Ca2+ and Zn2+-binding properties of nitrated S-100b protein from bovine brain.

The single tyrosine residue in S-100b protein was nitrated by treatment with tetranitromethane in 0.1 M-Tris/HCl buffer, pH 8.0, containing 2 mM-EDTA. The nitrated protein did not differ significantly in secondary structure from its native unmodified counterpart, as revealed by far-u.v. c.d. measurements. The effect of Ca2+ on the modified protein was different from that on the native protein, e.g. addition of Ca2+ resulted in a loss of helical content from 55 to 47% with the native protein whereas Ca2+ had no significant effect on the gross conformation of the nitrated derivative. Near-u.v. c.d. studies also indicated a very minimal effect on the tyrosine residue and this was also reflected in the u.v.-absorption difference spectrum. Polyacrylamide-gel electrophoresis in the absence of SDS showed the nitrated S-100b to move faster in the presence of EDTA compared with the calcium-bound state, suggesting that the modified protein does bind Ca2+ although it does not undergo a major conformational change in response to Ca2+ addition. In contradistinction, Zn2+ binding was not influenced by nitration, as demonstrated by aromatic c.d. and u.v.-difference spectroscopy. It is clear from this study that the single tyrosine residue in S-100b is critical to sense the Ca2+-induced conformational changes in the protein.     

Inhibition of pepsin by polyions and c.d. studies

The effects of poly(vinyl sulphate) (PVS), poly(vinyl pyrrolidone) (PVP) and protamine sulphate with the enzyme pepsin (EC 3.4.23.1) have been investigated. PVS, PVP and protamine acted as inhibitors of the enzyme pepsin at low concentrations, but at high concentrations of the polyions (with the exception of PVS) the inhibition was less pronounced. The catalytic effectiveness of several polyions has been shown experimentally; when used at pH 2.1 with haemoglobin and at pH 5.0 with azocasein thus acted as a weak proteolytic catalyst. The order of effectiveness was: polybrene greater than poly(L-lysine) (PLL) greater than spermine greater than spermidine greater than protamine greater than and PVP relative to pepsin. The hydrolysis of the substrates by the enzyme decreases in the presence of high concentrations of monovalent and/or divalent salts. The purity of the enzyme was assessed by determination of the molecular weight using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), phosphorus and nitrogen content. The circular dichroism (c.d.) spectrum of the enzyme in the wavelength range 240-310 nm, where the polycations did not have a c.d. spectrum has also been studied. Each of the polyions had a definite effect on the c.d. spectrum, showing strong binding to the enzyme. The neutral polymer PVP was also found to modify the c.d. spectrum, showing strong binding to the enzyme. The strengths of the interactions as indicated by the magnitudes of the changes in the c.d. spectrum of pepsin at 270 nm were: polybrene greater than protamine greater than PLL greater than PVP greater than spermidine greater than spermine.     

Circular dichroic, infrared and other studies on the protein component of pig brain thromboplastin.

1. A four-step procedure used to isolate the protein component (apoprotein III) of pig brain thromboplastin yielded approximately 25 mg from 500g of brain. 2. In the absence of detergent, apoprotein III had an apparent mol.wt. of 360 000 by gel-filtration, and, after electrophoresis on polyacrylamide gels in the presence of sodium docecyl sulphate, it appeared as a major protein band of mol.wt.59 000, suggesting the existence of polymeric and monomeric forms. 3. Chemical analyses of apoprotein III revealed that hydrophilic and hydrophobic amino acids were present in a ratio of 3:2, together with approx, 9% (w/w) of carbohydrate. 4. The far-u.v.c.d. and i.r. spectral data indicated that, like other membrane proteins, apoprotein III has a high percentage of unordered structure with lesser amounts of alpha and beta-forms. 5. Relipidation of apoprotein III to restore clotting activity caused no extensive alteration in the c.d. and i.r. spectra, indicating that the phospholipid associates with a comparatively small hydrophobic segment. The constrained unordered conformation, which makes the major contribution to the c.d. spectrum, probably forms a separate domain in the aqueous phase. The absence of any increase in the amplitude of both negative c.d. extrema, following relipidation, contrasted with the substantial increase observed in a helix-forming solvent and raises the possibility that the more stable polymeric form of apoprotein III is retained as the active form in the lipid phase. 6. We suggest that as a consequence of cell membrane damage, the recognition and activation of factor VII may involve minor changes of conformation that are dependent upon the flexibility inherent in an unordered secondary structure.     

