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The article presents some of the name of some of the papers that will be published in the forthcoming issues of the "European Journal of Biochemistry." Some of them are Membrane Proteins. The Amino Acid Composition of Membrane penetrating segments, On the role of the carbohydrate side chains of human plasminogen in its interaction with alpha-antiplasmin and fibrin, Respiratory and enzymatic properties of squid heart mitochondria, Self-Association of Bovine lung heparin sulphate's, Identification and characterization of contact zones and many others.

The article presents a list of topics, which are to be introduced in the forthcoming issue of the "European Journal of Biochemistry." Some of the them includes as: Effects of Cyclic Nucleotide Analogs on Intrachin Site 1 of Protein Kinase Isozymes; Direct Evidence that Cholestrol Ester Hydrolase from Adrenal Cortex is the Same Enzyme as Hormone-Sensitive Lipase from Adipose Tissue; Agonist-Mediated Confrontational Changes of Muscarinic Receptors in Rat Brain and The Reaction of Bovine Glutamate Dehydrogenase with Periodate-Oxidized ADP et al.

The transient-state kinetics of enzymic reduction of acetaldehyde and benzaldehyde by NADH, catalyzed by horse liver alcohol dehydrogenase, have been examined under single-turnover conditions, obtained by carrying out reactions either with limiting amounts of enzyme in the presence of 20 mM pyrazole or with limiting amounts of substrate. Analysis of the variation with substrate, coenzyme, and enzyme concentrations of amplitudes and time constants for the exponential transients observed at 328 nm and 300 nm shows that the kinetics of enzymic aldehyde reduction are qualitatively and quantitatively consistent with the relationships derived in the preceding paper for an ordered ternary-complex mechanism involving identical and independent catalytic sites. It is concluded that there is no evidence whatsoever for the kinetic significance of a half-of-the-sites reactivity or any other kind of subunit interaction in the liver alcohol dehydrogenase system. The biphasic transients observed at 328 nm for the reduction of aromatic aldehydes such as benzaldehyde are a normal kinetic characteristic of the ordered ternary-complex mechanism, being attributable to accumulation of the ternary enzyme · NAD · product complex when product dissociation from this complex is slow in comparison to its formation by ternary-complex interconversion. [ABSTRACT FROM AUTHOR]

A theory is proposed to explain the physical bases of the ionic control of the activity of an enzyme system, located on a plant cell surface, and probably involved in cell-wall synthesis and extension. The model, which is based on various previously published experimental results, involves several assumptions: a cell-wall pectin methyl esterase de-esterifies pectins and thus creates the fixed negative charges of the cell wall; various enzymes incorporate uncharged carbohydrates in cell-wall material; cell-wall extension implies the sliding of cellulose microfibrils; the enzymes responsible for carbohydrate incorporation are activated by protons in the pH range 4-8 and have very similar pH dependencies: the cell-wall pectin methyl esterase is inhibited by protons in the same pH range. The mathematical derivation of this model, written in the form of a hypercycle, indicates that it is equivalent to a set of two antagonistic enzyme reactions: an enzyme reaction conditioned by pectin methyl esterase which results in the increase of fixed charge density of the cell wall; a number of 'growth enzymes', which produce extension and building up of the cell wall and therefore a decrease of charge density. The mathematical study of this model shows it may display a very high co-operativity of its response to slight changes of pH. This co-operativity means that the cell wall charge density may dramatically increase or decrease, within a very narrow pH range. The steep response of this system appears to be the direct consequence of different pH sensitivities of pectin methyl esterase and of the other cell-wall enzymes involved in cell growth. Calcium, which tightly binds to the cell wall, may diminish or even suppress this abrupt charge transition. This model suggests a novel theory of the ionic control of cell-wall expansion. The very basis of this theory is the existence of an electrostatic potential difference, Δψ, between the inside and the outside of the cell wall. When this Δψ value is large, the local proton concentration is high. Therefore the enzymes involved in cell wall extension and building up are active, but pectin methyl esterase is not. Therefore, the cell wall extends and the charge density decreases. The Δψ value then declines, as well as the local proton concentration. Under these conditions, the pectin methyl esterase becomes activated, whereas the 'growth enzymes' are not. This activation of pectin methyl esterase restores the initial, or an even higher, clectrostatic potential difference, which in turn results in a decrease of local pH. In the following papers the basic ideas of this model are subjected to experimental control. [ABSTRACT FROM AUTHOR]

In this paper we describe the reduction of corticosteroid metabolites containing the 17β-aldol side chain (isocorticosteroids) by aldose and aldehyde reductase from human tissues. Aldose reductase catalyzed the reduction of the aldehydes derived from cortisol and corticosterone at about the same rate, whereas aldehyde reductase preferentially acted on the aldehydes derived Irom 1 7-deoxycorticosteroids. At comparable rates of reduction the Michaelis constants for the best steroid aldehydes were one order of magnitude lower than for the hitherto best substrates. We propose that aldose and aldehyde reductase participate in the conversion of the corticosteroid ketol side chain to the glycol side chain via an aldol intermediate by the `long loop' pathway proposed by Monder and Bradlow [(1977) J. Steroid Biochem. 8. 897 - 908]. [ABSTRACT FROM AUTHOR]

We have developed a semi-empirical method to determine the subunit molecular weight of a protein. The method is a minor modification of the Edman degradation and is based on a simple chemical procedure to modify specifically the N-terminal NH2 group. Initially, all NH2 groups, including the ε-NH2 groups of lysine residues and the N-terminal α-NH2 group, are reacted with phenylisothiocyanate and the protein derivative is subjected to one step of the Edman degradation. The newly exposed N terminus is then reacted with radioactively labelled phenylisothiocyanate. A value for the subunit molecular weight can be obtained from an analysis of the incorporated radioactivity and the amount of the protein in the sample. The molecular weight of five different proteins have been determined by this method. Our method is particularly useful for proteins containing lipid or sugar components and also for relatively small peptides. The procedure described in this paper for the specific modification of the N terminus has been found to be a powerful tool for protein sequencing. [ABSTRACT FROM AUTHOR]

A protein is usually classified into one of the following four structural classes: all α, all β, (a + B) and α/β. In this paper, based on the maximum correlation-coefficient principle, a new formulation is proposed for predicting the structural class of a protein according to its amino acid composition. Calculations have been made for a development set of proteins from which the amino acid compositions for the standard structural classes were derived, and an independent set of proteins which are outside the development set. The former can test the self consistency of a method and the latter can test its extrapolating effectiveness. In both cases, the results showed that the new method gave a considerably higher rate of correct prediction than any of the previous methods, implying that a significant improvement has been achieved by implementing the maximum-correlation-coefficient principle in the new method. [ABSTRACT FROM AUTHOR]

