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

Glycoproteins obtained from large amounts of highly purified synaptic vesicles isolated from adult rat brain were fractionated by sequential affinity chromatography in the presence of SDS on four different immobilized lectins: concanavalin A, Ulex europeus lectin, Ricinus sanguinis lectin and wheat germ agglutinin. 83% of the total protein-bound sugar of synaptic vesicles can be adsorbed on the lectins and separated from the bulk of carbohydrate free proteins. Nine fractions containing only glycoproteins and differing by their terminal sugars were separated and analysed for their carbohydrate composition and electrophoretic profiles. A considerable heterogeneity of the glycoprotein population was observed which cannot be explained solely by the microheterogeneity of the glycans of the synaptic vesicle glycoproteins.

Single relaxations for the equilibration of O₂ with monomeric and octameric deoxy forms of hemerythrin from Themiste zostericola have been observed at 25°C using the temperature-jump technique. At 25°C, pH 8.2 (Tris/H₂SO₄ and $\text{I = 0.10}\text{M}$ (Na₂SO₄), formation rate constants $\text{k}{}_{\mathrm{<mtext>on</mtext>}}$ are 7.8 · 10⁷ M⁻¹ · s⁻¹ and 7.5 · 10⁶ M⁻¹ s⁻¹, respectively. The procedure used does not give a precise measure (small intercepts) of dissociation rate constants, $\text{k}{}_{\mathrm{<mtext>off</mtext>}}$. These were determined instead by the stopped-flow method using dithionite to induce dissociation of the oxy protein. Values of $\text{k}{}_{\mathrm{<mtext>off</mtext>}}$ for the monomer (3.1 · 10² s⁻¹) and octamer (82 s⁻¹), in association with $\text{k}{}_{\mathrm{<mtext>on</mtext>}}$ values, lead to equilibrium constants for the formation of oxyhemerythrin of 2.5 · 10⁵ M⁻¹ and 0.9 · 10⁵ M⁻¹, respectively, at 25°C, pH 8.2 and $\text{I = 0.10}\text{M}$ (Na₂SO₄). These latter are in reasonable agreement with values (1.5 10⁵ M⁻¹ and 1.3 · 10⁵ M⁻¹) determined spectrally on the equilibrated solutions. Using the octameric protein, it was shown that replacement of $\text{SO}{}_4{}^{\mathrm{2-}}$ by $\text{ClO}{}_4{}^{\mathrm{-}}$ or Cl⁻ ions (at a constant $\text{I = 0.10}\text{M}$) led to an approximately 2-fold enhancement of k_on but had little effect on $\text{k}{}_{\mathrm{<mtext>off</mtext>}}$. The addition of Ca²⁺ or Mg²⁺ ions (0.01 M), with or without 0.50 M NaCl, also gives up to 4-fold increases in $\text{k}{}_{\mathrm{<mtext>on</mtext>}}$, but unchanged $\text{k}{}_{\mathrm{<mtext>off</mtext>}}$ values. Oxygen pulse experiments on the octamer show no effect on $\text{k}{}_{\mathrm{<mtext>off</mtext>}}$ of the degree of oxygenation of the protein. A comparison is made with similar data for hemoglobin, myoglobin and hemocyanin.

Biotin-binding and immunological methods were employed to demonstrate the similarity of oviduct and non-oviduct avidin in the chicken. Oviduct avidin was induced after oestrogen pretreatment by progesterone and non-oviduct avidin by intestinal tissue injury or by intraperitoneal actinomycin D administration. Avidin in the intestine, lung, bursa of Fabricius, plasma, pectoral muscle and liver after injury had biotin-binding activity similar to that of progesterone-induced oviduct avidin: (1) a temperature of 79–83°C was required for 50% of the maximum [¹⁴C]biotin uptake, (2) maximal exchange occurred only at 90 or 100°C and (3) denaturation of protein, i.e., loss of biotin-binding activity, was not yet observed at 100°C. Avidin in the intestine, lung, bursa of Fabricus, plasma and pectoral muscle also showed an identical cross-reaction with oviduct avidin. Furthermore, the increase in avidin-like biotin binding in the oviduct and most non-oviduct tissues was significantly correlated with the increase in avidin-like antigen in the tissue. This indicates that avidin induced in chicken non-oviduct tissues by injury or inflammation caused by actinomycin D administration is similar to progesterone-dependent oviduct avidin.

The first component of complement (C1q) coupled to Sepharose by cyanogen bromide was found not to bind aggregated human γ-globulin or immune complexes at room temperature, whereas at 4°C binding was nearly complete. The temperature sensitivity of solid phase C1q binding was reversible. Elution of aggregated human γ-globulin bound at 4°C was possible by raising the temperature to 23°C. However, free C1q or C1q adsorbed onto polystyrene balls could bind immune complex-like material at both 23 and 4°C. The conformational restraints of C1q covalently coupled to a solid support may not allow functional activity at elevated temperatures.

