Fractionation of yeast invertase isozymes and determination of enzymatic activity in sodium dodecyl sulfate-polyacrylamide gels

Three invertase isozymes extracted from derepressed yeast cell membranes with 1% deoxycholate (0.5 mg/mg protein) were separated on sodium dodecyl sulfate (0.25%)-polyacrylamide gels (Babczinski, P., and Tanner, W., 1978, Biochim. Biophys. Acta538, 426–434; Babczinski, P., 1980, Biochim. Biophys. Acta, in press). A dye-producing method for the location of isozymes after gel electrophoresis has been developed by using a coupled enzymatic reaction including the formazan reaction. By the incorporation of β-d-glucose dehydrogenase as the d-glucose-utilizing auxiliary enzyme into the test system the problem of contaminating sucrase activities in commercial glucose oxidase preparations has been overcome (the latter enzyme had often been used earlier in activity-staining procedures of glycosidases). Production of formazan dye is dependent on the presence of sucrose in the test system. By quantitative densitometry proportionality of dye intensity with incubation time, amount of deoxycholate extract, and amount of protein applied onto the gel were determined.     

Problems associated with models of cytoplasmic streaming in capillary tubes

Streaming of actomyosin solutions observed in glass capillary tubes has been attributed to the energy-tranducing interaction of actin and myosin in the presence of ATP (A. Oplatka and R. Tirosh, Biochim. Biophys. Acta 1973, 305, 684–688). Other results have indicated that this is a physical phenomenon of the system used (Y. Matsutake, J. Sagara, T. Yamada, and H. Shimizu, Biochim. Biophys. Acta 1975, 405, 347–352). Results presented here using a microcapillary system relatively free of artifacts confirm the indication that the structure of the capillary system used is responsible for the appearance of streaming in the solutions studied and further delineate the nature of the phenomenon.     

A simple micro-assay for inorganic phosphate

Modification of two assay procedures (Van Belle, H. (1970) Anal. Biochem.33, 132–142 and Itaya, K., and Ui, M. (1966) Clin. Chim. Acta14, 361) has allowed the development of a manual assay for inorganic phosphate of high simplicity and sensitivity. Total analysis requires only three reagents and is accomplished in less than 5 min, and smaples containing less than 1 μg/ml of inorganic phosphate may be detected. This assay retains a unique principle of the former two, complexation (instead of reduction) of the phosphomolybdate heteropoly complex with an appropriate triphenylmethane dye (malachite green, methyl green). Use of detergents has been eliminated and some further properties of the dyes, the assay, and the latter's applicability to a coupled enzyme system for phosphomonoester and phosphodiester analysis are discussed. Consideration is also given to the associated phenomena of transphosphorylation. (Dayan, J., and Wilson, I. B. (1964) Biochim. Biophys. Acta81, 620–623).     

Replacement of ethanolamine phosphate by 3-aminopropylphosphonate in the phospholipids of Tetrahymena

When Tetrahymena thermophila is grown on a medium containing up to 5 mm 3-aminopropylphosphonate, up to 90% of the ethanolamine phosphate in phosphatidylethanolamine is replaced by the 3-aminopropylphosphonate. No accompanying alteration of the phospholipid composition of Tetrahymena is observed. This contrasts with the results obtained when 2-aminoethylphosphonate, the naturally occurring compound, is added to the growth medium (Biochim. Biophys. Acta528, 394–398, 1978); the 2-aminoethylphosphonate causes a substantial increase in the 2-aminoethylphosphonolipid and a reciprocal decrease in phosphatidylethanolamine. Thus, there is apparently a one-way control system in Tetrahymena whereby 2-aminoethylphosphonate and its phosphonolipid may influence the level of phosphatidylethanolamine in the cell, but ethanolamine phosphate, as represented by its isosteric analog, does not influence the phospholipid levels. There is no effect of the 3-aminopropylphosphonate on de novo 2-aminoethylphosphonate biosynthesis indicating a strict specificity for 2-aminoethylphosphonate as its own feedback inhibitor.     

