteinyl residues are alkylated. During protein hydrolysis of the modified enzyme, S-carboxymethyl cysteine is formed which, when treated with performic acid in the presence of a strong acid and chloride ions, is oxidized only partially to yield open-chain S-carboxymethyl cysteine sulfone but, for the greater part, is transformed into S-carboxymethyl cysteine lactam sulfone (3-carboxy-5-oxo-tetrahydro-1,4-thiazine-1,1-dioxide), a novel derivative of S-carboxymethyl cysteine which cannot be detected with ninhydrin. If, on the other hand, the carbamoylmethylated enzyme is treated first with performic acid and hydrolyzed afterwards, a large part of the carbamoylmethyl residues is lost due to decarboxylation of the α-sulfonyl carboxylic acid S-carboxymethyl cysteine sulfone. When carboxamidomethylated fatty acid synthetase was digested with trypsin at 0 °C, two different carbamoylmethyl peptides were found which could be separated by gel filtration. The larger peptide TA was cleaved, during prolongated incubation with trypsin at 30 °C, to yield the smaller peptide TB which was purified and its amino acid sequence determined to be Thr-Pro-Val-Gly-Ala-Cys(carbamoylmethyl). By comparison of tryptic and peptic peptides from acetylated and carboxamidomethylated synthetase, respectively, it is demonstrated that the same cysteinyl residues are alkylated by iodoacetamide which are the acetyl-binding sites of the condensing enzyme component, i.e. the peripheral SH-groups. [ABSTRACT FROM AUTHOR]

The lytic enzyme from bacteriophage T7 has been purified from T7-infected Escherichia coli B cells and characterized. In the purification procedure advantage was taken of the reversible binding of the enzyme to chitin. Molecular weight determinations of the enzyme by Sephadex G-75 chromatography and sodium dodecyl sulphate gel electrophoresis gave estimates of 21000 and 18000 respectively. Because the activity of the enzyme falls rapidly to 1-3% of that observed immediately after lysis of the cells, various means to stabilize the enzyme have been tried. Of the methods attempted only binding to the substrate analogue chitin was successful. The enzymic activity was found to be markedly dependent on ionic strength and pH, maximum activity was observed using 5 mM phosphate buffer pH 7.5. MgCl2 was found to enhance the lytic activity 2–3-fold, the optimal concentration being about 0.5 mM. The enzyme was not sensitive to penicillin G or spermine, but was specifically inhibited by p-chloromercuribenzoate. Following digestion of E. coli peptidoglycan the cell wall peptides L-Ala-D-Glu(A2pm), L-Ala-D-Glu(A2pm-D-Ala) and the dimer of the latter were formed, together with paper chromatographically immobile glycan strands. The enzyme, therefore, acts as an N-acetylmuramyl-L-alanine amidase by cleaving the bond between N-acetylmuramic acid and L-alanine in the E. coli peptidoglycan. The enzyme has apparently no effect on the peptidoglycan from Micrococcus luteus, and seems to have a preference for high-molecular-weight substrates. Thus the disaccharide-tetrapeptide GlcNAc-MurNAc-L-Ala-D-Glu(A2pm-D-Ala) and its dimer are not substrates for the enzyme. [ABSTRACT FROM AUTHOR]

It was demonstrated that an enzyme system(s) extracted from etiolated seedlings of Stizolobium hassjoo catalyzed the conversion of L-dihydroxyphenylalanine into stizolobinic acid. α-amino-6-carboxy-2-oxo-2H-pyran-3-propionic acid, and stizolobic acid, α-amino-6-earboxy-2-oxo-2H-pyran-4-propionic acid, in the presence of NADP+ or NAD+ under aerobic condition. Enzymically synthesized radioactive stizolobinic acid and stizolobic acid isolated from the reaction mixtures were purified and confirmed to have constant specific radioactivities by cocrystallization with authentic samples. Maximal activity of the enzyme preparation was obtained by using an insoluble polyphenol adsorbent (Polyclar AT) and a reducing agent (araboascorbic acid) in the extraction medium and by subsequent fractionation of the extract with ammonium sulfate followed by Sephadex G-25 gel filtration. Catalytic activity of the enzyme preparation was more unstable under aerobic condition than anaerobic, Attempts to stabilise the enzyme activity were made by the use of many substances which are known to stabilise other enzymes or expected to arrest the inactivation. Evidence is provided in this paper that the previously proposed biosynthetic pathways of stizolobinic acid and stizolobic acid from dihydroxyphenylalanine proceeded in the cell-free system from etiolated seedlings of S. hassjoo. [ABSTRACT FROM AUTHOR]

