Reaction of chicken egg white lysozyme with 7-chloro-4-nitrobenz-2-oxa-1,3-diazole. II. Sites of modification.

1. Procedure for the isolation of peptides from proteins bearing the chemically labile aromatic ether, O-tyrosyl-4-nitrobenz-2-oxa-1,3-diazole group, is described. 2. The tyrosyl residue reactive towards 7-chloro-4-nitrobenz-2-oxa-1,3-diazole in chicken egg white lysozyme (Aboderin, A. A., Boedefeld, E. and Luisi, P. L., (1973) Biochim. Biophys. Acta 328. 20-30) is tyrosine-23. The amino group in the protein whose reaction with the reagent is dependent on the prior reaction of tyrosine-23 is the epsilon-amino group of lysine-33.     

Monte Carlo studies of phospholipid lamellae. Effects of proteins, cholesterol, bilayer curvature, and lateral mobility on order parameters

In this paper we present the results of a Monte Carlo study of the effects of protein, cholesterol, bilayer curvature, and mobility on the chain order parameters of a lipid layer. The Monte Carlo method used is identical to the version developed earlier (Scott, Jr., H.L. (1977) Biochim. Biophys. Acta 469, 264–271). Simulations of protein and cholesterol effects are accomplished by insertion of a rigid stationary cylinder into the lipid matrix. The protein studies show the presence of boundary lipid (Jost, P., Griffith, O.H., Capaldi, R.H. and Vanderkooi, G. (1973) Biochim. Biophys. Acta 311, 141–152). The effect of cholesterol is dependent upon the length of the lipid hydrocarbon chains relative to the cholesterol depth of penetration. Our computer studies of bilayer curvature show the manner in which this curvature disrupts chain packing and are consistent with experimental results (Chrzeszczyk, A., Wishnia, A. and Springer, C.S. (1977) Biochim. Biophys. Acta, 470, 161–171). We also find that restricting lateral motion in chains, the simplest manner in which head group interactions can affect hydrocarbon chain order, does not measurably alter the order parameters. We argue that this provides some support for an earlier hypothesis by Scott (Scott, Jr., H.L. (1975) Biochim. Biophys. Acta 406, 329–346) regarding head group-chain interaction in monolayer experiments.     

An improved large scale procedure for the purification of porcine pancreatic lipase

A modified procedure for purifying porcine pancreatic lipase (triacyglycerol acyl-hydrodrolase, EC 3.1.1.3) is described. In comparison to the previous procedure reported by Verger, R., de Hass, G.H., Sarda, L and Desnuelle, P. (1969) Biochim. Biophys. Acta 188, 272--282) it is more rapid, more reproducible and results in a purer enzyme preparation. No colipase could be detected in the mixture of isoenzymes and, naturally, in the different separated lipases. In this process, butanol treatment is omitted. After pancreas powder extraction, a batch procedure was used for adsorption of DEAE-cellulose. Sephadex filtration (pH 8.0) was made in a largeer size column. Finally the isoenzymes were separated on CM-cellulose as in the Verger procedure, but under slightly modified conditions. Lipase LB was fully homogeneous as judged by end group determination, gel electrophoresis (in the presence of absence of sodium dodecyl sulfate) and sedimentation equilibrium.     

Steady-state kinetics of laccasse from Rhus vernicifera.

The steady-state kinetics of laccasse (monophenol, dihydroxyphenylalanine: oxygen oxidoreductase, EC 1.14.18.1) from the lacquer tree Rhus vernicifera is investigated using the respirograph method to produce Lineweaver-Burk plots of oxygen consumption rate against oxygen concentration. A ping-pong mechanisms is established. The kinetic constants obtained according to the model is in close agreement with the corresponding values obtained from earlier studies on the transient reactions between the reduced enzyme and oxygen (Andréasson, L.E., Br?ndén, R. and Reinhammar, B. (1976) Biochim. Biophys. Acta 438, 370--379) and between the oxidized enzyme and reducing substrates (Andréasson, L.E. and Reinhammar, B. (1976) Biochim. Biophys. Acta 445, 579--597).     