Light-induced oxygen reduction as a probe of electron transport between respiratory and photosynthetic components in membranes of Rhodopseudomonas capsulata

Membrane vesicles prepared from photosynthetically grown cells of Rhodopseudomonas capsulata strain M7 are able to perform light-induced oxygen uptake. In contrast, chromatophores from mutant strain M6, lacking the cytochrome b₂₆₀-containing pathway of oxygen reduction, are completely devoid of oxygen uptake induced by light. Therefore, cytochrome b₂₆₀ (cyt b₂₆₀), previously demonstrated to be the alternative oxidase in photosynthetic and aerobic membranes of R. capsulata (La Monica, R. F., and Marrs, B. L. (1976) Biochim. Biophys. Acta449, 431–439; Zannoni, D., Melandri, B. A., and Baccarini-Melandri, A. (1976) Biochim. Biophys. Acta449, 386–400), also appears to be involved in light-induced oxygen uptake. Light-driven oxygen reduction activity is inhibited by high concentrations of cyanide (5 × 10^?3m) and by carbon monoxide in accord with the sensitivity of cyt b₂₆₀ to these inhibitors reported in aerobic membranes (Zannoni, D., Melandri, B. A., and Baccarini-Melandri, A. (1976) Biochim. Biophys. Acta423, 413–430). Further evidence for a direct involvement of the cyt b₂₆₀-containing pathway in light-induced oxygen uptake has been obtained by comparing membranes from semiaerobically grown cells to those from photosynthetic cells of R. capsulata M7. The substrate-dependent dark oxidase activity associated with the cyt b₂₆₀-containing pathway is 4.6 times higher in semiaerobic than in photosynthetic membranes, and a parallel enhancement of light-induced oxygen uptake is observed. The data presented are in agreement with and extend previous results on the composition and function of the respiratory and photosynthetic apparatus, supporting an association of cytochrome b₂₆₀ with both the aerobic and photosynthetic systems present in membranes of R. capsulata. These findings clearly demonstrate that respiratory electron carriers have access to electrons flowing from the photosynthetic reaction center, i.e., the two systems are “electrically connected” in membrane fragments from this organism.     

Recognition intensities of submolecular structures,mammalian glyco-structural units,ligand cluster and polyvalency in abrin-a-carbohydrate interactions

Abrin-a is the most toxic fraction of lectins isolated from Abrus precatorius seeds and belongs to the family of type 2 ribosome inactivating proteins (RIP). This toxin may act as a defense molecule in plants against viruses, fungi and insects, where attachment of abrin-a to the exposed glycans on the surface of target cells is the crucial and initial step of its cytotoxicity. Although it has been studied for over four decades, the recognition factors involved in abrin-a-carbohydrate interaction remains to be clarified. In this study, roles of mammalian glyco-structural units, ligand clusters and polyvalency in abrin-a recognition were comprehensively analyzed by enzyme-linked lectinosorbent binding and inhibition assays. The results indicate that: (i) this toxin prefers oligosaccharides having α-anomer of galactose (Gal) at the non-reducing terminal than the corresponding β-anomer; (ii) Galα1-3Galα1- (B_α), Galα1-4Gal (E), Galβ1-3GalNAc (T) and Galβ1-3/4GlcNAc (I/II) related oligosaccharides were the active glyco-structural units; (iii) tri-antennary II_β, prepared from N-glycan of asialo fetuin, played a dominant role in recognition; (iv) many high-density polyvalent I_β/II_β and E_β glycotopes enhanced the reactivity; (v) the carbohydrate recognition domain of abrin-a is proposed to be a combination of a small cavity type of Gal as major site and a groove type of additional one to tetrasaccharides as subsites with a preference of α1-3/4/6Gal, β1-3GalNAc, β1-3/4/6GlcNAc, β1-4/6Glc, β1-3DAra and β1-4Man as subterminal sugars; (vi) size of the carbohydrate recognition domain may be as large enough to accommodate a linear pentasaccharide and complementary to Galα1-3Galβ1-4GlcNAc β1-3Galβ1-4Glc (gailipenta) sequence. A comparison of the recognition factors and combining sites of abrin-a with ricin, another highly toxic lectin, was also performed to further understand the differences in recognition factors between these two type 2 RIPs.     

A simple statistical method for use in kinetic analysis based on Lineweaver-Burk plots

Statistical methods for the analysis of initial-velocity and/or inhibition data are described. They involve application of F tests (i) to determine goodness of fit to the first-order Michaelis-Menten equation, (ii) to predict the reaction mechanism by assessing slope and y-intercept effects in Lineweaver-Burk plots according to the inspection rules of Cleland [Cleland, W. W. (1963) Biochim. Biophys. Acta67, 188–196], (iii) to test the linearity of the replots of slopes or y-intercepts versus the reciprocal of the substrate concentration or the inhibitor concentration, and (iv) to estimate the true K_m or K_i values from these replots. The method serves to fill a gap in the kinetic analysis methodology between the antiquated graphical method and the sophisticated direct computer-fitting of data to a variety of possible rate equations. The entire theoretical and computational format is provided to allow the investigator to apply these statistical tests to his data using only a desk-top calculator.     

