Biochimica et Biophysica Acta (BBA) - Enzymology最新文献

Pepsin treatment of ascitic fluid from patients with neoplasia generated a cysteine (thiol) proteinase activity which resembles cathepsin B (EC 3.4.22.1) in its requirements for thiol activators, susceptibility to inhibitors and specificity for synthetic substrates. As judged by gel filtration, pepsin reduced the molecular size of the latent enzyme from an M_r of 41 000 to 33 000 after activation. Both forms are larger than human liver cathepsin B. In addition to its presence in ascitic fluid, the pepsin-activated species was found in the medium of ascites cells maintained in culture. The latent enzyme may be an enzyme-inhibitor complex or an inactive precursor of a cathepsin B-like proteinase.

With the aim of confirming our previous spectrophotometric binding studies ((1978) Eur. J. Biochem. 85, 345–350 and (1980) Eur. J. Biochem. 104, 249–254) and of ascertaining the full physiological significance of ion binding, we investigated the effects of ions and thiol reagents on the proteolysis of yeast phosphoglycerate kinase (ATP : 3-phospho-d-glycerate 1-phosphotransferase, EC 2.7.2.3). The single non-essential thiol of the enzyme was modified with 5,5′-dithiobis(2-nitrobenzoic acid) or 2-chloromercuri-4-nitrophenol. Both modifications greatly increased the susceptibility of the kinase to inactivation by trypsin or yeast proteinase A, when compared with that of the native kinase. Electrophoresis in sodium dodecyl sulfate (SDS) revealed that limited proteolysis had occurred. The time courses for the proteolysis and loss of catalytic activity were followed and the active and inactive fragments identified. The molecular masses of the major proteolytic fragments differed with the two endopeptidases. Substrate and non-substrate anions in a concentration-dependent fashion, protected the native and mercurial-labelled kinase from inactivation by trypsin or yeast proteinase A. However, Zn²⁺, in a concentration-dependent fashion, increased the susceptibility of the native kinase to inactivation by each endopeptidase. The time courses for the inactivation and for the proteolysis allowed the active and inactive fragments to be identified. Zn²⁺ decreased the rate of inactivation of the mercurial-labelled kinase by proteinase A. The effects of these ions were detected at concentrations compatible with occupancy of an anion binding site and a low affinity Zn²⁺ binding site, both of which have been indicated from our previous binding studies.

D. discoideum amoebae were found to phosphorylate plasma membrane proteins when intact cells were incubated with either [γ-³²P]ATP or [³²P]phosphate. In the first case, the incorporation was largely a consequence of the hydrolysis of [γ-³²P]ATP, cellular uptake of the generated [³²P]phosphate and its subsequent incorporation into ATP. When the contribution of this process to the phosphorylating activity of intact cells was eliminated, an ecto-protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) activity could be demonstrated. As amoebae progressed through their aggregation program, they showed a decreased ability to phosphorylate their plasma membrane when incubated with [γ-³²P]ATP or [³²P]phosphate. Analysis of ATPase activity, permeability properties and the pattern of proteins phosphorylated by intact cells and isolated plasma membranes lead to the following conclusions: the lower levels of phosphorylation observed with starved cells reflected an altered uptake of [³²P]phosphate by these cells rather than a significant change in the plasma membrane protein kinase activity. Neither the substrates nor the activity of the ecto-protein kinase was dramatically altered during starvation.

Similarity of the protein tertiary structures of the native horseradish peroxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) and protoporphyrin-apoperoxidase complex has been shown on the basis of identity of the tryptophan fluorescence parameter at pH 2.0–8.0 and of the circular dichroism spectra of the two proteins. Absorption and fluorescence spectra have been obtained for protoporphyrin in the complex in the pH range 7.0–1.6. A shift in the apparent pK by 4 units has been observed for protonation of the protoporphyrin pyrrolic ring in the complex. From this shift, the dielectric constant has been evaluated for the heme pocket of the peroxidase (approx. 20). Fluorescence quantum yield of protoporphyrin in the complex increased with pH decreasing from 5.0 to 3.5, whereas the spectrum pattern and fluorescence lifetime did not change. The ions, I⁻ and [Fe(CN)₆]⁻⁴, peroxidase substrates, did not quench the protoporphyrin fluorescence in the complex at about neutral pH, whereas the quenching markedly enhanced with lowering pH. The bimolecular constant for the I⁻-quenching of the porphyrin fluorescence in the complex showed a pH-dependence similar to that of the bimolecular rate constant for the reaction of peroxidase compound I with I⁻. A mechanism for I⁻ oxidation at an acid pH in the presence of peroxidase has been proposed.

Two classes of inhibitors of trypsin (EC 3.4.21.4) have been studied, viz. active site-directed agents such as ovomucoid and active site titrants such as 4-methylumbelliferyl-4-guanidinobenzoate. The kinetics of β-naphthylamidase inhibition by an active site-directed agent were markedly different from simultaneous assays of the availability of the active site towards active site titrants in the presence of the active site-directed agents. Analysis of these data indicated an exchange of active site-directed agent by subsequent addition of active site titrant. One class of trypsin inhibitor could be displaced by another from the trypsin active centre. Competitive chase experiments were designed to measure this exchange in which the active site-directed agent was first equilibrated with trypsin, then partially displaced by incremental additions of an active site titrant; the degree of active sites occupied by these two agents was then determined by active site titration with a second reagent.