Response of rat liver glutaminase to pH. Mediation by phosphate and ammonium ions

The activity of rat liver glutaminase from sedimented fractions of freeze-thawed mitochondria is strongly affected by variation in pH over a physiologically relevant range at approximate physiological concentrations of activators. As pH increases from 7.1 to 7.7 at 0.7 mM ammonium and 10 mM phosphate, the S0.5 for glutamine decreases 3.5-fold, from 38 to 11 mM. This results in an 8-fold increase in reaction velocity at 10 mM glutamine. In addition, the M0.5 for phosphate activation decreases from 21 to 8.9 mM as pH increases from 7.1 to 7.7. This apparent effect of pH on the affinity of glutaminase for phosphate is similar to previous reports of the pH effect on activation by ammonium (Verhoeven, A. J., Van Iwaarden, J. F., Joseph, S. K., and Meijer, A. J. (1983) Eur. J. Biochem. 133, 241-244; McGivan, J. D., and Bradford, N. M. (1983) Biochim. Biophys. Acta 159, 296-302). Glutaminase does not respond to variation in pH between 7.1 and 7.7 when phosphate and ammonium are saturating. The effects of the two modifiers are additive. Each is still effective, as is pH, when the other is saturating. Therefore, it appears that the effects of pH on the apparent affinity of the enzyme for ammonium and phosphate account for the enzyme's response to pH. These results may help explain previous reports of minimal effects of pH on glutaminase at saturating concentrations of related substances (McGivan, J. D., Lacey, J. H., and Joseph, K. (1980) Biochim. J. 192, 537-542; Horowitz, M. L., and Knox, W. E. (1968) Enzymol. Biol. Clin. 9, 241-255; McGivan, J. D., and Bradford, N. M. (1983) Biochim. Biophys. Acta 759, 296-302). Glutaminase binds glutamine cooperatively with Hill coefficients ranging from 1.7 to 2.2, which suggests at least two and probably three or more interacting binding sites for glutamine. The strong response of liver glutaminase to pH and the fact that the reaction can supply metabolites for urea synthesis suggest a possible regulatory role of glutaminase in ureagenesis.     

The reaction of cytochrome oxidase with cyanide. Preparation of the rapidly reacting form and its conversion to the slowly reacting form

The magnitude of the slow phase of reaction of cytochrome oxidase with cyanide has been correlated with the size of the epr signal at g' = 12. This epr signal was not found in submitochondrial particles, and significant g' = 12 epr was only observed late in the purification of solubilized enzyme. The Hartzell-Beinert procedure for the purification of cytochrome oxidase (Hartzell, C.R., and Beinert, H. (1974) Biochim. Biophys. Acta 368, 318-338) has been modified so that the purified enzyme reacts in a single rapid phase with potassium cyanide and lacks the g' = 12 epr signal. This enzyme could be converted to the slowly reacting form upon incubation at low pH and/or low enzyme concentration. No procedure for the stable reversal of the process could be found. Some physical and chemical properties of the two forms of the enzyme are compared.     

Kinetic studies on the reaction of compound II of myeloperoxidase with ascorbic acid. Role of ascorbic acid in myeloperoxidase function

Ascorbic acid is known to stimulate leukocyte functions. In a recent publication it was suggested that the role of ascorbic acid is to reduce compound II of myeloperoxidase back to the native enzyme (Bolscher, B. G. J. M., Zoutberg, G. R., Cuperus, R. A., and Wever, R. (1984) Biochim. Biophys. Acta 784, 189-191). In this paper we report rapid spectral scan and transient state kinetic results on the reaction of three myeloperoxidase compounds II, namely, human neutrophil myeloperoxidase, canine myeloperoxidase, and bovine spleen heme protein with ascorbate. We show by rapid scan spectra that compound II does not pass through any other intermediate when ascorbic acid reduces it back to native form. We also show that the reactions of all three compounds II involve a simple binding interaction before enzyme reduction with an apparent dissociation constant of 6.3 +/- 0.9 x 10(-4) to 2.0 +/- 0.3 x 10(-3)M and a first-order rate constant for reduction of 12.6 +/- 0.6 to 18.8 +/- 1.3 s-1. The optimum pH is 4.5, and at this pH the activation energy for the reaction is 13.2 kJ mol-1. Results of this work lend further evidence that the spleen green heme protein is very similar if not identical to leukocyte myeloperoxidase based on a comparison of spectral scans, pH-rate profiles, and kinetic parameters. We demonstrate that chloride cannot reduce compound II whereas iodide reduces compound II to native enzyme at a rate comparable to that of ascorbate. This explains why ascorbate accelerates chlorination but inhibits iodination. Formation of compound II is a dead end for the generation of hypochlorous acid; ascorbate regenerates more native enzyme to enhance the chlorination reaction namely: myeloperoxidase + peroxide----compound I followed by compound I + chloride----HOCl. On the other hand, ascorbate is a competitor with iodide for both compounds I and II and so inhibits iodination.     