Proteolysis in eucaryotic cells: Aminopeptidases and dipeptidyl aminopeptidases of yeast revisited

Using nine different l-aminoacyl-4-nitroanilides and four different dipeptidyl-4-nitroanilides, aminopeptidases and dipeptidyl aminopeptidases active at pH 7.5 and (or) pH 5.5 in logarithmically growing and stationary-phase cells of Saccharomyces cerevisiae were searched for. Ion-exchange chromatography was used to separate the proteins of the soluble cell extract. Besides the three already-characterized aminopeptidases—aminopeptidase I (P. Matile, A. Wiemken, and W. Guyer (1971) Planta (Berlin)96, 43–53; J. Frey and K. H. Röhm (1978) Biochim. Biophys. Acta527, 31–41), aminopeptidase II (J. Frey and K. H. Röhm (1978) Biochim. Biophys. Acta527, 31–41; J. Knüver (1982) Thesis, Fachbereich Chemie, Marburg, FRG), and aminopeptidase Co (T. Achstetter, C. Ehmann, and D. H. Wolf (1982) Biochem. Biophys. Res. Commun.109, 341–347)—12 additional aminopeptidase activities are found in soluble cell extracts eluting from the ion-exchange column. These activities differ from the characterized aminopeptidases in one or more of the parameters such as charge, size, substrate specificity, inhibition pattern, pH optimum for activity and regulation. Also, a particulate aminopeptidase, called aminopeptidase P, is found in the nonsoluble fraction of disintegrated cells. Besides the described particulate X-prolyl-dipeptidyl aminopeptidase (M. P. Suarez Rendueles, J. Schwencke, N. Garcia-Alvarez and S. Gascon (1981) FEBS Lett.131, 296–300), three additional dipeptidyl aminopeptidase activities of different substrate specificities are found in the soluble extract.     

On “Entropy consumption in primary photosynthesis” by Jennings et al.

The preceding paper [R.C. Jennings, E. Belgio, A.P. Casazza, F.M. Garlaschi, G. Zucchelli, Entropy consumption in primary photosynthesis, Biochim. Biophys. Acta (in press)] is criticized on several grounds.     

Multivalent human blood group ABH and Lewis glycotopes are key recognition factors for a lFuc>Man binding lectin from phytopathogenic Ralstonia solanacearum

Ralstonia solanacearum lectin (RSL), that might be involved in phytopathogenicity, has been defined as lFuc?Man specific. However, the effects of polyvalency of glycotopes and mammalian structural units on binding have not been established. In this study, recognition factors of RSL were comprehensively examined with natural multivalent glycotopes and monomeric ligands using enzyme linked lectin-sorbent and inhibition assays. Among the glycans tested, RSL reacted strongly with multivalent blood group A_h (GalNAcα1–3[Fucα1–2]Gal) and H (Fucα1–2Gal) active glycotopes, followed by B_h (Galα1–3[Fucα1–2]Gal), Le^a (Galβ1–3[Fucα1–4]GlcNAc) and Le^b (Fucα1–2Galβ1–3[Fucα1–4]GlcNAc) active glycotopes. But weak or negligible binding was observed for blood group precursors having Galβ1–3/4GlcNAcβ1- (I_β/II_β) residues or Galβ1–3GalNAcα1- (T_α), GalNAcα1-Ser/Thr (Tn) bearing glycoproteins. These results indicate that the density and degree of exposure of multivalent ligands of α1–2 linked lFuc to Gal at the non-reducing end is the most critical factor for binding. An inhibition study with monomeric ligands revealed that the combining site of RSL should be of a groove type to fit trisaccharide binding with highest complementarity to blood group H trisaccharide (H_L; Fucα1–2Galβ1–4Glc). The outstandingly broad RSL saccharide-binding profile might be related to the unusually wide spectrum of plants that suffer from R. solanacearum pathogenicity and provide ideas for protective antiadhesion strategies.     

Vanadate affects glucose metabolism of human erythrocytes

Vanadate causes a rapid breakdown of 2,3-bisphosphoglycerate in intact erythrocytes. This metabolite is nearly stoichiometrically transformed into pyruvate, which changes the cell redox state and enhances the glycolytic flux. The results show that the vanadate effect on 2,3-bisphosphoglycerate, also evident in hemolysates, is attributable to the stimulation of a phosphatase activity of the phosphoglycerate mutase. In agreement with others (J. Carreras, F. Climent, R. Bartrons and G. Pons (1982) Biochim. Biophys. Acta705, 238–242), vanadate is thought to destabilize the phosphoryl form of this enzyme which shows competitive inhibition between the ion and 2,3-bisphosphoglycerate in the mutase reaction. A competitive inhibition between vanadate and glucose 1,6-bisphosphate is also found for phosphoglucomutase, without evidence for phosphatase activity toward the bisphosphate cofactor.     

