Biochimica et Biophysica Acta (BBA) - Protein Structure最新文献

C1s and C1r proenzymes and enzymes (C1s, C1r) and C1q were labeled with ¹²⁵I. The distribution of the ¹²⁵I label between H- and L-chain of C1s was only slightly dependent on the state of activation of C1s, and approx. 90% of the label was found in the H-chain. In the Clr proenzyme molecules 50% of the label was incorporated into the H-chain. The C1r H-chain label was reduced to 10% on activation of C1r to C1r, while the L-chain label increased to 90% of the total label. The presence of either C1s, C1q or C1qs during labeling reduced the C1r H-chain label, although C1r remained in the proenzyme form. The presence of C1s or C1rs enhanced the ¹²⁵I uptake of C1q in Ca²⁺ or EDTA medium. This was unexpected because one would have anticipated a diminution of the C1q label due to the apposition of C1r and C1s, similarly as it occurs during C1rs complex and C1s dimer formation for the H-chain label of C1s. The results show that C1r and C1q after their conformation during activation and C1 complex formation.

‘Thermitase’ (EC 3.4.21.14), a thermostable extracellular serine protease from Thermoactinomyces vulgaris, binds one calcium ion with a dissociation constant of about 10⁻⁴ M at 25°C and pH 7.5 to 3.5. In addition, two calcium ions are bound more tightly to the enzyme, as shown by experiments with a calcium-selective electrode. The single most weakly bound calcium ion causes a slight quenching of the protein fluorescence emission, accompanied by a stabilization against thermal denaturation or autolysis and an increase of esterolytic activity of approx. 10%. The tightly bound calcium ions have only a slight influence on activity or on thermal denaturation or autolytic degradation. The activation parameters of thermal denaturation indicate that ‘thermitase’ belongs to the class of thermostable enzymes with a high intrinsic stability.

Isoelectric focusing was performed on bacteriorhodopsin isolated from Halobacterium halobium, R₁ strain, solubilized in Nonidet P-40, on sucrose density gradient stabilized columns. When 560 nm absorbance was monitored, four forms of bacteriorhodopsin were observed, having isoelectric points (pI) of: A, 3.93; B, 4.34; C, 5.03; D, 5.49. Instability of some of the isoelectic forms during the very process of electrofocusing was observed. When focused over a period of 6 days, the relative abundance of the forms changed although their pI values remained constant. Refocusing of the isolated forms A or B led to the production of form C. The latter species was stable to refocusing. Form B was unstable to storage either as an aqueous suspension of the purple membrane or as a detergent extract. Each of the forms had the absorption spectrum typical for bacteriorhodopsin and each showed identical patterns after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

α-Actinin increases the ATPase activity of actin by up to 84%, depending on pH, divalent cations present and the added Mg²⁺: ATP ratio. Dithiothreitol decreases actin ATPase activity approx. 20% but does not reduce the ability of α-actinin to increase actin ATPase activity. Increasing amounts of added α-actinin up to 1 mol α-actinin to 49 mol actin cause an increasing increment in actin ATPase activity, but adding α-actinin beyond 1 mol α-actinin to 49 mol actin elicits only small additional increments in activity. Actin ATPase activity ranges from approx 100 nmol P_i/mg actin per h (4.3 mol P_i/mol actin per h) at high levels (10 mM) of ATP in the presence of lower amounts (1 mM) of added Mg²⁺ to approx. 12.5 nmol P_i/mg actin per h (0.52 mol P_i/mol actin per h) at high pH (8.5) or at low levels (0.5–1.0 mM) of ATP in the presence of higher amounts (10 mM) of added Mg²⁺. ATP uncomplexed with Mg²⁺ inhibits the ability of α-actinin to increase F-actin ATPase activity. Activities with different divalent cations showed that the actin ATPase in these studies, which was 1/100 as great as Mg²⁺-modified actomyosin ATPase activity, was not due to trace amounts of myosin contaminating the actin preparations. The results are consistent with the concept that α-actinin can alter the structure of actin monomers.

A carbohydrate-rich, water-soluble glycoprotein has been isolated in pure form from delipidated lung lavage fluid from a patient with pulmonary alveolar proteinosis, in a three-step procedure involving ion-exchange and gel filtration chromatography. The molecular weight of the glycoprotein was determined to be 45 900 by sedimentation equilibrium analysis in the analytical ultracentrifuge and 45 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, indicating a single polypeptide chain. Nearly half of the mass of the glycoprotein is comprised of carbohydrate that is contributed by 24 residues sialic acid, 23 residues N-acetylglucosamine, 6 residues N-acetylgalactosamine, 19 residues galactose, 4 residues mannose, 1 residue fucose and 1 residue glucose per mol. Unlike a number of collagen-related glycoproteins that have been isolated by others from insoluble lung contents in pulmonary proteinosis, the water-soluble glycoprotein described in the present report does not contain hydroxyproline or hydroxylysine and contains less than 10% of its amino acid residues as glycine. Using rabbit antibodies directed against our purest preparation of material and an immunoperoxidase staining procedure, the 45 000 molecular weight glycoprotein was localized to the thin film of fluid lining the surfaces of alveoli in normal human lungs.