The interaction of nitrotyrosine-83 plastocyanin with cytochromes f and c: pH dependence and the effect of an additional negative charge on plastocyanin.

Spinach plastocyanin was selectively modified using tetranitromethane which incorporates a nitro group ortho to the hydroxyl group of tyrosine 83 (Anderson, G.P., Draheim, J.E. and Gross, E.L. (1985) Biochim. Biophys. Acta 810, 123-131). This tyrosine residue has been postulated to be part of the cytochrome f binding site on plastocyanin. Since the hydroxyl moiety of nitrotyrosine 83 is deprotonated above its pK of 8.3, it provides a useful modification for studying the effect of an extra negative charge on the interaction of plastocyanin with cytochrome f. No effect on cytochrome f oxidation was observed at pH 7 under conditions in which the hydroxyl moiety is protonated. However, the rate of cytochrome f oxidation increased at pH values greater than 8, reaching a maximum at pH 8.6 and decreasing at still higher pH values. The increase was half-maximal at pH 8.3 which is the pK for the hydroxyl moiety on nitrotyrosine 83. In contrast, the rate of cytochrome f oxidation for control plastocyanin was independent of pH from pH 7 to 8.6. These results show that increasing the negative charge on plastocyanin at Tyr-83 increases the ability to react with cytochrome f, supporting the hypothesis that cytochrome f interacts with plastocyanin at this location. In contrast, the reaction of Ntyr-83 plastocyanin with mammalian cytochrome c was independent of pH, suggesting that its mode of interaction with plastocyanin is different from that of cytochrome f. A comparison of the effects of Ntyr-83 modification of plastocyanin with the carboxyl- and amino-group modifications reported previously suggests that plastocyanin binds to cytochrome f in such a way that electrons could be donated to plastocyanin at either of its two binding sites.     

Slow fluorescence quenching of type A chloroplasts. Resolution into two components.

The divalent-cation-specific ionophore A23187 is used to define two components of the slow fluorescence quenching of type a spinach chloroplasts: ionophore-reversible and ionophore-resistant quenching. Ionophore-reversible quenching predominates at relatively low light intensities and approaches saturation as light levels are increased. It is sensitive to uncouplers and to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and is dark reversible. At high light intensities the bulk (greater than 80%) of slow fluorescence quenching is ionophore-resistant. Ionophore-resistant quenching is stimulated by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at pH 7.6 and by both CCCP and methylamine at pH 9.0. It is insensitive to DCMU and is not reversed in subsequent darkness. Taken together, the two components account for all quenching observed in Type A chloroplasts. Ionophore-reversible quenching is identified with the Mg2+-mediated fluorescence quenching described by Krause (Biochim. Biophys. Acta (1974) 333, 301-313) and by Barber and Telfer (in Membrane Transport in Plants (Dainty, J., AND Zimmermann, U., eds.), pp. 281-288, Springer-Verlag, Berlin, 1974). Ionophore-resistant quenching, a first-order process requiring high light, resembles the quenching reported by Jennings et al. (Biochim. Biophys. Acta (1976) 423, 264-274). The resolution of the fluorescence quenching phenomenon into two distinct components reconciles the apparently contradictory observations of these earlier investigations.     