A number of N-alkylated-l,3-diaminopropane derivatives [H2N-(CH2)3-NH-(CH2)nH, where n = 1 -9] have been tested as potential inhibitors of partially purified rat hepatoma (HTC) cell or pure bovine spleen spermine synthase. Among the compounds described in this paper, the most potent competitive inhibitor of spermine synthase, with respect to spermidine, is N-butyl-l,3-diaminopropane with Ki values of 11.9 nM and 10.4 nM for the HTC cell and bovine spleen enzymes respectively. Inhibition of spermine synthase by this alkylated amine is selective since spermidine synthase activity is not affected up to 100 µM N-butyl-l,3-diaminopropane at a range of 5200 µM putrescine. Added to the culture medium of growing HTC cells, N-butyl-l,3-diaminopropane causes the expected changes in the polyamine levels with a marked decrease of spermine and an increase of spermidine. Under these conditions cell growth continues unabated. Such N-alkylated-l,3-diaminopropane derivatives may have considerable potential as tools for studying the role of polyamines and in particular the functions of spermine in cell multiplication and differentiation. [ABSTRACT FROM AUTHOR]

The native lipoteichoic-acid-like amphiphite from Bifidobacterium bifidum subspecies pennsylvanicum and its basic glycan moiety, obtained by alkaline hydrolysis, were studied by 1H-, 13C-, and 31P-NMR, DEPT (dis- torsionless enhancement by polarization transfer), 1H-1H correlation spectroscopy and 13C-1H shift correlation NMR spectroscopy. The results are consistent with the structure elucidated by chemical analysis (Fischer, W., preceding paper in this journal): ... and establish the structures of the repeating units independently. In addition to characteristic shifts in the 13C-NMR spectrum, the sites of phosphodiester and alanine ester bonds are also manifest in the 1H-NMR spectrum. Substitution of the glycerophosphate residues can be recognized in the 31P-NMR spectrum also. [ABSTRACT FROM AUTHOR]

[sup31]P and [sup13]C magnetic resonance spectra of fructose 2,6-bisphosphate, prepared as described by Van Schaftingen and Hers (preceding paper), and of commercial fructose 1,6-bisphosphate were obtained. Analysis of these spectra has confirmed the structure of fructose 2,6-bisphosphate; this ester was present as a single anomeric form to which the β configuration could be assigned. [ABSTRACT FROM AUTHOR]

This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach. 1. The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two-step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one. 2. α-Chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxytic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of α-chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll-400/Dextran-70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65-98 °C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date. [ABSTRACT FROM AUTHOR]

The structure of the carbohydrate moiety of the lipopeptidophosphoglycan from Trypanosoma cruzi was studied by 13C NMR spectroscopy and by methylation analysis of the original and of an acid-degraded sample. An oligosaccharide, consisting of 2-O-substituted and 6-O-substituted mannoses, which is linked to the ceramide, was separated by partial acid hydrolysis from an external chain that contained 3-O-substituted mannopyranosyl residues. β-D-Galactofuranosyl terminal units are attached to position 3 of (1 → 2)-linked mannopyranose. Besides the previously reported monosaccharide components (mannose, galactose, glucose and glucosamine), ribose was identified in a partial acid hydrolysate of the lipopeptidophosphoglycan. The last three sugars are minor components and their oganization into the overall structure of the lipopeptidophosphoglycan has not been determined. [ABSTRACT FROM AUTHOR]

Inhibitor-2, purified by an improved procedure, was used to identify protein phosphatases capable of catalyzing its dephosphorylation. The results showed that, under our experimental conditions, protein phosphatases-1, 2A and 2B were the only significant protein phosphatases in rabbit skeletal muscle extracts acting on this substrate. Protein phosphatase-1 and 2A accounted for all the inhibitor-2 phosphatase activity in the absence of Ca2+ (resting muscle), and the potential importance of these enzymes in vivo is discussed. Protein phosphatase-2B, a Ca2+-calmodulin-dependent enzyme, could account for up to 30% of the inhibitor-2 phosphatase activity in contracting muscle. The Km of protein phosphatase-1 for inhibitor-2 (40 nM) was 100-fold lower than the Km for phosphorylase a (4.8 µM). This finding, coupled with the failure of inhibitor-2 to inhibit its own dephosphorylation, suggests that inhibitor-2 is dephosphorylated at one of the two sites on protein phosphatase-1 involved in preventing the dephosphorylation of other substrates. The dephosphorylation of inhibitor-2 by protein phosphatase-1 was also unaffected by inhibitor-1, suggesting that the phosphorylation state of inhibitor-2 is unlikely to be controlled by cyclic AMP in vivo. [ABSTRACT FROM AUTHOR]

A rat heart extract synthesizes glycogen-like material from UDP-glucose in the absence of added primer. Most of this material is precipitable by dilute trichloroacetic acid, in contrast to glycogen synthesized in presence of added glycogen primer. It is postulated that the endogenous initiation of glycogen synthesis occurs by apposition of glucose residues to a protein and that the material so synthesized in vitro corresponds to the protein-bound glycogen reported to exist in rat heart. The ability to label the putative protein primer in vitro with [14C]glucose should assist the isolation of the primer and the elucidation of the nature of the association between the carbohydrate and protein moeities. [ABSTRACT FROM AUTHOR]

Inverted membrane vesicles from Klebsiella aerogenes containing the Na+-activated oxaloacetate decarboxylase accumulated 22Na+ in response to the decarboxylation of oxaloacetate. Complexation of the biotin prosthetic group of the decarboxylase with avidin completely prevented the oxaloacetate-dependent Na+ uptake, whereas avidin saturated with biotin was without effect. The rapid accumulation of 22Na+ inside the vesicles following oxaloacetate addition was succeeded by a slower efflux of 22Na+ after exhaustion of oxaloacetate. Other substrates, e.g, D-lactate, were unable to replace oxaloacetate in accumulating 22Na+ to a comparable extent, with the exception of (S)-malate, which acted through its conversion to oxaloacetate. The amount of Na+ which was transported into the vesicles by a limited amount of oxaloacetate critically depended on the rate of oxaloacetate decarboxylation. This rate was retarded by using a low temperature (0 °C), by inhibiting the enzyme with oxalate or by using oxaloacetate-generating systems, where the activity of the enzyme catalyzing oxaloacetate formation was rate-determining. With these retarded systems of oxaloacetate decarboxylation the efficiency of the Na+ transport was significantly increased. Under optimized conditions Na+ could be concentrated inside the inverted vesicles eight-times higher than in the incubation medium. The Na+-carrying ionophores nigericin and trinactin effectively prevented the oxaloacetate-dependent Na+ accumulation in a K+-free incubation medium. In the presence of valinomycin and K+, however, the amount of Na+ which was taken up by the vesicles following oxaloacetate addition increased by a factor of two, which indicated limitation of Na+ uptake by electrical parameters. The generation of a membrane potential was determined from the distribution of the lipophilic anion [14C]SCN- between interior and exterior vesicular spaces. The oxaloacetate-decarboxylase-driven Na+ transport into inverted vesicles at 0 °C generated a membrane potential of 65 mV, an Na+ gradient equivalent to 49 mV and a total driving force of 114 mV. The proton carrier carbonylcyanide-p-trifluoromethoxy phenylhydrazone had essentially no effect on the oxaloacetate-decarboxylase-driven Na+ uptake, indicating that the generation of a proton gradient is not involved. It is concluded from these results that oxaloacetate decarboxylase acts as a primary active Na+ pump which generates an electrochemical gradient of Na+ ions. [ABSTRACT FROM AUTHOR]