The acyl carrier protein of citrate lyase contains adenine, phosphate, sugar, cysteamine, β-alanine and pantoic acid in a molar ratio of 1:2:2:1:1:1. Peptides containing these components in the same stoichiometric relationship were isolated after proteolytic digestion of acyl carrier protein. All components were linked together in a single prosthetic group. This was released from the peptide by mild alkaline hydrolysis. Under these conditions a phosphodiester bond is cleaved which links the prosthetic group to a serine residue of the peptide. Incubation of the prosthetic-group-containing peptide with phosphodiesterase I yielded 4′-phosphopantetheine and adenylic acid. The 5′-AMP was not free but was substituted by presumably an acidic sugar residue, which was released by mild acid hydrolysis yielding free 5′-AMP. It was concluded from these results that the prosthetic group of citrate lyase acyl carrier protein consists of a substituted isomeric dephospho-CoA. This is bound to the protein by the 5′-phosphate group of adenylic acid. The 4′-phosphopantetheine residue is bound by a phosphodiester linkage to the 2′ or 3′ position of ribose and the remaining hydroxyl group of ribose is substituted with presumably an acidic sugar residue. The structural similarities of this prothetic group and coenzyme A are discussed and related to the catalytic properties of citrate lyase [ABSTRACT FROM AUTHOR]

The enzymic formation of guanosine diphosphate 2-deoxy-D-glucose and uridine diphosphate 2-deoxy-D-glucose from synthetically prepared 2-deoxy-D-glucose 1-phosphate is described. Incubation of 2-deoxy-D-glucose 1-phosphate with an enzyme preparation from bovine mammary glands and either GTP or UTP gives rise to the corresponding nucleoside-diphosphate derivative of 2-deoxy-o-glucose. Uridine diphosphate 2-deoxy-D-glucose could also be obtained by incubation of 2-deoxy-D-glucose 1-phosphate with UTP and UDP glucose p yrophosphorylase from beef liver. [ABSTRACT FROM AUTHOR]

A strong synergistic response was observed between the five endo-1,4-β-glucanases and the exo-1,4-β-glucanase obtained from culture solutions of the rot fungus Sporotrichum pulverulentum (formerly called Chrysosporium lignorum), when these enzymes were allowed to degrade de-waxed cotton and Avicel. No synergism was observed if Walseth cellulose, an acid-swollen cullulose, was used. If de-waxed cotton was pretreated with endo-1,4-β-glucanases, the exo-1,4-β-glucanase enzyme released much more degradation products than from an untreated cotton. This strongly supports the theory that the endo-1,4-β-glucanases, acting randomly over the cellulose chain, go in first and open up chain ends the exo-enzymes can act. In an earlier paper [Eriksson, K.-E. & Pettersson, B. (1975) Eur. J. Biochem. 51, 193-206] physicochemical differences between the endo-1,4-β-glucanases were demonstrated. Functional differences have now been found. The five endo-enzymes give rise to different degradation products from Avicel, show differences in preferred mode of attack on cellodextrins, and exhibit pronounced differences in synthetic activity. The exo-1,4-β-glucanase released its products in the α-configuration. A method for the separation of cellodextrins on a Bio-Sil A Column is also described. [ABSTRACT FROM AUTHOR]

Several large oligonucleotides obtained by partial digestion of tRNAIIArg from brewers' yeast with ribonnclease T1 have been isolated and their sequences have been established. These results and those described in the preceding paper permitted the derivation of the complete primary structure of this tRNA. [ABSTRACT FROM AUTHOR]

Lysozyme from bacteriophage T4 was found to digest a soluble, uncrosslinked peptidoglycan which is secreted by cells of Micrococcus luteus when incubated in the presence of penicillin G. Analysis of the enzymatic degradation products shows that T4 acts as an endo-acetylmuramidase capable of cleaving glycosidic bonds only at muramic acid residues that are substituted with peptide side-chains. The results indicate that the secreted peptidoglycan may consist of a mixture of chains, approximately half of which are substituted by peptide side chains on most of their muramic acid residues, while the other half is made up of chains in which the muramic acid moieties are unsubstituted. [ABSTRACT FROM AUTHOR]