Further characterization of human eosinophil peroxidase.

The large and the small subunits (Mr 50 000 and 10 500 respectively) of human eosinophil peroxidase were isolated by gel filtration under reducing conditions. The subunits were very strongly associated but not apparently cross-linked by disulphide bridges. During storage, the large subunit tended to form aggregates, which required reduction to dissociate them. Amino acid analysis of the performic acid-treated large subunit showed the presence of 19 cysteic acid residues. The small subunit of eosinophil peroxidase had the same Mr value as the small subunit of myeloperoxidase. However, although these subunits have very similar amino acid compositions, they showed different patterns of peptide fragmentation after CNBr treatment. The carbohydrate of eosinophil peroxidase seemed associated exclusively with the large subunit and comprised mannose (4.5%, w/w) and N-acetylglucosamine (0.8%, w/w). The far-u.v.c.d. spectrum of the enzyme indicated the presence of relatively little ordered secondary structure.     

Ligand-induced conformational transitions and secondary-structure composition of chicken liver pyruvate carboxylase

Apparent conformational transitions induced in chicken liver pyruvate carboxylase by substrates, KHCO(3) and MgATP, and the allosteric effector, acetyl-CoA, were studied by using the fluorescent probe, 8-anilinonaphthalene-1-sulphonic acid and c.d. Fluorescence measurements were made with both conventional and stopped-flow spectrophotometers. Additions of acetyl-CoA and/or ATP to the enzyme-probe solutions quenched fluorescence of the probe by the following cumulative amounts regardless of the sequence of additions: acetyl-CoA, 10-13%; ATP, 21-24%; acetyl-CoA plus ATP, about 35%. Additions of KHCO(3) had no effect on the fluorescence. The rates of quenching by acetyl-CoA and MgATP (in the presence of acetyl-CoA) were too rapid to measure by stopped-flow kinetic methods, but kinetics of the MgATP effect (in the absence of acetyl-CoA) indicate three unimolecular transitions after the association step. The negligible effect of the probe on enzyme catalytic activity, a preservation of the near-u.v. c.d. effect of MgATP and acetyl-CoA in the presence of the probe and no observable unimolecular transitions after binding of the probe to the enzyme indicate that the probe had no deleterious effect on the enzyme. In contrast with results with 8-anilinonaphthalene-1-sulphonic acid, fluorescence of the epsilon-derivative of acetyl-CoA or ATP [fluorescent analogues; Secrist, Barrio, Leonard & Weber (1972) Biochemistry11, 3499-3506] was not changed when either one was added to the enzyme. Secondary-structure composition of chicken liver pyruvate carboxylase estimated from the far-u.v. c.d. spectrum of the enzyme is 27% helix, 7% beta-pleated sheet and 66% other structural types.     

Purification and properties of galactokinase from Saccharomyces cerevisiae.

Galactokinase (EC 2.7.1.6; ATP:D-galactose-1-phosphotransferase) was purified to homogeneity with a 50% yield from cells of Saccharomyces cerevisiae which were fully induced for the production of the galactose metabolizing enzymes. The purification was accomplished by:(a) ammonium sulfate fractionation, (b) streptomycin sulfate precipitation. (c) DEAE-cellulose chromatography, (d) hydroxylapatite chromatography, and finally (e) Bio-Gel A-0.5 m gel filtration. The resulting preparation of galactokinase was judged to be at least 95% pure by the following criteria: (a) sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (b) ultracentrifuge analysis, (c) nondissociating polyacrylamide gel electrophoresis, and (d) Bio-Gel A-0.5 m gel filtration. The purified enzyme preparation was used to determine the Km values for the two substrates, galactose and ATP, which were found to be 0.60 and 0.15 mM, respectively. Vmax was also determined and found to be 3.35 mmol/h/mg. This corresponds to a turnover rate of 3350 molecules of galactose phosphorylated/min/enzyme molecule. The effect of pH on the galactokinase-catalyzed phosphorylation of galactose was determined; the results showed the pH optimum of the reaction to be in the range of pH 8.0 to 9.0. The enzyme is highly specific for galactose since galactokinase did not appear to phosphorylate any of the other sugars tested at a rate greater than 0.5% of the rate of galactose phosphorylation. Amino acid analysis was performed on the enzyme preparation and the results were used to calculate the partial specific volume (v) of 0.736. The NH2-terminal sequence was determined for the first 3 residues. The molecular weight and subunit composition were determined by ultracentrifugation and polyacrylamide gel electrophoresis under dissociating and nondissociating conditions. The data obtained indicated that galactokinase is a monomeric protein of molecular weight 58,000.     