The cell-wall mutant of a hydrocarbon-assimilating yeast, Candida sp. M-7002 and its wild type have shown a significant difference in mannose content. Each mannan was isolated from the mutant and the wild-type cells by fractionation with Cetavion and copper reagent. Both mannans contain D-mannose, D-glucose and phosphate. The mutant mannan has a relatively high content of protein (1870 in weight bases) whereas the wild-type mannan has a low protein content (5.1%) with a high amount of carbohydrate (>90%). Structural analyses by enzymatic and chemical methods showed that both mannans had an mannosidic (α1 -6)-linked back bone substituted at O-2 by side chains of varying length. The side chains of the mutant mannan were shown to consist of single niannose units and disaccharide units whose linkages were predominantly α1 -2. while the wild-type mannan had two additional side chains of disaccharides. These additional side chains had α1-3 linkages which were scarcely found in the mutant mannan. β-Elimination reaction demonstrated that the mannans also contain mannosyl oligosaccharides linked to protein through O-glycosidic linkage. The chemical properties of the mannan of the Candida mutant indicates that the mutation might occur not only in the side chain structure but also in the (α1 -6)-linked mannan back bone. [ABSTRACT FROM AUTHOR]

The enzyme transferring the oligosaccharide from DolPP-(GlcNAc)2(Man)9(Glc)3 to asparagine residues of glycoproteins has been solubilized from yeast membranes by extraction with detergents. Enzyme activity was tested by measuring transfer of the glycosyl moiety from 20 min, with about 40% of dolichyl-diphosphate-bound radioactivity transferred to the peptide. The solubilized enzyme has been characterized as follows: 1. The enzyme is most efficiently solubilized (60% of the membrane-associated activity) by 0.5% Nonidet P40 at a protein/detergent ratio of 2. Octylglucoside solubilizes one third of the activity, but strongly inhibits the reaction of present in the test at a concentration of 1%. 2. Divalent cations are absolutely required. 1 mM Mn2+is optimal; Mg2+ at a concentration of 10 mM yields one third the activity observed with Mn2+. 3. The enzyme transfers besides doluchyl-diphosphate-bound (GlcNAc)2(Man)9(Glc)3 also (GlcNAc(2(Man)1 and GlcNAc)2; the rate decreases in this order. No transfer is observed from DolPP-(GlcNA)2(Man)9 and from DolPP-GlcNAc. The Km value for DolPP-(GlcNAc)2(Man)9(Glc)3 of 0.5 µm does not differ significantly from that for DolPP-(GlcNAc_2 of 1.2 µM. A broad pH-optimum for the reaction with both substrates was found between 6.5 and 7.7. 5. However, a clear difference in Km values for the hexpeptide was observed with different dolichol-linked sugar derivatives. With DolPP-(GlcNAc)2 a peptide concentration of 0.6 mM resulted in half-maximal transfer rate, whereas 0.05 mM peptide were dufficient with DolPP-(GlcNAc)2(Man)9(Glc)3 as donor. [ABSTRACT FROM AUTHOR]

Fatty acid synthetase was covalently labelled with [14C]palmitic acid from [14C]palmityl-CoA. Tryptic and peptic digestion of the [14C]palmityl enzyme resulted in the formation of radioactive palmityl peptides carrying the long-chain acyl residue both in oxygen-ester and thio-ester linkage. The lipophilic palmityl peptides were purified by column and thin-layer chromatography using organic solvent systems. Peptides arising from the acyl carrier protein, the condensing enzyme and the palmityl transferase were identified and characterized. The amino acid sequence of a 4'-phosphopantetheine- containing peptide was established. It comprises 13 residues and shows a high degree of homology with the acyl carrier protein from Escherichia coli. A heptapeptide and an octapeptide from the palmityl transferase active site were partially sequenced. The identical amino acid composition of palmityl transferase and malonyl transferase core peptides is briefly discussed. [ABSTRACT FROM AUTHOR]

The morphology and structural organisation of the complexes formed from the apoprotein of porcine high-density lipoprotein and dimyristoyl phosphatidylcholine (lecithin) have been studied using the technique of small-angle X-ray scattering. Scattering measurements made in solvents of varying electron density were interpreted in terms of a scattering-equivalent model for the structure of the complex. This model is described by an oblate ellipsoidal morphology with dimensions at 20 °C: major axis 11.0 nm, minor axis 5.5 nm. Within this overall shape the lipid hydrocarbon chains are organised in an apolar core whilst the lipid polar head groups and protein are located in a outer shell 0.85 nm in thickness. The oblate morphology demonstrates that the structure of the complex is directed by the fundamental bilayer organisation of the lecithin. The dimension of the minor axis (5.5 nm) indicates that phospholipid hydrocarbon chains are orientated perpendicular to the interface. [ABSTRACT FROM AUTHOR]

High-pressure NMR experiments performed on the histidine-containing phosphocarrier protein (HPr) fromStaphylococcus carnosushave shown that residue Ile14, which is located in the active-centre loop, exhibits a peculiarly small pressure response. In contrast, the rest of the loop shows strong pressure effects as is expected for typical protein interaction sites. To elucidate the structural role of this residue, the mutant protein HPr(I14A), in which Ile14 is replaced by Ala, was produced and studied by solution NMR spectroscopy. On the basis of 1406 structural restraints including 20 directly detected hydrogen bonds, 491HN-15N, and 251HN-1Hα residual dipolar couplings, a well resolved three-dimensional structure could be determined. The overall fold of the protein is not influenced by the mutation but characteristic conformational changes are introduced into the active-centre loop. They lead to a displacement of the ring system of His15 and a distortion of the N-terminus of the first helix, which supports the histidine ring. In addition, the C-terminal helix is bent because the side chain of Leu86 located at the end of this helix partly fills the hydrophobic cavity created by the mutation. Xenon, which is known to occupy hydrophobic cavities, causes a partial reversal of the mutation-induced structural effects. The observed structural changes explain the reduced phosphocarrier activity of the mutant and agree well with the earlier suggestion that Ile14 represents an anchoring point stabilizing the active-centre loop in its correct conformation. [ABSTRACT FROM AUTHOR]

The penicillin-binding proteins (PBPs) are ubiquitous bacterial enzymes involved in cell wall biosynthesis, and are the targets of the β-lactam antibiotics. The low molecular mass Neisseria gonorrhoeae PBP 4 (NG PBP 4) is the fourth PBP revealed in the gonococcal genome. NG PBP 4 was cloned, overexpressed, purified, and characterized for β-lactam binding, dd-carboxypeptidase activity, acyl-donor substrate specificity, transpeptidase activity, inhibition by a number of active site directed reagents, and pH profile. NG PBP 4 was efficiently acylated by penicillin (30 000 m−1·s−1). Against a set of five α- and ε-substituted l-Lys- d-Ala- d-Ala substrates, NG PBP 4 exhibited wide variation in specificity with a preference for Nε-acylated substrates, suggesting a possible preference for crosslinked pentapeptide substrates in the cell wall. Substrates with an Nε-Cbz group demonstrated pronounced substrate inhibition. NG PBP 4 showed 30-fold higher activity against the depsipeptide Lac-ester substrate than against the analogous peptide substrate, an indication that k2 (acylation) is rate determining for carboxypeptidase activity. No transpeptidase activity was apparent in a model transpeptidase reaction. Among a number of active site-directed agents, N-chlorosuccinimide, elastinal, iodoacetamide, iodoacetic acid, and phenylglyoxal gave substantial inhibition, and methyl boronic acid gave modest inhibition. The pH profile for activity against Ac2- l-Lys- d-Ala- d-Ala ( kcat/ Km) was bell-shaped, with p Ka values at 6.9 and 10.1. Comparison of the enzymatic properties of NG PBP 4 with other dd-carboxypeptidases highlights both similarities and differences within these enzymes, and suggests the possibility of common mechanistic roles for the two highly conserved active site lysines in Class A and C low molecular mass PBPs. [ABSTRACT FROM AUTHOR]