400 fold purification of GDP glucose pyrophosphorylase from pea seedlings concomitant with stabilization of enzyme activity has been accomplished by (NH4)2SO4 fractionation, followed by chromatography on Sephadex G200 and DEAE-cellulose columns. In this last step the enzyme is eluted with 0.20 M NaCl. The enzyme is specifically activated by Mn2+. The apparent Km value for glucose-1-phosphate (37°, pH 7.5) is about 3 × 10-4 M. The GDP glucose biosynthesis is found to be reversible, with the equilibrium in favour of pyrophosphorolysis. The purified enzyme is not active with GTP plus mannose-1-phosphate or galactose-1-phosphate. No formation of nucleotide sugar is observed in the presence of glucose-1-phosphate plus ATP, ITP, dTTP, or CTP. A small residual UDPG pyrophosphorylase activity is activated by Mg2+. [ABSTRACT FROM AUTHOR]

The maximal potential activity of UDP-glucuronyltransferase is constrained by the structure of the phospholipid environment in intact microsomal membranes. This constraint can be relieved by treatment of microsomes with phospholipase A. As shown by the data in this paper, however, relief of constraint is associated with a loss of specificity in the binding of UDP-sugars at the UDP-glucuronic acid site of UDP-glucuronyltransferase. As a result, several UDP-sugars which have no effect on the activity of the untreated enzyme act as inhibitors of the unconstrained, phospholipase A-treated form of UDP-glucuronyltransferase. In addition to the loss of specificity of substrate binding, the phospholipase-A-treated form of UDP-glucuronyltransfease cannot be activated by UDP-N-acetylglucosamine, which is a positive K-type of allosteric effector for the untreated form of the enzyme. UDP-N-acetylglucosamine, in fact, is an inhibitor of the phospholipase-A-treated form of the enzyme. In contrast to the effects of other UDP-sugars, inhibition by UDP-N-acetylglucosamine seems to result from the binding of UDP-N-acetylglucosamine at an allosteric site rather than the active site. Inhibition of the phospholipase-A-treated form of UDP-glucuronyltransferase by UDP-N-acetylglucosamine and other UDP-sugars is additive. [ABSTRACT FROM AUTHOR]

Trehalose synthase (TreS) catalyzes the reversible interconversion of trehalose (glucosyl-α,α-1,1-glucose) and maltose (glucosyl-α1-4-glucose). TreS was purified from the cytosol ofMycobacterium smegmatisto give a single protein band on SDS gels with a molecular mass of≈ 68 kDa. However, active enzyme exhibited a molecular mass of≈ 390 kDa by gel filtration suggesting that TreS is a hexamer of six identical subunits. Based on amino acid compositions of several peptides, thetreSgene was identified in theM. smegmatisgenome sequence, and was cloned and expressed in active form inEscherichia coli.The recombinant protein was synthesized with a (His)6 tag at the amino terminus. The interconversion of trehalose and maltose by the purified TreS was studied at various concentrations of maltose or trehalose. At a maltose concentration of 0.5 mm, an equilibrium mixture containing equal amounts of trehalose and maltose (42–45% of each) was reached during an incubation of about 6 h, whereas at 2 mmmaltose, it took about 22 h to reach the same equilibrium. However, when trehalose was the substrate at either 0.5 or 2 mm, only about 30% of the trehalose was converted to maltose in≥ 12 h, indicating that maltose is the preferred substrate. These incubations also produced up to 8–10% free glucose. TheKm for maltose was≈ 10 mm, whereas for trehalose it was≈ 90 mm. Whileβ,β-trehalose, isomaltose (α1,6-glucose disaccharide), kojibiose (α1,2) or cellobiose (β1,4) were not substrates for TreS, nigerose (α1,3-glucose disaccharide) andα,β-trehalose were utilized at 20 and 15%, respectively, as compared to maltose. The enzyme has a pH optimum of about 7 and is inhibited in a competitive manner by Tris buffer.[3H]Trehalose is converted to[3H]maltose even in the presence of a 100-fold or more excess of unlabeled maltose, and[14C]maltose produces[14C]trehalose in excess unlabeled trehalose, suggesting the possibility of separate binding sites for maltose and trehalose. The catalytic mechanism may involve scission of the incoming disaccharide and transfer of a glucose to an enzyme-bound glucose, as[3H]glucose incubated with TreS and either unlabeled maltose or trehalose results in formation of[3H]disaccharide. TreS also catalyzes production of a glucosamine disaccharide from maltose and glucosamine, suggesting that this enzyme may be valuable in carbohydrate synthetic chemistry. [ABSTRACT FROM AUTHOR]