Energy requirements for biosynthesis of DNA in Escherichia coli. Role of membrane-bound energy-transducing ATPase (coupling factor).

A mutant of Escherichia coli missing energy-transducing ATPase and known to be defective in a variety of membrane functions from earlier studies (Yamamoto, T. H., Mével-Ninio, M. and Valentine, R. C. (1973) Biochim. Biophys. Acta 314, 267-275; Thipayathasana, P. and Valentine, R. C. (1974) Biochim. Biophys. Acta 347, 464-468; Mével-Ninio, M. and Yamamoto, T. (1974) Biochim. Biophys. Acta 357, 63-66) has been found to be blocked for anaerobic DNA synthesis. The rate of anaerobic DNA synthesis in the mutant, measured as radioactive adenine incorporation into the alkali-resistant fraction of whole cells, is about 1/6 the rate of DNA synthesis in the wild type culture under similar conditions. Addition of NO-3- or O-2 restores DNA biosynthesis in the mutant. The entry of radioactive adenine is not appreciably affected in the mutant by anaerobiosis. It is concluded that coupling factor plays a role in some step(s) of DNA biosynthesis.     

Membrane-bound states of alpha-lactalbumin: implications for the protein stability and conformation.

alpha-Lactalbumin (alpha-LA) associates with dimyristoylphosphatidylcholine (DMPC) or egg lecithin (EPC) liposomes. Thermal denaturation of isolated DMPC or EPC alpha-LA complexes was dependent on the metal bound state of the protein. The intrinsic fluorescence of thermally denatured DMPC-alpha-LA was sensitive to two thermal transitions: the Tc of the lipid vesicles, and the denaturation of the protein. Quenching experiments suggested that tryptophan accessibility increased upon protein-DMPC association, in contrast with earlier suggestions that the limited emission red shift upon association with the liposome was due to partial insertion of tryptophan into the apolar phase of the bilayer (Hanssens I et al., 1985, Biochim Biophys Acta 817:154-166). On the other hand, above the protein transition (70 degrees C), the spectral blue shifts and reduced accessibility to quencher suggested that tryptophan interacts significantly with the apolar phase of either DMPC and EPC. At pH 2, where the protein inserts into the bilayer rapidly, the isolated DMPC-alpha-LA complex showed a distinct fluorescence thermal transition between 40 and 60 degrees C, consistent with a partially inserted form that possesses some degree of tertiary structure and unfolds cooperatively. This result is significant in light of earlier findings of increased helicity for the acid form, i.e., molten globule state of the protein (Hanssens I et al., 1985, Biochim Biophys Acta 817:154-166). These results suggest a model where a limited expansion of conformation occurs upon association with the membrane at neutral pH and physiological temperatures, with a concomitant increase in the exposure of tryptophan to external quenchers; i.e., the current data do not support a model where an apolar, tryptophan-containing surface is covered by the lipid phase of the bilayer.     

Purification of 5'-nucleotidase from human seminal plasma.

5'-Nucleotidase from human seminal plasma was purified to electrophoretic homogeneity and some of its kinetic and molecular properties compared with those of 5'-nucleotidase from bull seminal plasma. The purification of the enzyme was achieved by using the same affinity chromatography media (Con A-Sepharose and AMP-Agarose or ADP-Agarose) previously used for the purification of bull seminal plasma 5'-nucleotidase (Fini, C., Ipata, P.L., Palmerini, C.A. and Floridi, A. (1983) Biochim. Biophys. Acta 748, 405-412). However, in the present purification procedure no detergent was used as it had been necessary for the purification of the bovine enzyme. The experimental data reveal some main differences between these two enzymes; first, the human enzyme seems to be constituted of a single polypeptide chain of about 71 kDa, while the 5'-nucleotidase of bull seminal plasma, in non denaturing detergent solutions, is a homodimer of about 160 kDa. Another most remarkable difference is that the human enzyme does not seem to contain a phosphatidylinositol anchoring system like the one present in the bovine enzyme and in 5'-nucleotidase of different sources (Low, M.G. (1987) Biochem. J. 244, 1-13). Finally, the AMPase activity of 5'-nucleotidase from human seminal plasma is not affected by dithiothreitol which, on the contrary, is a powerful inhibitor of the bovine enzyme causing the dissociation of its subunits which are held together by disulphide bridges (Fini, C., Minelli, A., Camici, M. and Floridi, A. (1985) Biochem. Biophys. Acta 827, 403-409).     