Effects of oxidative stress on some physiochemical properties of caeruloplasmin.

We report the effects of oxidative stress generated by low-intensity u.v. irradiation (366 and 254 nm), dialysis against ascorbate and isolated stimulated neutrophils on some physicochemical properties of caeruloplasmin. Low-intensity u.v. irradiation resulted in a loss of ferroxidase activity and 610 nm absorption, changes previously reported to occur during storage and manipulation of caeruloplasmin. These alterations were found to correspond to aggregation of the protein, induction of visible fluorescence (excitation, 360 nm; emission, 454 nm), changes in c.d. spectra which were indicative of alterations in protein conformation, loss of half-cystine, tryptophan and tyrosine content and loss immunoreactivity. The changes in the far-u.v. c.d. spectrum of caeruloplasmin were more pronounced than those observed for u.v.-irradiated IgG. Similar c.d. changes and induction of fluorescence were observed following dialysis of caeruloplasmin against ascorbate or exposure to stimulated neutrophils. It is concluded that the lability of caeruloplasmin may arise from oxidative modification, in addition to the previously described susceptibility of this protein to proteolysis.     

Effect of physical environment on the conformation of ricin. Influence of low pH.

The molecular properties of ricin (the toxic lectin from Ricinus communis seeds, RCA II or RCA 60) were evaluated by analytical ultracentrifugation, viscosimetry, c.d., fluorescence and equilibrium dialysis. Measurements of sedimentation (S0(20,W) = 4.60 S) and viscosity (eta = 2.96 X 10(-2) dl/g) indicated that, at neutral pH, the ricin molecule is very compact. Various transitions were explored, and a pH-triggered change in the ricin conformation was observed between pH 7 and 4. In this range, the sedimentation coefficient, far-u.v. c.d. and fluorescence altered simultaneously without unfolding. Below pH 7 the change in the ricin conformation was accompanied by a decrease in the affinity of ricin for galactosides, and at pH 4.0 by an alteration in its binding capacity. These effects of low pH are discussed in relation to the physical conditions encountered by ricin molecules during their entry into living cells.     

Denaturation studies on natural and recombinant bovine prochymosin (prorennin).

1. Prochymosin in solution in the presence of 8 M-urea is fully unfolded, as indicated by its fluorescence spectrum, fluorescence quenching behaviour and far-u.v.c.d. spectrum. 2. Equilibrium studies on the unfolding of prochymosin and pepsinogen by urea were carried out at pH 7.5 and pH 9.0. The results indicate that the stabilization energies of the two proteins are identical at pH 7.5, but that at pH 9.0 pepsinogen is significantly less stable than prochymosin. 3. Kinetic studies on the unfolding of prochymosin and pepsinogen indicate that the processes can be described by a single first-order rate constant, and that at any given value of denaturant concentration and pH the rate of unfolding of prochymosin is significantly greater than that of pepsinogen. 4. Unfolding of prochymosin by concentrated urea is not fully reversible, unlike that of pepsinogen. Kinetic analysis of the refolding of the proteins suggests the presence of a slow process following unfolding in urea; for pepsinogen this process leads to a slowly refolding form, whereas for prochymosin the slow process in urea leads to a form that cannot refold on dilution of the denaturant. 5. The results provide a rationale for an empirical process for recovery of recombinant prochymosin after solubilization of inclusion bodies in concentrated urea. 6. In all respects studied here, natural and recombinant bovine prochymosin were indistinguishable, indicating that the refolding protocol yields a recombinant product identical with natural prochymosin.     