A large number of proteins, found experimentally to have different optimum pH of maximal stability, were studied to reveal the basic principles of their preferenence for a particular pH. The pH-dependent free energy of folding was modeled numerically as a function of pH as well as the net charge of the protein. The optimum pH was determined in the numerical calculations as the pH of the minimum free energy of folding. The experimental data for the pH of maximal stability (experimental optimum pH) was reproducible (rmsd = 0.73). It was shown that the optimum pH results from two factors – amino acid composition and the organization of the titratable groups with the 3D structure. It was demonstrated that the optimum pH and isoelectric point could be quite different. In many cases, the optimum pH was found at a pH corresponding to a large net charge of the protein. At the same time, there was a tendency for proteins having acidic optimum pHs to have a base/acid ratio smaller than one and vice versa. The correlation between the optimum pH and base/acid ratio is significant if only buried groups are taken into account. It was shown that a protein that provides a favorable electrostatic environment for acids and disfavors the bases tends to have high optimum pH and vice versa. [ABSTRACT FROM AUTHOR]

The C-5 of 5-aminolaevulinate, a tetrapyrrole precursor which accumulates when inhibitory laevulinate is present, is derived from either the C-2 of glycine by the 5-aminolaevulinate-synthase-mcdiated Shemin pathway or the C-1 of 2-oxogjutarate by the C5 pathway. Thin-layer-radiochromatographic procedures are described for determining whether [2-14C]glycine or 2-[1-14C]oxoglutarate labelled the macrocycle of bacteriochiorophyll a, in addition to or rather than the methyl ester or phytyl ester moieties of the side-chains. The method was also used for detecting whether the same substrates label the formaldehyde (C-5) or the succinate (Cal to C-4) fragments, obtained by periodate cleavage of 5-aminolaevulinate. These methods therefore can readily distinguish between the Shemin and C5 pathways as was demonstrated by using Rhodopseudomonas spherokks and Zea mays (maize), respectively, as examples of each pathway. Both [2-14C] glycine and, to a lesser extent 2-[l-14C]oxoglutarate Labelled the macrocycle of bacteriochiorophyll a formed during adaptation of respiring R. spheroides cells to photosynthetic (anaerobic, illuminated) conditions. This and earlier evidence suggested augmentation of the Shemin pathway by a minor C5 pathway contribution. The present studies revealed only Shemin pathway activity: with laevulinate present, [2-14C]glycine formed 5[5-14C]aminolaevulinate as proved by H14CHO production during periodate cleavage. These methods were sufficiently sensitive also to detect the incorporation of 14CO2, from degradation of either substrate, into 5-aminolaevulinate via the Shemin pathway thus labelling the succinate fragment produced with periodate: this explains bacteriochiorophyll a labelling by 2-[1-14C]oxoglutarate and proves double labelling of 5-aminolaevulinate by [2-14C]glycine. The same techniques were applied to etiolated maize leaves exposed to aerobic illuminated conditions with laevulinate and either 2-[1-14C]oxoglutarate or [2-14C]glycine as substrates. Only the C5 pathway was detected: 2[1l4CJoxoglutarate was converted to 5-[5-14C]aminolaevu1inate, which yielded H14CHO on periodate cleavage. This is not inconsistent with our earlier 13C-NMR studies [Porra, R. I., Klein, 0. and Wright, P. E. (1983) Eu,'. J. Biochetn. 130, 509- 516] showing that the C5 pathway formed all the 5-aminolacvulinate for chlorophyll biosynthesis. No conversion of [2-14C]glycine to 5-[5-14claminolaevulinate could be detected; thus, if 5-aminolaevlalinate synthase occurs in plant leaf mitochondria for mitochondrial haem formation it does so in quantities too small to be detected by these methods, or the expression of the gene for this Shemin pathway enzyme may be repressed in greening maize leaves. Absorption coefficients of the metbanolysis products of bacteriophaeophytin a and of phacophytins a and b are given for solutions in diethyl ether. [ABSTRACT FROM AUTHOR]

A novel enzyme, formaldehyde dismutase, was purified and crystallized from the cell extract of an isolated bacterium, Pseudomonas putida F61. The enzyme catalyzes the dismutation of aldehydes and alcohol : aldehyde oxidoreduction in the absence of an exogenous electron acceptor. The enzyme is composed of four identical subunits with a Mr of 44000. Each subunit contains 1 mol NAD(H) and 2 mol zinc/mol. The ratio of NAD+ and NADH in a crystalline preparation of the enzyme was about 7:3. The enzyme-bound coenzyme was completely reduced and oxidized on the addition of a large amount of an alcohol and an aldehyde respectively. Both the oxidized and reduced enzymes catalyzed the dismutation reaction to the same extent. Steady-state kinetics of the enzyme were investigated using an oxidoreduction reaction between an alcohol and p-nitroso-N,N-dimethylaniline. The enzyme obeys a ping-pong mechanism and is competitively inhibited by an alcoholic substrate analogue, pyrazole, but not coenzyme analogues, such as AMP, N-methylnicotinamide. These results indicate that NAD(H) binds firmly (but not covalently) at each active site. The enzyme-bound NAD(H) was reduced and oxidized only by the added second substrates, alcohol and aldehyde respectively, and not by exogenous electron acceptors [including NAD(H)]. [ABSTRACT FROM AUTHOR]

Experimental approaches to modelling the enzymatic function of biological membranes are discussed. Emphasis is given to pseudohomogeneous systems such as proteolipid complexes and enzymes in organic solvents; the latter are solubilized with phospholipids or synthetic surfactants. Methods for producing and studying such micellar systems are considered. The key research problems of micellar enzymology are formulated and its relation to enzyme membranology is discussed. Finally, the new potentialities are noted of applied enzymology (biotechnology) offered by application of a colloidal solution of water in organic solvents as a microheterogeneous medium for enzymatic reactions. [ABSTRACT FROM AUTHOR]