A low-molecular-weight acid phosphatase which contains iron

A simple, large scale purification procedure for the violet “phosphoprotein phosphatase” from beef spleen [Glomset, J. and Porath, J., Biochim. Biophys. Acta, 39, 1 (1960)] has been devised. The procedure involves a 24-hr extraction of a tissue homogenate at pH 3, ammonium sulfate fractionation, CM-cellulose chromatography, and Sephadex G-75 chromatography. The highly purified enzyme shows a constant ratio of both enzymatic activity and absorbance at 550 nm to iron concentration. The data strongly indicate that the enzyme contains iron, and moreover, support a 1:1 molar ratio of iron to enzyme.     

Enzymatic degradation of succinyl-coenzyme A by rat liver homogenates.

When a dilute suspension of the mitochondrial fraction of rat liver homogenates was incubated with chemically synthesized succinyl-CoA, a product was rapidly formed which was retained at pH 3.9 on Dowex 50 (H+). Although its acid-base properties were indistinguishable from those of epsilon-aminolevulinic acid, the product did not form a pyrrole with acetylacetone, nor was its enzymatic formation dependent on added glycine. The enzyme which cleaved succinyl-CoA to the epsilon-aminolevulinic acid-like product was inhibited by phenylmethyl sulfonylfluoride. The first substance formed by the peptidase was the unstable thioester of succinic acid and cysteamine which underwent rearrangement to the more stable N-succinyl cysteamine above pH 4.0. It is apparent that the assay of epsilon-aminolevulinic acid synthetase (EC 2.3.1.37) by the ion-exchange method of Ebert et al. (Ebert, P.S., Tschudy, D.P., Choudhry, J.N. and Chirigos, M.A. (1970) Biochim. Biophys. Acta 208, 236--250) can yield erroneous results with succinyl-coenzyme A as substrate, especially when incubations are carried out for less than 25 min.     

Reflection coefficients of permeant nonelectrolytes for dog and beef red cell membranes.

The reflection coefficient, sigma, for several small permeant nonelectrolytes was determined for dog and beef red blood cell membranes. Our sigma values were considerably higher than those previously reported for dog cells; e.g., out sigma urea was 87% higher than the sigma urea of Rich, Sha'afi, Barton and Solomon (J. Gen. Physiol. 50: 2391, 1967). Our sigma values for urea were only slightly greater in beef cells than previously reported by Farmer and Macey (Biochim. Biophys. Acta 290: 290, 1972). We found that a trend exists when (1 - sigma) is plotted against the log of the permeability coefficient, omega. This observation is also consistent with our previously reported sigma data for human red cell membranes (Owen & Eyring, J. Gen. Physiol. 66: 241, 1972). This trend suggests that small hydrophilic molecules interact highly with cell membrane water. The exceptions to this trend were lipophilic molecules, indicating they do not interact with water while penetrating the red cell membrane.     

Human seminal ribonuclease. A tool to check the role of basic charges and glycosylation of a ribonuclease in the action of the enzyme on double-stranded RNA

Human seminal ribonuclease (a basic protein occurring in a glycosylated and in a non-glycosylated form) is very active against double-stranded RNAs (De Prisco, R., Sorrentino, S., Leone, E. and Libonati, M. (1984) Biochim. Biophys. Acta 788, 356-363). The action of the two enzyme forms on single-stranded and double-stranded substrates was studied as a function of pH and ionic strength. Results indicate (1) that glycosylation of the RNAase molecule does not affect enzyme action on single-stranded RNAs, while (2) degradation of double-stranded RNAs is moderately increased by the presence of carbohydrates in the enzyme molecule. Human seminal RNAase shows a marked helix-destabilizing activity on poly(dA-dT) X poly(dA-dT). Under various conditions, this action (1) is definitely stronger than that of bovine RNAase A, and (2) seems to be less dependent on the glycosylation than on the basicity of the enzyme protein. The remarkable activity of human seminal RNAase on double-stranded RNA may, at least partly, be related to the enzyme properties mentioned above.     