Autophagy in Tobacco Suspension-Cultured Cells in Response to Sucrose Starvation

The response of tobacco (Nicotiana tabacum) suspension-cultured cells (BY-2) to nutrient starvation was investigated. When the cells that were grown in Murashige-Skoog medium containing 3% (w/v) sucrose were transferred to the same medium without sucrose, 30 to 45% of the intracellular proteins were degraded in 2 d. An analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that proteins were degraded nonselectively. With the same treatment, protease activity in the cell, which was measured at pH 5.0 using fluorescein thiocarbamoyl-casein as a substrate, increased 3- to 7-fold after 1 d. When the cysteine protease inhibitor (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methyl-butane (10 [mu]M) was present in the starvation medium, both the protein degradation and the increase in the protease activity were effectively inhibited. Light microscopy analysis showed that many small spherical bodies accumulated in the perinuclear region of the cytosol 8 h after the start of the inhibitor treatment. These bodies were shown to be membrane-bound vesicles of 1 to 6 [mu]m in diameter that contained several particles. Quinacrine stained these vesicles and the central vacuole; thus, both organelles are acidic compartments. Cytochemical enzyme analysis using 1-naphthylphosphate and [beta]-glycerophosphate as substrates showed that these vesicles contained an acid phosphatase(s). We suggest that these vesicles contribute to cellular protein degradation stimulated under sucrose starvation conditions.     

Structural and circular-dichroism studies on the interaction between human C1-esterase inhibitor and C1s.

The reaction between complement factor C1s and C1-esterase inhibitor has been investigated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, N-terminal amino acid analysis and c.d. studies. It is confirmed that a very stable stoichiometric 1:1 complex with a molecular weight of about 180000 is formed, involving the light chain of C1s. On the sodium dodecyl sulphate/polyacrylamide gels a small peptide with a molecular weight of about 5000 can be seen, which may be released from the C-terminal portion of the inhibitor moiety in a manner analogous to that occurring in other similar proteinase-inhibitor reactions. By N-terminal amino acid analysis, a newly formed threonine residue is found in the complex, suggesting that the inhibitor peptide chain is cleaved in the complex between C1s and C1-esterase inhibitor. The stabilizing bond may therefore be an ester bond. C.d. studies of the native C1-esterase inhibitor indicated the presence of about 38% alpha-helix, about 24% beta-structure and about 38% unordered structure. By gradual cleavage of the disulphide bridges under non-denaturating conditions, gradual changes in the c.d. spectra occurred, suggesting loss of ordered secondary structures. The c.d. spectra of the complex between C1s and C1-esterase inhibitor indicate that tryptophan residues are affected by the complex-formation.     

Action spectrum for the induction of single-strand breaks in DNA in buffered aqueous solution in the wavelength range from 150 to 272 nm: dual mechanism

The induction of single-strand breaks by vacuum-u.v. and far-u.v. radiations (150 nm-272 nm) in plasmid Col E1 DNA in buffered aqueous solution was studied by agarose gel electrophoresis. The results indicated that the mechanisms of DNA strand-break induction are different in the two u.v. regions: above 210 nm the direct excitation of DNA seemed to be the main cause; below 180 nm the photolysis of solvent water seemed to be essential. The demonstration of a systematic protection at 160 nm and not at 209 nm by OH scavengers, such as ethyl alcohol, potassium iodide and D-mannitol, supports the involvement of OH radicals in the vacuum-u.v. region.     

Circular-dichroic properties and secondary structure of Pseudomonas aeruginosa soluble cytochrome c oxidase.

The c.d. spectra of Pseudomonas aeruginosa cytochrome c oxidase in the oxidized state and the reduced state are reported in the visible- and u.v. absorption regions. In the visible region the comparison between the spectra of reduced cytochrome c oxidase and ferrocytochrome c-551 allows the identification of the c.d. bands mainly due to the d1 haem chromophore in cytochrome c oxidase. In the near-u.v. region the assignment of some of the observed peaks to the haem groups and to the aromatic amino acid residues is proposed. A careful analysis of the data in the far-u.v. region leads to the determination of the relative amounts of alpha-helix and beta-sheet in the enzyme, giving for the first time a picture of its secondary structure. A significant difference in this respect between the reduced and the oxidized species is observed and discussed in the light of similar conclusions reported by other workers.     