We have characterized the binding parameters of renal receptors (Scatchard analysis revealed the presence of two binding sites: site I, Ka1 = 1.29 X 109 M-1, number of binding sites = 9.9 X 106/μg protein; site II, Ka2 = 0.93 X 108 M-1, number of binding sites = 4.27 X 108/μg protein) and studied the effect of solubilization. The high-affinity sites are preserved during affinity chromatography and the process results in a 6080-fold purification of those sites. The lower-affinity sites are also preserved but the overall purification factor is about 40% lower than that obtained using molecular sieving. The purification of the renal calcitonin receptor by molecular sieving (Sephacryl S-200) is accompanied by total loss of the high-affinity site; however, the low-affinity site is enriched over 1642-fold. Binding parameters were obtained for the purified fractions. Synthetic salmon calcitonin was also bound to renal membranes using the bifunctional reagent disuccinimidyl suberate and photo-affinity cross-linking using hydroxysuccinimidyl azidobenzonate reagent. Cross-linked receptor eluted in the same volume as solubilized membranes specifically binding salmon calcitonin (S-200 chromatography). Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of purified fractions showed several protein bands with apparent molecular masses ranging from 18000 Da to 100000 Da in the presence or absence of a reducing agent (2-mercaptoethanol). Autoradiography of polyacrylamide gels of cross-linked calcitonin receptor showed only three protein bands specifically binding salmon calcitonin, Their molecular masses were 70000 Da, 40000 Da and 33000 Da respectively. The 40000-Da molecule represents a major band (47% total binding species). This suggests that these three proteins are the principal components of the calcitonin receptor and that S-S bonds are not involved in the assembly of the receptor subunits. [ABSTRACT FROM AUTHOR]

A cell-free system from the protozoon Tetrahymena pyriformis capable of cyclizing squalene into tetrahymanol cyclizes all-trans pentaprenyl methyl ether to a scalarane-type sesterterpene and all-trans hexaprenyl methyl ether to bicyclo-, tricyclo-, tetracyclo- and pentacyclohexaprenyl methyl ethers, each corresponding to a possible cationic intermediate. The structures of the cyclization products have been determined by spectroscopic methods and are compatible with a biogenetic scheme involving polyprenyl ether cyclization. This is the first direct proof of an enzymatic cyclization of higher isoprenic alcohol derivatives, and we assume it was performed by the squalene-to-hopane cyclase of the protozoon. The formation of a scalarane-type sesterterpene from C25 polyprenyl methyl ether suggests that these terpenoids, whose presence is restricted to a few sponges, might be in fact microbial metabolites. Tricyclopolyprenyl derivatives have been identified in the organic matter from numerous sediments and they were interpreted as being chemical fossils of still unidentified microorganisms. The cyclization of hexaprenyl methyl ether is the first attempt of identification of these tricyclopolyprenol derivatives in living organisms. [ABSTRACT FROM AUTHOR]

Carbohydrate moieties were released from human thyroglobulins prepared from normal and transformed, thyroid tissues by hydrazinolysis. The oligosaccharides thus prepared were fractionated by DEAE-cellulose, Bio-Gel P-4, concanavalin-A - Sepharose and Ricinus-communis-agglutinin- Sepharose columns and were characterized by exo-glycosidase and endo-glycosidase digestions, methylation analysis and 400-MHz 1H-NMR spectroscopy. These studies showed that the alterations accompanying malignancy occurred in complex type carbohydrate chains and exhibited the following structural features: (a) a complex-type carbohydrate chain containing I phosphate in a diester linkage was present in all the malignant thyroglobulins. (b) Asialo-carbohydrate chains with biosynthetically incomplete structures were found in all of the malignant thyroglobulins. (c) Carbohydrate chains of higher molecular mass, which have repeating Gal-GlcNAc disaccharides and peripheral α-fucosyl residues were present in metastatic papillary carcinoma. [ABSTRACT FROM AUTHOR]

Cytosolic glutamine synthetase (GS1) was purified to homogeneity from etiolated barley leaves by DEAE-Sephacci and hydroxyapatite chromatography, gel filtration and polyacrylamide get electrophoresis. Specific antibodies against the purified protein were raised by the immunization of rabbits. Immunoprecipitation experiments demonstrated that cytosolic glut a mine synthetases isolated from the leaves of different plant species were very similar proteins. Good recognition of other cytosolic glutamine synthetases from roots, root nodular tissue and seeds by barley GS1 antibodies was obtained, suggesting that they too are all quite similar proteins In contrast, chloroplast glut amine synthetase (GS2) was considered to be a different protein in view of its low level of recognition by barley GS1 antibodies. [ABSTRACT FROM AUTHOR]

In Salmonella minnesota lipopolysaccharide the lipid A backbone, a substituted diphosphorylated β, 6-linked D-glucosamine disaccharide molecule, carries approximately seven residues of fatty acids: one each of dodecanoic, hexadecanoic, n-3-hydroxytetradecanoic and D-3-O-(tetradecanoyl)-tetradecanoic acid in ester linkage and two of D-3-hydroxytetradecanoic acid in amide linkage. In the present study it is shown that treatment of the lipopolysaccharide with alkali at elevated temperature leads, through a β-elimination reaction, to the generation of amide- bound Δ2-tetradecenoic acid. This suggested that the 3-hydroxyl group of amide-bound hydroxy fatty acids carried a substituent. To elucidate the nature of the substituent, free Salmonella lipid A was methylated with methyl iodine in the presence of silver salts followed by mild acid hydrolysis, a procedure which is known to cleave amide (and not ester) bonds selectively. In the hydrolysate, by means of combined gas-liquid chromatography/mass spectrometry the methyl esters of 3-O-dodecanoyl)-tetradecanoic and 3-O-hexadecanoyl)-tetradecanoic acid were identified, This shows that in Lipid A amide-linked 3-hydroxytetradecanoic acid residues are 3-O-acylitted by dodecanoic and hexadecanoic acid, respectively. Quantitative analyses suggest that the Salmonella lipid A backbone is substituted by four D-3-hydroxytetra- decanoyl residues., two being present as esters and two as amicles. The nonhydroxylated fatty acids are not bound directly to the backbone. Rather, they are attached to hydroxyl groups of 3-hydroxytetradecanoyl residues: specifically, tetradecanoic acid substitutes ester-bound and dodecanoic and hexadecanoic acid amide-bound 3-hydroxytetradecanoic acid. [ABSTRACT FROM AUTHOR]

Anti-(P1-450) and anti-(P-448) are two antibodies developed against distinctly different Forms of 3-methyl- cholanthrene-induced cytochrome P-450 in C57BL/6N mouse liver microsomes [Negishi and Nebert, J. Blot C/win. 254, 11015-11023 (1979)]. The effects of these antibodies on long-Evans and Sprague-Dawley rat liver microsomes and rat `minimal deviation' hepatoma 7777 microsomes were studied. Rat microsomal aryl hydrocarbon hydroxylase activity, induced by polycyclic aromatic compounds, is preferentially inhibited by anti-(Pi-450) more so than anti-(P-448). Polycyclic-aromatic-induced rat liver microsomal acetanilide 4-hydroxylase activity is preferentially inhibited by anti-(P-448) more so than anti-(P,-450). Anti (P1 -450) precipitates a 56-kDa moiety in polycyclic aromatic-induced rat liver microsomes. Anti-(P-448) precipitates a 55-kDa moiety in control, phenobarbital-treated, or polycyclic-aromatic-induced rat liver microsomes. A heterogeneity was found amongst individual Morris `minimal deviation' hepatomas 7777. indicating the 3-metliylcholanthrene-induced aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase activities must represent different forms of rat P1-450. Clone 46 DNA, a portion of the mouse cloned P1-450 structural gene. hybridizes to one gene (or a small number of genes) in rat liver and to 3-niethylcholanthrene-induced rat 23-S P1-450 mRNA. As has already been shown in the mouse [Tukey, Nebert and Negishi, J. Biol. Chem. 256, 6969-6974 (1981)], rat liver P1-450 induction is under transcriptional control, and there is no evidence for gene amplification or some gross form of DNA rearrangement. These data demonstrate striking similarities between rat and mouse for the P1-450 structural gene, the P1-450 mRNA, the P1-450 protein and the P-448 protein. Polycyclic-aronlatic-induced P1-450, and its association with polycyclic-aromatic-induced aryl hydrocarbon hydroxylase activity, therefore does not appear to be closely correlated with polycyclic-aromatic-induced P-448 in either rat or mouse. [ABSTRACT FROM AUTHOR]