Mechanism of binding of the competence substance to the cell wall receptor sites

The induction of competence by the competence substance is strongly inhibited in the presence of phytohemagglutinins. A great inhibition effect is also found when the competence substance is preincubated with some amino sugars. Both the phytohemagglutinins and the competence substance exhibit a similar binding affinity to cross-linked dextran (Sephadex) and to the cell surfaces, as both are inhibited by some sugars in their interaction with the sensitive cells. It is proposed that they are bound to similar or identical cell receptor sites. These seem to be some specific sugar molecules forming a part of cell wall structures of the transformable and to competence inducible bacterial strains. This paper is part XIV of the series “Studies on phytohemagglutinins”; part XIII:Biochim. Biophys Acta,304, 93 (1973)     

Synthetic studies on the carbohydrate moiety of the antigen from the parasite Echinococcusmultilocularis

Stereocontrolled syntheses of branched tri-, tetra-, and pentasaccharides displaying a Galβ1→3GalNAc core in the glycan portion of the glycoprotein antigen from the parasite Echinococcusmultilocularis have been accomplished. Trisaccharide Galβ1→3(GlcNAcβ1→6)GalNAcα1-OR (A), tetrasaccharide Galβ1→3(Galβ1→4GlcNAcβ1→6)GalNAcα1-OR (D), and pentasaccharides Galβ1→3(Galβ1→4Galβ1→4GlcNAcβ1→6)GalNAcα1-OR (E) and Gal β1→3(Galα1→4Galβ1→4GlcNAcβ1→6)GalNAcα1-OR (F) (R = 2-(trimethylsilyl)ethyl) were synthesized by block synthesis. The disaccharide 2-(trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl-(1→3)-2-azido-4-O-benzyl-2-deoxy-α-d-galactopyranoside served as a common glycosyl acceptor in the synthesis of the branched oligosaccharides. Moreover, linear trisaccharide Galβ1→4Galβ1→3GalNAcα1-OR (B) and branched tetrasaccharide Galβ1→4Galβ1→3(GlcNAcβ1→6)GalNAcα1-OR (C) were synthesized by stepwise condensation.     

Development of the cation-induced stacking capacity during the biogenesis of higher plant thylakoids

We studied the capacity of the thylakoid membrane to form grana stacks in the presence of cations, monovalent or divalent, added to N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine “low-salt” disorganized plastids during their greening. Grana stacking was monitored by the yield of heavy subchloroplast fractions separated by differential centrifugation after digitonin disruption of plastids (J. H. Argyroudi-Akoyunoglou, 1976, Arch. Biochem. Biophys., 176, 267–274). Primary thylakoids of the agranal protochloroplasts formed in periodic light do not show the cation-induced stacking capacity of the mature green chloroplast thylakoids. Similarly, the cation effect saturates at lower cation concentrations in mature chloroplasts than in plastids of the early stages of greening. The capacity for cation-induced stacking and for saturation of the effect at low cation concentrations appears gradually after exposure to continuous light and parallel to the appearance of chlorophyll b and the polypeptides of the 25,000–30,000 molecular weight range of lipid-free thylakoids, probably derived from the chlorophyll b-rich chlorophyll protein Complex II. The thylakoid peripheral stroma proteins ribulosediphosphate carboxylase and the coupling factor protein are not involved in the cation-induced stacking, since their removal (H. Strotmann, H. Hesse, and K. Edelmann, 1973, Biochim. Biophys. Acta, 314, 202–210) does not affect the thylakoid aggregation.     

Changes in oxygen evolution induced by a long preillumination at 650 nm with Chlorella pyrenoidosa

The relationship between the rate of O₂ evolution and the corresponding concentration of System II active centers E has been studied in “State 1” and in “State 2”. No variation of α (fraction of absorbed light captured by System II) could be observed. The phenomena shown by Bonaventura and Myers (Biochim. Biophys. Acta, 189 (1969) 366) may be interpreted by an increase of the apparent equilibrium constant between the two photoreactions II and I when the algae are illuminated at 650 nm.     

Interaction of poly(L-lysine) and copolymers of lysine with immobilized DNA.