Inhibition of activated factor XII by antithrombin-heparin cofactor.

The activation of Factor XII occurs via fragmentation of this zymogen into a diverse spectrum of enzymatically potent molecular species. To study the interaction of antithrombin-heparin cofactor and heparin with activated Factor XII, we have employed two forms of this enzyme with widely differing physical characteristics and biologic potencies. Antithrombin-heparin cofactor was found to be a progressive, time-dependent inhibitor of both forms. The addition of heparin dramatically accelerated the rates of these interactions. Furthermore, sodium dodecyl sulfate gel electrophoresis of reduced proteins has indicated that antithrombin-heparin cofactor functions by forming an undissociable complex with either species of the enzyme. This complex represents a 1:1 stoichiometric combination of activated Factor XII and inhibitor. In the presence of heparin, both species undergo virtually instantaneous complex formation with antithrombin-heparin cofactor without exhibiting alterations in dissociability or stoichiometry.     

A re-evaluation of some basic structural and functional properties of Pseudomonas cytochrome oxidase

Determinations of iron content and dry-weight measurements on samples of Pseudomonas cytochrome oxidase were coupled with sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis studies of both the native protein and covalently cross-linked oligomers in order to estimate the enzyme's molecular weight and spectral absorption coefficients. A value of epsilon(ox.) (410)=282x10(3) litre.mol(-1).cm(-1) was calculated for a dimeric protein molecule having a total molecular weight of 122000 (based on iron analysis). Steady-state kinetic observations of the enzyme-catalysed oxidation of reduced azurin by nitrite indicated a marked increase in enzyme inactivation as the pH was raised from 5.7 to 7.2. Since NO, a product of the nitrite reductase activity of Pseudomonas cytochrome oxidase, is known to bind to the enzyme, a study was undertaken to try to assess the potential of NO as a product inhibitor. Investigations showed that samples of the oxidized protein at pH values 4, 5 and 6 bound NO to both haem c and d(1) components, but oxidized enzyme samples at pH7 and above formed their reduced ligand-bound forms when placed under an atmosphere of the gas. Ascorbate-reduced enzyme samples at pH4, 5, 6 and 7 were also found to bind NO at both haem components, although at pH7 the rate of haem c binding was very slow. At pH8 and 9 only the ferrohaem d(1) bound NO. Titration experiments on the reduced protein over the pH range 5-7, with nitrite as a precursor of NO, showed that the haem d(1) had a much higher affinity than the haem c: experiments at pH5.2 and 5.9 with NO-equilibrated solutions revealed the same pattern of behaviour with the oxidized enzyme.     

The reaction of Pseudomonas aeruginosa cytochrome c-551 oxidase with oxygen.

The reaction of ascorbate-reduced Pseudomonas cytochrome oxidase with oxygen was studied by using stopped-flow techniques at pH 7.0 and 25 degrees C. The observed time courses were complex, the reaction consisting of three phases. Of these, only the fastest process, with a second-order rate constant of 3.3 X 10(4) M-1.S-1, was dependent on oxygen concentration. The two slower processes were first-order reactions with rates of 1.0 +/- 0.4s-1 and 0.1 +/- 0.03s-1. A kinetic titration experiment revealed that the enzyme had a relatively low affinity constant for oxygen, approx. 10(4)M-1. Kinetic difference spectra were determined for all three reaction phases, showing each to have different characteristics. The fast-phase difference spectrum showed that changes occurred at both the haem c and haem d1 components of the enzyme during this process. These changes were consistent with the haem c becoming oxidized, but with the haem d1 assuming a form that did not correspond to the normal oxidized state, a situation that was not restored even after the second kinetic phase, which reflected further changes in the haem d1 component. The results are discussed in terms of a kinetic scheme.     