Sarcoplasmic reticulum Ca2+ -ATPase is slowly inactivated by the β,γ-bidentate of the chromium(llI) complex of ATP (CrATP). The rate of inactivation depends on the presence of calcium ions in the incubation medium. Half-maximal inactivation rate is reached at 80 nm Ca2+. The dissociation constant of enzyme-CrATP complex at 26 °C is 31 μM. Inactivation of the enzyme by CrATP is protected competitively by ATP. From this a dissociation constant of the enzyme ATP complex of 6.2 μM is calculated. The inactivation of Ca2+-ATPase is proportional to the incorporation of 32P from [γ-32P]CrATP into the 100 000-Mr protein. Concomitant with the inactivation there is also an incorporation of 45Ca2+ into the purified Ca2+-ATPase. A ratio of 45Ca2+ binding to phosphorylation of 1.7-1.9 is calculated from the capacities of both processes as well as from their kinetics. When Ca2+ uptake into sarcoplasmic reticulum is fuelled by ATP, treatment of the vesicles with the Ca2+ ionophore X-537-A or the production of leaks by adding deoxycholate leads to a loss of already accumulated 45Ca2+ Such treatment, however, leaves the 45Ca2+ in the vesicles of Ca2+ATPase unaffected, if ATP had been replaced by CrATP during the Ca2+ uptake. It is possible to localize 45Ca2+ together with the phospho- intermediate in the dodecylsulfate electrophoresis on the 100000-Mr protein and to a minor degree on the 200000-Mr dimer. This apparently occluded 45Ca2+ is rapidly lost by treatment of the enzyme with trichioro- acetic acid. It is concluded, that CrATP acts as an ATP analogue on Ca2+ATPase of sarcoplasmic reticulum. Hydrolysis of CrATP, as in (Na+ + K+)-ATPase [Pauls. H., Bresenbröcker, B., and Schoner, W. (1980) Eur. J. Biochem. 109, 523 - 533] leads to a stable phosphointermediate, which appears to bind 45Ca2+ in a stable occluded form. Both processes apparently lead to the suicide of the Ca2+-ATPase. [ABSTRACT FROM AUTHOR]

Aldehyde reductase I from pig liver is strongly inhibited by cyclized NADP-2-oxodiacid adducts. This result, in conjunction with those showing a strong inhibitory effect of certain diacid derivatives, such as (±)- dimethylsuccinic acid, towards aldehyde reductase I, suggests the presence of two anion sites in or near the substrate binding site. As previously shown by our group [Eur. J. Biochem. 116, 505 - 512 (1981)], at least one of the two anion sites is an arginine residue. The fact that (2R,3R)-dimethylsuccinic acid is bound more tightly to aldehyde reductase I than the meso and (2S,3S)-dimethylsuccinic acids is indicative of a particular spatial orientation of the two anion sites relative to those of the coenzyme and substrate binding sites of the reductase. This result also supports the idea that one of the two diastereomers of the cyclized adducts, corresponding to the A-side addition product, should exert a stronger inhibitory effect on aldehyde reductase than the other. On the basis of the NMR study made recently by L. J. Arnold et al. [Biochemistry, 18, 2787-2793 (1979)], who determined the conformation and configuration of cyclized pyridine nucleotide adducts, a tentative design of the hydrophobic pocket of the substrate binding site of aldehyde reductase I, containing the two anion sites, is presented. [ABSTRACT FROM AUTHOR]

Protein-lipid complexes were transferred directly from mitochondria and submitochondrial particles into hexane and ether. The protein-lipid residue left after solvent removal from these extracts was used to form liposomes which display low-temperature-resistant ATPase activity. Centrifugation experiments indicate that the ATPase activity is associated to the vesicles. Most of the F1-ATPases appear to be accessible to the external water phase of the liposomes. The ATPase activity of these particles was insensitive to dicyclohexylcarbodiimide and oligomycin. Incubation of these vesicles at room temperature activated (4 - 10-fold) the ATPase through a process that is partially sensitive to phenylmethylsulfonyl fluoride. The results with purified ATPase-inhibitor protein and (F1- ATPase)-inhibitor complex indicate that the activation process in the liposomes is due to the abolition of the inhibitory action of the inhibitor protein bound to a large fraction of the extracted ATPases. Liposomes prepared from hexane extracts obtained from submitochondrial particles having different levels of ATPase activity displayed an activation ratio which correlated with the number of ATPases that are inhibited by the inhibitor protein in the submitochondrial particles. The extraction of mitochondrial ATPase and its incorporation into liposomes followed by activity measurements may be used to judge the number of ATPases that in a given preparation contain the inhibitor protein in its inhibiting site. [ABSTRACT FROM AUTHOR]

Relationships between nitrogenase activity and individual components of the proton electrochemical potential gradient (ΔμH) in Anabaena variabilis have been investigated. The ionophore nigericin was found to collapse ΔpH-in favour of the membrane potential (Δψ); hyperpolarization of Δψ was correlated with an increase in nitrogenase activity. A positive relationship between nitrogenase activity and membrane potential was also observed using the ionophore valinomycin and the uncoupler carbonylcyanide m-chlorophenylhydrazone. Furthermore, using the energy transfer inhibitor N,N′-dicyclohexylcarbodiimide, nitrogenase activity appeared to be limited by the supply of reductant rather than ATP. The data suggest, for intact filaments and isolated heterocysts, that Δψ may be important in regulating nitrogenase activity in vivo. [ABSTRACT FROM AUTHOR]