We have examined the α-actinin-F-actin interaction by measuring the effect of highly purified α-actinin on bound nucleotide exchange in F-actin. Exchange was followed by measuring the release of actin-bound [¹⁴C]ADP in the presence of ATP using an ultrafiltration technique. α-Actinin increases by about 60 to 70% the rate of release of F-actin bound nucleotide when incubated for 1 h in the presence of 1 mM ATP/1 mM MgCl₂/0.05 mM CaCl₂/0.5 mM dithioerythritol/100 mM KCl/20 mM Tris-acetate, pH 7.5, at 37°C. The ability of α-actinin to enhance nucleotide exchange was maximal when α-actinin was added at a level near 10% of actin present by weight (molar ratio of 1 α-actinin to 49 actin monomers). The potentiating effect of α-actinin on the nucleotide exchange rate of F-actin was not highly related to the Mg²⁺: ATP ratio present in the incubation mixture. α-Actinin also increased the rate of bound nucleotide exchange of F-actin when the actin was present in a reconstituted actomyosin suspension. The results are consistent with the possibility that one α-actinin can affect the structure of multiple actin monomers present in an actin filament.

The lengths of the carbonyl as well as of the adjacent C-N and C-C bonds in peptides are shown to vary systematically with the central C-N bond length. Results of ab initio calculations on N-methylacetamide and its Li⁺, Na⁺ and Mg²⁺ complexes are also discussed.

A cyclic AMP-dependent protein kinase, its regulatory (R) and catalytic (C) protein were isolated from bovine liver. The cyclic AMP-dependent protein kinase showed two protein bands on SDS-polyacrylamide gel electrophoresis with molecular weights of 54 000 and 40 000. They correspond to the data for the separately isolated R- and C-protein. The molecular weight of the holoenzyme ranged from 172 000–179 000, depending on the estimation method. The molecular weight of the R-protein ranged from 97 000–98 000. This, and the results of the SDS-polyacrylamide gel electrophoresis, demonstrates a dimeric structure. The frictional ratios ($\text{f}\text{f}{}_0$) of 1.67–1.7 for the holoenzyme and 1.9 for the R-protein correspond to highly asymmetric shapes. Assuming a prolate form, the axial ratios are 13–14 and 17, respectively. The C-protein is globular ($\text{f}\text{f}{}_0$ 1.1–1.26, axial ratio 3–5). The seondary structure with 35% α-helix, 19% β-sheet and 49% aperiodic form of the holoenzyme is similar to the R-protein with 35, 19 and 46%, respectively. The C-protein contains 29% α-helix, 21% β-sheet and 50% aperiodic form. The dimeric R-protein binds 4 mol cyclic AMP and can be phosphorylated in the presence of the C-protein. An absorption coefficient, A_280nm^1.0%, of 5.4 was calculated for the R-protein and of 13.6 for C-protein. The data for C-protein, e.g., molecular weight, heterogeneity in isoelectrofocusing, phosphate content, etc., are in good agreement with those found by Sudgen, P.H. and Corbin, J.D. (1976) Biochem. J. 159, 423–437.

Ferredoxin II from Desulphovibrio gigas is a tetrameric protein containing a novel iron-sulphur cluster consisting of three iron atoms. The low-temperature magnetic circular dichroism (MCD) spectra of the oxidized and dithionite-reduced forms of ferredoxin II have been measured over the wavelength range approx. 300–800 nm. Both oxidation levels of the cluster are shown to be paramagnetic, although only the oxidized form gives an EPR signal. MCD magnetization curves have been constructed over the temperature range approx. 1.5–150 K and at fields between 0 and 5.1 Tesla. The curve for the oxidized protein can be fitted to a ground state of spin $\text{S =}\text{1}\text{2}$ with an isotropic g factor of 2.01. There is evidence for the thermal population of a low-lying electronic state above 50 K. The reduced protein gives a distinctive set of magnetization curves that are tentatively assigned to a ground state of S = 2, with a predominantly axial zero-field distortion that leaves the doublet M_s = ±2 lowest in energy. The zero-field components have a maximum energy spread of approx. 15 cm⁻¹. which places an upper limit of 4 cm⁻¹ on the axial zero-field parameter D. The MCD spectra of the oxidized and reduced forms of the cluster are quite distinctive from one another. The spectra of the oxidized state are also different from those of oxidized high-potential iron protein from Chromatium and should provide a useful criterion for distinguishing between four- and three-iron clusters in their highest oxidation levels.

In the marine terebellid worm Amphitrite ornata the vascular fluid contains a high molecular weight erythrocruorin, while cells of the coelom contain a monomeric hemoglobin. The structural integrity of the erythrocruorin molecule is known to be dependent on the presence of a minimal concentration of divalent cations (1–3 mM) in the medium. The functional properties of Amphitrite erythrocruorin are also affected by cations. The oxygen affinity tends to increase with increasing cation concentration and the degree of cooperative interactions, expressed in the kinetics and equilibria of ligand binding, goes through a maximum. Maximal Hill coefficients of 3–4 are observed with 50 mM CaCl₂, 50 mM MgCl₂ or 1 M NaCl in measurements at the physiological pH of 7.75. Only 2 mM CaCl₂ is required for maximal cooperativity at pH 8.5. This suggests partial deprotonation of the cation binding site at high pH. It is somewhat unusual that pH effects on cooperativity are reversible, since this is not a common feature of the giant erythrocruorin molecules. The oxygen binding experiments revealed a marked effect of divalent cations of Amphitrite erythrocruorin at high pH and cation concentration. Above pH 8.5, at 50 mM CaCl₂ and 12°C, the erythrocruorin will form a polymer upon deoxygenation. This polymerization is readily reversible by bringing the temperature from 12 to 20°C or by oxygenation. Under physiological conditions of pH and cation concentration and at 12°C, the ervthrocruorin and the monomeric coelomic hemoglobin require a similar oxygen pressure for half saturation. However, the allosteric regulation of function is absent for the coelomic protein.