Energy requirement for biosynthesis of DNA in Escherichia coli: Role of membrane-bound energy-transducing ATPase (coupling factor)

A mutant of Escherichia coli missing energy-transducing ATPase and known to be defective in a variety of membrane functions from earlier studies (Yamamoto, T. H., Mével-Ninio, M. and Valentine, R. C. (1973) Biochim. Biophys. Acta 314, 267–275; Thipayathasana, P. and Valentine, R. C. (1974) Biochim. Biophys. Acta 347, 464–468; Mével-Ninio, M. and Yamamoto, T. (1974) Biochim. Biophys. Acta 357, 63–66) has been found to be blocked for anaerobic DNA synthesis. The rate of anaerobic DNA synthesis in the mutant, measured as radioactive adenine incorporation into the alkali-resistant fraction of whole cells, is about 1/6 the rate of DNA synthesis in the wild type culture under similar conditions. Addition of NO₃^- or O₂ restores DNA biosynthesis in the mutant. The entry of radioactive adenine is not appreciably affected in the mutant by anaerobiosis. It is concluded that coupling factor plays a role in some step(s) of DNA biosynthesis.     

Spin-label studies of the sulfhydryl environment in bovine plasma albumin. 1. The N--F transition and acid expansion.

The environment of the sulfhydryl group in plasma albumin was previously characterized by employing spin-labels of varying chain lengths (Hull, H. H., Chang, R., & Kaplan, L. J. (1975) Biochim. Biophys. Acta 400, 132). It was established that the sulfhydryl is in a crevice approximately 10 A deep but this crevice was not identified further. We now report the results of titrating albumin through the acidic conformational transitions while monitoring the electron-spin resonance of the bound nitroxide. With short spin-labels a general change is observed as the pH is lowered but the N--F transition is not discernible. However, with a spin label previously shown to project to the lip on the crevice a clear N--F transition as well as the subsequent acid expansion are observed. These results indicate that the sulfhydryl is in the crevice, formed by the domains of albumin, which opens during the N--F transition. Further results indicate that bound fatty acids do not influence the integrity of the sulfhydryl environment at neutral pH.     

Studies on phytohemagglutinins. XXIV. Isoelectric point and hybridization of the pea (Pisum sativum L.) isophytohemagglutinins.

The isoelectric point of the two pea isophytohemagglutinins varies from pH 5.7 to pH 8.4 depending on the composition of the buffer used. Isoelectric focusing reveals three main molecular species with pI at pH 5.90, 6.35 and 7.00. Molecular species with pI at pH 5.9 and 7.0 correspond to the two pea isophytohemagglutinins which can be obtained by DEAE-cellulose chromatography (Entlicher, G., Kostír, J.V. and Kocurek, J. (1970) Biochim. Biophys. Acta 221, 272-281). A molecular species with pI at pH 6.35 is formed from the two pea isophytohemagglutinins by hybridization. According to the hybridization pattern and subunit composition of the pea isophytohemagglutinins the subunit composition AABB, AACC and AABC can be proposed for the three molecular species with respect to ionic properties of the subunits.     

Post-translational modification of human erythrocyte pyruvate kinase

Upon storage, partially purified human erythrocyte pyruvate kinase (ATP: pyruvate-phosphotransferase, E.C. 2.7.1.40) from normal individuals was found to undergo a spontaneous oxidation to a form which displayed markedly reduced activity. This modified form of the enzyme exhibited kinetic patterns similar to those frequently reported for the enzyme in cases of nonspherocytic hemolytic anemia. The data are discussed in relation to the recently proposed theory that post-translational modification of pyruvate kinase is responsible for the abnormal kinetic patterns frequently encountered for this enzyme in the disease state. [Van Berkel, T. J. C., Koster, J. F., Kruyt, J. K. and Staal, G. E. J. 1973 $\text{Biochim. Biophys. Acta 321}$, 496–502].