A number of biomolecules were coupled covalently by nucleophilic displacement to agarose preparations substituted with tosyl groups. In one series of experiments 6-(6-aminohexyl)-adenosine 5ʹ-monophosphate and 6-(6-aminohexyl)adenosine-2ʹ,5ʹ bisphosphatewere bound by their terminal amino groups to the polysaccharide support. It could be shown that from a mixture of lactate and 6-phosphogluconate dehydrogenase the immobilized monophosphate showed bio-affinity only for NA +-dependent lactate dehydrogenase, whereas the immobilized bisphosphate showed affinity only for the NADP+-dependent 6 phosphogluconate dehydrogenase. Furthermore, the immobilized monophosphate (5 μmol/g wet gel) was applied for the single-step purification of lactate dehydrogenase from crude beef heart extract. To demonstrate the immobilization of proteins, soybean trypsin inhibitor (75 mg/g dry support) was immobilized to tosylated agarose, tested as affinity chromatography material and shown to bind 60 mg trypsin/g dry gel. Horseradish peroxidase and horse liver alcohol dehydrogenase were used as model enzymes. Although no optimization had been attempted, the former (approximately 70 mg/g dry support) had a coupling yield of approximately 18% with a specific activity (relative to soluble enzyme) of approximately 10% whereas approximately 60% of alcohol dehydrogenase was couple (approximately 100 mg/g dry support) with a specific activity of approximately 25%. [ABSTRACT FROM AUTHOR]

p-Amidinophenyl esters of an enantiomeric pair of N-(2,4-dinitrophenyl)alanine (N2Ph-Ala) were both efficiently hydrolyzed by trypsin. The acylation and deacylation rate constants for the D-isomer are 1/2.5 of those for the L-isomer. Slow rates of deacylation of the two substrates made is possible to prepare the pair of enantiomeric acyl-trypsins. Circular dichroic (CD) spectra of the purified acyl-typsins revealed that the two extrinsic chromophores are somewhat differently oriented in the chiral environment of the active site, although both chromophores could couple intermolecularly with similar intrinsic chromophores near the active site. When p-amidinophenol was added, not only was the deacylation rate of N2Ph-DAla-trypsin noticeably increased, but also the transient CD sprectrum of the enzyme derivative changed markedly in comparison with that of the L-derivative. The observations indicate that the two enantiomeric acyl groups at the active site are situated in different microenvironments. [ABSTRACT FROM AUTHOR]

The proton and charge stoichiometry of cytochrome c oxidase reconstituted into phospholipid vesicles has been analysed using a fast responding oxygen electrode and a system for the simultaneous measurement of H-, K+ or lipophilic cations, and O2. From initial rate measurements after addition of reductant (ascorbate,′′N,N,N′,N′-tetramethyl-p-phenylenediamine) the following stoichiometries could be extrapolated: K+/e-, between 1.85 and 2.05, H+/e-, between 0.58 and 0.75. Lipophilic cations can replace valinomycin potassium as the charge compensating system. Net H+ extrusion was observed for up to 11 turnovers of the enzyme (11 O2/cytochrome aa3). The variation of the internal pH buffering capacity of the vesicles has an influence on the number of turnovers during which net proton translocation can be observed, but has no influence on the determined stoichiometries. [ABSTRACT FROM AUTHOR]

Presents a study concerning the recommendations for the nomenclature of tetrapyrroles. Basis for the development of a semisystematic tetrapyrrole nomenclature on few trivial names selected mainly from the Fischer system; Establishment of international rules for naming organic compounds and natural products; Designation given to compounds closely related to the trivially named porphyrins of Rule TP-2.

When calf lens crystallin mRNA and rabbit globin mRNA are competing for factors limiting protein synthesis in living Xenopus oocytes, no mRNA species is preferentially selected for translation. Differences in the intrinsic translational efficiency of the mRNA species exist, but the relative efficiencies are the same at high and low mRNA concentrations. mRNAs already being translated, in particular endogenous oocyte mRNAs, are less sensitive to competitive inhibition by injected mRNAs. As injected mRNAs gradually become incorporated into the protein-synthesizing machinery of the oocyte, they acquire the same status as the oocyte's own active mRNAs. Exogenous mRNAs thus become endogenous mRNAs. These results, together with previous estimates of the translational efficiency of injected heterologous mRNA species, are compatible with the assimiption that a large proportion of the endogenous mRNAs is not competing for the translational apparatus of the oocyte and, therefore, probably is present in a temporarily inactivated form. [ABSTRACT FROM AUTHOR]

Methyl α-D-glucoside and α,α'-D-trehalose acylated on their primary hydroxyl group by α-alkyl-β-hydroxy acids (mycolic acids) with 18, 32 and more than 80 carbon atoms were studied. These cord factors and analogues when dispersed in water gave hydrated and organized phases which bore resemblances and differences to phospholipid liposomes. Phase transition temperature was determined by fluorescence for the C32 derivatives and by differential scanning calorimetry for the C80 derivatives. A narrow temperature interval between the gel and the fluid states was observed, indicating that this transition was a strongly cooperative phenomenon. The activity on mitochondria of these diacyl trehaloses was studied with succinate as substrate. The fluid suspensions were more active than the rigid ones. There was no influence of the relative configuration of the mycoloyl residue. Acyl chains with 18, 32 and more than 80 carbon atoms had rather similar activities on phosphorylation, but differences appeared when considering effects on respiration. Electron microscopy suggested that cord factor weakened the peripheral part of the inner membrane, but cristae looked unchanged. These observations, together with the results of our previous monolayer studies of these glycolipids, led us to propose a model for interaction of cord factor with mitochondria. [ABSTRACT FROM AUTHOR]

UDP-galactose : N-acetylglucosamine galactosyltransferase was isolated from pooled human milk, pooled amniotic fluid and from two different individual samples of malignant ascites. The purification procedure involving two successive affinity chromatography steps on N-acetylglucos- amine - agarose and -lactalbumin - agarose yielded an enzyme preparation homogeneous by size. Under non-denaturing conditions the ascites and amniotic fluid enzymes had identical electrophoretic mobility, but they moved laster than the milk enzyme. Isoelectric analysis in the presence and absence of urea resolved the milk enzyme into at least 13 different forms, nine of which had the same isoelectric points after refocusing. All enzyme forms showed similar activity when free N-acetyl- glucosamine, ovalbumin. sialic-acid-free ovine submaxillary mucin and glucose, in the presence of a-lactalbumin, were used as acceptor substrates. Comparative isoelectric focusing of the three galactosyltransferases revealed identical patterns of the amniotic and ascites enzymes, but only partial overlap with the milk enzyme, which was less negatively charged. Neuraminidase treatment of ascites and milk galactosyltransferases produced very similar focusing patterns. The possible structural basis for this charge heterogeneity is briefly discussed. [ABSTRACT FROM AUTHOR]

C-6 esters of methyl α-D-glucoside and C-6, C-6' diesters of α,a'-D-trehalose with C18 and C32 threo and erythro mycolic acids (from chemical source) and of C80-erythro-mycolic acid(from natural source) have been synthesized. Esters of a C32 deoxy analogue were prepared as well. Throughout a monolayer study at the air-water interface, these glycolipids are shown to form well organized phases in which the two hydrocarbon chains of mycoloyl residues must be in interaction. Compression isotherms of C32 esters suggested a transition between liquid-expanded and liquidcondensed states. Latent heats Qc and entropy changes Δ S associated with these phase transitions as well as the critical temperature at which they occur have been measured. Within the monolayer, the molecular packing of these glycolipids depends on the presence of the hydroxyl group of mycoloyl residues and on its stereochemistry. In particular, intermolecular hydrogen bonds between these groups are postulated in the case of the bis(C32-erythro-mycoloyl)-trehalose. On the other hand, short chain C18 esters form fluid phases (t > 10°C) whereas very long chain C80 mycoloyl esters of trehalose exist in a condensed state (t = 20 °C). These glycolipids were found to interact strongly with dipalmitoylphosphatidylcholine and egg yolk lecithins (3-sn-phosphatidylcholine). Their phase behaviours are discussed in connection with hypotheses concerning the way they can interact with mitochondrial membranes. [ABSTRACT FROM AUTHOR]