The hydrogenase of Rhodopseudomonas capsulata is an intrinsic membrane protein extractable from the membrane by detergents. Triton X-100 produces stable soluble extracts. Stability of solubilized hydrogenase depends drastically on two factors: temperature and gas-phase. The solubilised hydrogenase is more stable at 20°C than in the cold and is further stabilised under an H₂ atmosphere. The kinetic properties of the membrane-bound and Triton-solubilised forms of the enzyme have been compared. Both forms of the enzyme show a pH optimum for the reduction of benzyl or methyl viologen at 8.5–9.0, for H₂ production with methyl viologen semiquinone at 5.7 and for H²H exchange at 4.5. In vitro, the hydrogenase functions as a reversible enzyme although at a slower rate for H₂ evolution than for H₂ uptake. The apparent K_m for H₂ (uptake) is 0.25 μM. The artificial electron acceptors having the highest affinity for hydrogenase are methylene blue (K_m = 60 μM) and benzyl viologen (K_m = 100 μM). Methyl viologen has a higher affinity in the semiquinone form (K_m = 450 μM) than in the oxidized dicationic form (K_m = 3.6 mM). The Arrhenius plot of the activity of hydrogenase in the membrane and in the solubilised extract shows a break at 13°C. This transition temperature of 13°C is probably linked to a change of protein conformation. The activation energy is 110 kJ/mol (26.4 kcal/mol) adnd 38 kJ/mol (9.1 kcal/mol) below and above the transition temperature, respectively. While hydrogenase is a cold labile enzyme, it is remarkably resistant to heat inactivation, for example the membrane-bound form can withstand heating at 80°C for 3 h without loss of activity.

The total -SH content of purified NADPH-cytochrome P-450 reductase (NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) from rabbit liver microsomes accessible to an excess equivalent of PCMB was 7.0 ± 0.3 mol thiol groups/mol protein. The modification of four -SH groups at low concentrations of PCMB stimulated the activity of the enzyme. On the other hand, further blocking of -SH groups (6–7 mol -SH groups/mol protein) with an excess amount of PCMB completely inhibited cytochrome c (or DCPI) reductase activity. The fluorescence quenching of the flavin was rapidly removed by binding of PCMB to a fifth and sixth -SH group during a gradual titration. Kinetic and fluorimetric analyses confirmed the suggestion that these two -SH groups essential for catalytic function were partly protected by NADP⁺ or 2′-AMP against the reaction with PCMB. Excess PCMB begins to compete with the ligand preincubated with the enzyme. The spectral perturbation on the addition of approx. 6–7 equiv. PCMB/mol enzyme is accompanied by a slight blue shift of the absorbance maximum at 380 nm, with the appearance of a pronounced shoulder at 475 nm. In contrast to the native enzyme, 3-electron-reduced semiquinone form of PCMB-treated enzyme showed the same absorption spectrum as 1-electron-reduced semiquinone which has an absorption maximum at 585 nm with a broad shoulder around 635 nm. An inhibitory effect may be attributable to the fact that NADPH is less accessible to the FAD binding site as well as the pyridine nucleotide binding site, since the rate of FAD reduction becomes extremely slow after complete modification.

A steady-state differential equation that describes the kinetics of suicide substrate was derived for a scheme presented by Walsh et al. (Walsh, C., Cromartie, T., Marcotte, P. and Spencer, R. (1978) Methods Enzymol. 53, 437–448). Using its analytical solutions, the progress curves of substrate disappearance, product formation and enzyme inactivation were calculated for a hypothetical model system, and were compared with the exact solutions which were obtained by the numerical computation on a set of rate equations. The results obtained with the present analytical solutions were much more consistent with the exact solutions than those obtained using Waley's solutions (Waley, S.G. (1980) Biochem. J. 185, 771–773). The most important factor for a system of suicide substrates was found to be the term (1 + r)μ instead of rμ as proposed by Waley, where r is the ratio of the rate constant of product formation to that of enzyme inactivation and μ is the ratio of initial concentration of enzyme to that of suicide substrate. In cases where this term has a value greater than unity, all the molecules of suicide substrate are used up leaving some enzyme molecules still active. To the contrary, in cases where the term has a value smaller than unity, all the enzyme molecules are inactivated with some molecules of suicide substrate being left unreacted. When the term is equal to unity, then all the enzyme molecules are inactivated and all the molecules of the suicide substrate are converted. Practical methods for estimating kinetic parameters are described.

Aspergillus niger ATCC 6274 was selected as an aldose 1-epimerase (EC 5.1.3.3) producer from 45 stock cultures of A. niger. The aldose 1-epimerase was purified 115-fold to apparent homogeneity from cell extracts with a yield of 2.6%. The molecular weight was calculated to be 260 000 and that of the subunit to be 130 000. The enzyme preparation was active at pH 5–7. The K_m value was 50 mM and the V value was 1200 units/mg toward α-d-glucose. This enzyme catalyzed mutarotation of the following substrates; α-d-glucose, β-d-fructose, β-l-arabinose and β-d-galactose. The time required for glucose determination with a glucose oxidase reagent was significantly shortened by the addition of aldose 1-epimerase.

Cathepsin D (EC 3.4.23.5) was purified from rabbit skeletal muscle using acetone-dried muscle powder as starting material. After the acetone-dried powder was extracted with 0.2 mM ATP, the extract was fractionated with acetone and subjected to DEAE-Sephadex A-50 and Sephadex G-100 column chromatography. Rechromatography on a Sephadex G-100 column resulted in a purified preparation. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed one major band of 42 000 daltons and some bands of contaminants. Since gel filtration also indicated a value of 42 000 daltons for the enzyme, it was concluded that muscle cathepsin D has no subunit structure. The enzyme acted optimally towards myofibrils around pH 3, resulting in the degradation of the myosin heavy chain and production of a 30 000-dalton component.