Sonicated DNA has been covalently attached to Sepharose 4B by a carbodiimide method [Rickwood, P. (1972) Biochim. Biophys. Acta269, 47–50] which minimizes modification of the DNA and matrix. Columns of this material have been used to study the interaction between cationic polypeptides and DNA. When poly(l-lysine) is bound to such columns at low ionic strength and then eluted with a linear salt gradient the polypeptide elutes over a broad range of salt concentration, presumably reflecting different strengths of interaction with various sites on the DNA. The broadness of the elution profile cannot be attributed to heterogeneity in the poly(l-lysine) sample but rather to the $\text{AT}\text{GC}$ content of various DNA sites. Studies with other polypeptides, poly(l-Lys⁷⁹, l-Leu²¹) and poly-(l-Lys-l-Ala-Gly), as well as studies at different temperatures, have helped to clarify the possible roles of conformational mobility, polypeptide hydrophobicity, and the presence of contiguous lysines in determining the strength of interaction of polypeptides and proteins with DNA sites.     

Novel and facile synthesis of furanodictines A and B based on transformation of 2-acetamido-2-deoxy-d-glucose into 3,6-anhydro hexofuranoses

A novel synthesis of furanodictines A [2-acetamido-3,6-anhydro-2-deoxy-5-O-isovaleryl-d-glucofuranose (1)] and B [2-acetamido-3,6-anhydro-2-deoxy-5-O-isovaleryl-d-mannofuranose (2)] is described starting from 2-acetamido-2-deoxy-d-glucose (GlcNAc). The synthetic protocol is based on deriving the epimeric bicyclic 3,6-anhydro sugars [2-acetamido-3,6-anhydro-2-deoxy-d-glucofuranose (4) and 2-acetamido-3,6-anhydro-2-deoxy-d-mannofuranose (5)] from GlcNAc. Reaction with borate upon heating led to a facile transformation of GlcNAc into the desired epimeric 3,6-anhydro sugars. The C5 hydroxyl group of the 3,6-anhydro compounds 4 and 5 was regioselectively esterified with the isovaleryl chloride to complete the synthesis of furanodictines A and B, respectively. The targets 1 and 2 were synthesized in only two steps requiring no protection/deprotection.     

Synthesis of a heptasaccharide fragment of the mannan from Candida guilliermondii cell wall and its conjugate with BSA

The 3-aminopropyl glycoside of a heptasaccharide fragment of the cell wall mannan from Candida guilliermondii18, which corresponds to the antigenic Factor 9, has been synthesized by a convergent approach based on glycosylation of a tetrasaccharide acceptor with a trisaccharide donor as the key step to give a protected heptasaccharide 17. Subsequent two-step deprotection of 17 afforded the heptamannoside 18, which was then conjugated with BSA using the squarate procedure.     

Effect of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone on the secondary electron acceptor B of photosystem II

Measurements of chlorophyll fluorescence have been used to monitor electron transport from the primary electron acceptor of photosystem II, Q, to the secondary acceptor, B, in chloroplasts in either the presence or the absence of the plastoquinone analog 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). Electron transport is markedly slower from Q^? to either B or B^? in the presence of DBMIB. Binary oscillations in the rate of reoxidation of Q^? (equivalent to the reactions Q^?B → QB^? and Q^?B^? → QB^2?) after each of a series of flashes were of a phase opposite to those observed in the absence of DBMIB (J. M. Bowes, and A. R. Crofts, (1980) Biochim. Biophys. Acta590, 573–584). The results confirm that inhibition of electron transport by DBMIB in chloroplasts is not restricted to an inhibition of electron transfer from the plastoquinone pool, but that there is also a specific interaction between the reduced form of the inhibitor and the secondary electron acceptor B. Models are discussed to account for the mechanism of this interaction.     

Amino acid sequence of the coelomic C globin from the sea cucumberCaudina (Molpadia) arenicola

The sequence of a globin from a marine invertebrate, the sea cucumberCaudina (Molpadia) arenicola (Echinodermata), is reported. This globin, chain C, is one of four major globins found in coelomic red cells in this organism and is the second to be sequenced. Chain C consists of 157 residues, is amino-terminally acetylated, and has an extended amino-terminal region. This globin shares a 60% sequence identity with the other sequencedC. arenicola globin, D chain (Mauriet al., Biochem. Biophys. Acta 1078, 63–67, 1991), but has a 93.6% identity with a globin from another sea cucumber,Paracaudina chilensis (Suzuki,Biochem. Biophys. Acta, 998, 292–296, 1989).