About 50% of the carbohydrate moiety of ovomucoid was destroyed by periodate oxidation. The oxidation was carried out for 6 h or 24 h. The data obtained showed that in the carbohydrate chain 2–5 glucosamines and 1 – 2 neutral sugar residues were decomposed with the consumption of 16 mol and 29 mol of periodate respectively. Periodate oxidation slightly changed the inhibitory activity of the ovomucoid, but altered its spectral properties. An increase of the absorption maximum at 278 nm was noted, as well as a tendency for normalization of phenolic ionization and an increase of the relative fluorescence. The reactivity of tyrosine residues towards tetranitromethane is also changed. It was suggested that even in native ovomucoid the tyrosines could be regarded as ‘dissolved’ in the ‘carbohydrate solvent’. This contact could be achieved by the hydrogen bonds in the formation of which the NHCOCH3 groups of the glucosamine residues play an essential role. Peroxidate oxidation seems to lead to an alteration of the nature of the ‘sugar solvent’ and disturbs the conformation of the sugar chain. [ABSTRACT FROM AUTHOR]

Ceruleotoxin, a toxin component of Bungarus caeruleus venom, blocks in vivo the depolarisation caused by carbamyicholine on the isolated electroplaque from Electrophorus electricus and in vitro the increase of 22Na+ and 42K+ efflux caused by cholinergic agonists on excitable receptor-rich microsacs prepared from Torpedo marmorata electric organ. However, at variance with the typical α-toxins, ceruleotoxin does not interfere with the binding of [³H]acetylcholine and of α-[³H]toxin to the cholinergic receptor site. Ceruleotoxin also slightly modifies 35SO42- and [14C]glucose efflux. Some of its effects on the postsynaptic membrane may be related to the presence of a phospholipase activity present in the preparation and are discussed in those terms. It is proposed that ceruleotoxin selectively blocks the electrogenic action of acetylcholine on the postsynaptic membrane without binding to the cholinergic receptor site, and might serve as an useful tool to characterise the ion translocating device, the ionophore, associated with the cholinergic receptor site. [ABSTRACT FROM AUTHOR]

The calcium accumulated by the fast uptake has an apparent association constant of 0.8 × 106 M-1 and a maximum of 80 nmol/mg protein. The fast uptake and the initial rate of the slow uptake show a similar dependence on the calcium concentration when the latter ranges from 5 to 50 μM. The fast uptake is a linear and the slow uptake rate an exponential function of the reticulum concentration. Both uptakes of calcium display a fast and nearly total isotopic equilibration between intra- and extravesicular calcium. After depletion of ATP the calcium accumulated during the slow uptake is released, while that sequestered during the fast uptake is retained by the vesicles, though it remains rapidly exchangeable. After depletion of ITP or acetylphosphate, or addition of EGTA, the release is more substantial, but is almost complete only after addition of ionophore X 537A or deoxycholate. The presence of oxalate strongly reduces the rates of these releases. It is concluded that in the steady state observed after the depletion of ATP, a Ca2+ gradient exists through the sarcoplasmic membrane, and the sarcoplasmic pump works at equilibrium. The fast uptake is an active transport and not an active binding. The slow uptake represents an extension of the calcium capacity of the vesicles due to the phosphate liberated by the sarcoplasmic ATPase. [ABSTRACT FROM AUTHOR]

Functional arginyl residues of porcine carboxypeptidase B were modified with phenylglyoxal, decreasing activity towards peptide substrates and the ester hippurylphenyllactate while enhancing activity towards the ester hippurylargininic acid. Both affinity chromatography on Sepharose-6aminohexanoyl-D-arginine and analysis of the chymotryptic peptides from the modified enzyme reveal heterogeneity in the modification. At least two arginyl, residues are modified, and the sequences of the purified chymotryptic peptides containing these residues are Glu-Leu-Arg-Asp-Lys-Gly-ArgTyr and His-Ala-Arg-Glu-Trp. These peptides are homologous with the peptides containing Arg-276 and Arg-71 of carboxypeptidases A and B, but are not homologous to the sequence around Arg-145 in that enzyme. A tentative explanation is provided for the effect of modification of these residues on the activity of carboxypeptidase B. [ABSTRACT FROM AUTHOR]

Proton magnetic resonance, circular dichroism and other studies of whole and cleaved calf thymus histone H1 (formerly F1) reveal the presence of specific folded structures in the region approximately from residue 40-115. Ionic, hydrogen-bond and hydrophobic interactions all appear to contribute to the stability of the structure, which is predicted to contain a-helices in regions 42-55 and 58-75. No evidence was found for β-structures, either inter or intramolecular, or for any structure formation outside the region 40-115. At 18 °C and a protein concentration of 2 mM the first-order exchange rate between random-coil and structured forms is slower than 80 s-1; at 40°C the exchange rate is faster than 330 s-1. [ABSTRACT FROM AUTHOR]

Limited tryptic digestion of native sucrase. isomaltase complex produced a more rapid destruction of isomaltase activity than sucrase activity. It was possible to isolate a partially fragmented sucrase subunit in high yields with a specific activity twice that of the native complex. Amino acid and carbohydrate analyses are reported and compared with the results obtained for sucrase isomaltase complex and isomaltase subunit obtained by a different method. [ABSTRACT FROM AUTHOR]

Purple acid phosphatase of the common bean Phaseolus vulgaris (KBPase), a dimeric 110-kDa glycoprotein related to the mammalian purple acid phosphatases with a two-metal cluster at the active site contains five oligosaccharide side chains/monomer. The N-linked glycan structures were characterized by selective enzymic degradation in combination with matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS). The purified protein was cleaved by cyanogen bromide. One 30-kDa large methionine-free fragment required a further tryptic digest. The peptides were separated by HPLC and the glycosylated species were identified both by their heterogeneous mass spectra and by an immunoassay. None of the glycopeptides proved to have more than one glycosylation site. The composition of the carbohydrate moieties were calculated by comparing the mass spectra of the glycopeptides before and after enzymic deglycosylation. These results were complemented by data from a carbohydrate composition analysis. In four of the five peptides an al-3 fucose attached to the asparagine-linked N-acetylglucosamine prevented removal of the glycan by peptide N-glycosidase F: peptide N-glycosidase A removed all carbohydrates from the peptides. To reveal the sequence of the carbohydrate moiety including the linkage positions between the different saccharides, one of the glycopeptides was degraded by specific exoglycosidases. The enzymic degradations by these hydrolases were monitored by mass spectrometry of small aliquots taken at intervals during the reaction. The detailed structure of this one glycan in conjunction with the respective mass spectra and the composition analysis were used to infer the structure of the other four glycans. All glycans of the KBPase have a complex-type xylose-containing structure with four of the five having an additional fucose. [ABSTRACT FROM AUTHOR]