Serine hydroxymethyltransferase from spinach leaf mitochondria. Purification and characterization

A mitochondrial serine hydroxymethyltransferase (EC 2.1.2.1) has for the first time been purified close to homogeneity from a photosynthetically active tissue, spinach ( Spinacea oleracea L. cv Viking II) leaves. The specific activity of the enzyme was 7.8 μmol (mg protein)⁻¹ min⁻¹ using L-serine as substrate. The enzyme was stable for at least 8 weeks at 4°C in the presence of folate. The pH optimum was at pH 8.5 where the enzyme had a K_m for L-serine of 0.9 m M . Carboxymethoxylamine was a strong competitive inhibitor with a K₁ of 1.4 μM. An absorption spectrum taken of the enzyme in the presence of glycine and tetrahydrofolate showed a peak at 492 nm, probably originating from a substrate-enzyme complex. The molecular weight obtained by gel filtration was 209 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified enzyme showed that the apparent molecular weight of the subunit was 53 kDa, indicating four subunits.     

Purification and characterisation of a malto-oligosaccharide-forming amylase active at high pH from Bacillus clausii BT-21

Bacillus clausii BT-21 produced an extracellular malto-oligosaccharide-forming amylase active at high pH when grown on starch substrates. The enzyme was purified to homogeneity by affinity and anion-exchange chromatography. The molecular weight of the enzyme estimated by sodium dodecyl sulfate polyacrylamide electrophoresis was 101 kDa. The enzyme showed an optimum of activity at pH 9.5 and 55 degrees C. Maltohexaose was detected as the main initially formed starch hydrolysis product. Maltotetraose and maltose were the main products obtained after hydrolysis of starch by the enzyme for an extended period of time and were not further degraded. The enzyme readily hydrolysed soluble starch, amylopectin and amylose, while cyclodextrins, pullulan or dextran were not degraded. The mode of action during hydrolysis of starch indicated an exo-acting type of amylolytic enzyme mainly producing maltohexaose and maltotetraose. Amino acid sequencing of the enzyme revealed high homology with the maltohexaose-forming amylase from Bacillus sp. H-167.     

Clavulanate inactivation of Staphylococcus aureus beta-lactamase.

The interaction of clavulanic acid with beta-lactamase from Staphylococcus aureus was investigated, particularly with a view to determining whether conformational effects are involved. The inactivation at neutral pH is essentially stoichiometric, leading to an inactive species with an enamine chromophore. Two forms of the enamine were observed, the first-formed having a positive ellipticity with a maximum near 290 nm. This species slowly converted into the stable form of the inactivated enzyme that had a negative ellipticity with a minimum at 275 nm. This change in sign of the ellipticity of the enamine is consistent with the previously proposed cis-trans isomerization of the enamine [Cartwright & Coulson (1979) Nature (London) 278, 360-361). Both the far-u.v.c.d. and the intrinsic viscosity of the inactivated enzyme indicated that negligible change in conformation of the enzyme accompanied inactivation. The rates of inactivation and enamine formation were compared at low temperatures, where the initial rates were slow enough to be monitored. The rate of loss of 95% of the catalytic activity was almost 100-fold faster than the rate of formation of the first-formed enamine species. The remaining 5% activity was lost with a rate comparable with that for formation of the initial enamine. The simplest explanation of these results is that a relatively stable acyl-enzyme intermediate builds up initially and more slowly partitions between turnover (hydrolysis) and enamine formation. The initially formed enamine is in the cis conformation but slowly isomerizes to the more stable trans form.     

Comparison of pepsins isolated from porcine, bovine and Penicillium jathiuellum

1. The reactivities of pepsins, isolated from three different sources (porcine, bovine, and Penicillium jathiuelum), toward ester and peptide substrates were compared. 2. Porcine pepsin showed the highest activity followed by penicillopepsin with bovine pepsin being the least active. 3. The esterase activity of penicillopepsin was greater than that of porcine pepsin with bovine pepsin again showing the least activity. 4. The CD spectra indicate that porcine and bovine pepsin have similar conformations, even though bovine pepsin shows less ellipticity at 220 nm. 5. Penicillopepsin showed a completely opposite sign in the near-u.v. region of the CD spectrum. 6. The far-u.v. region of the CD spectrum of penicillopepsin strongly suggests a beta-sheet structure. 7. Previously reported X-ray crystallographic data suggest that porcine pepsin has a compact three-dimensional structure, while the structures of bovine and penicillopepsins are partially unfolded.