Endogenous phosphorylation of intact cells was studied with four mouse, hamster and human cell lines using [γ-³²P]ATP and [γ-³²P]GTP as exogenous substrates. With all four cell lines distinct differences in the phosphoprotein patterns could be demonstrated for cells grown in suspension culture compared to cells grown in monolayers. Two major, apparently ubiquitous phosphoproteins with molecular weights of 135 000 (128 000 in HeLa eells) and 105 000, representing up to 60% of total phosphorylation, were phosphorylated only in cells grown in suspension. These phosphoproteins and the kinase(s) were located on the surface of the suspension cells. Evidence showed that phosphorylation was apparently not a true endogenous reaction, that rather it occurred by cell-cell collision, showing exponentially increasing ³²P incorporation with increasing cell population density. Phosphorylation of pp135 and pp105 was established with ATP as well as with GTP and was not dependent on cyclic nucleotides cyclic AMP, cyclic GMP and cyclic CMP. The substrate-attached cells of all four cell lines have protein kinases on the cell surface. The lack of pp135 and pp105 phosphorylation may be due to the fact that these phosphoproteins are not expressed at all on the surface of substrate-attached cells or that these phosphoproteins are already fully phosphorylated.

By the use of a stopped-flow light-scattering device, in combination with a stopped-flow fluorescence (or ultraviolet absorption) measurement, one can examine a correlation between an intermolecular association (or dissociation) reaction and an intramolecular conformation change of a macromolecule involved.

Chromatographically purified Hb F₀, Hb A₀ and Hb S₀ fractions were incubated with 70 μM [¹⁴C]acetyl-CoA for 3 h in 20 mM Tris-HCl, pH 7.6 and 8.6. The acid-precipitable radioactivity was monitored and the hemoglobins were separated by Biorex 70 chromatography. The pH dependence of acetylation showed an increase in acetylation with an increase in pH from 7.6 to 8.6, whereas an increase in ionic strength decreased acetylation. Incubation of Hb F₀ with [¹⁴C]acetyl-CoA resulted in modified hemoglobin and γ chains that co-chromatographed with Hb F_1c and γ_Ic chains, respectively. Acetylation of Hb A₀ and Hb S₀ produced minor hemoglobins whose chromatographic mobilities were slightly faster than those of Hb A_Ic and Hb S_Ic, respectively. Radioactivity peaks also appeared at the leading edges of the major hemoglobin zones as well, which indicates that, like non-enzymatic glycosylation, non-enzymatic acetylation of hemoglobins involves both specific and nonspecific reactions.

The EPR spectrum at 15 K of Pseudomonas cytochrome $\text{c}$ peroxidase, which contains two hemes per molecule, is in the totally ferric form characteristic of low-spin heme giving two sets of $\text{g-}\text{values}$ with $\text{g}{}_{\mathrm{z}}$ 3.26 and 2.94. These vaues indicate an imidazole-nitrogen : heme-iron : methionine-sulfur and an imidazole-nitrogen : heme-iron : imidazole-nitrogen hemochrome structure, respectively. The spectrum is essentially identical at pH 6.0 and 4.6 and shows only a very small amount of high-spin heme iron ($\text{g 5–6}$) also at 77 K. Interaction between the two hemes is shown to exist by experiments in which one heme is reduced. This induces a change of the EPR signal of the other (to $\text{g}{}_{\mathrm{z}}\text{2.83, g}{}_{\mathrm{y}}\text{2.35}$ and $\text{g}{}_{\mathrm{x}}\text{1.54}$), indicative of the removal of a histidine proton from that heme, which is axially coordinated to two histidine residues. If hydrogen peroxide is added to the partially reduced protein, its EPR signal is replaced by still other signals ($\text{g}{}_{\mathrm{z}}\text{3.5}$ and 3.15). Only a very small free radical peak could be observed consistent with earlier mechanistic proposals. Contrary to the EPR spectra recorded at low temperature, the optical absorption spectra of both totally oxidized and partially reduced enzyme reveal the presence of high-spin heme at room temperature. It seems that a transition of one of the heme $\text{c}$ moieties from an essentially high-spin to a low-spin form takes place on cooling the enzyme from 298 to 15 K.

A soluble immunoactive peptide with a molecular weight of 16 000 was isolated and purified from the cyanogen bromide digest of the insoluble 50 000 dalton glial fibrillary acidic protein by Sephacryl S-200 gel filtration followed by DEAE-Bio-gel A chromatography. The homogeneity of the peptide was established by SDS-polyacrylamide gel electrophoresis and isoelectric focusing. The peptide from several species showed immunocrossreaction with rabbit antibody to intact glial fibrillary acidic protein. The peptide has a $\text{p}\text{I}$ value of 5.32. The amino acid sequence of 28 residues from the amino terminus of the calf peptide has been determined.

Interactions between human collagens type I, II and III with human C1q or its collagen-like fragment (CLF) were investigated with different techniques. It was found that in solution both C1q and CLF form stable complexes with the native collagens. No preferential binding to a specific collagen type was observed. If C1q (CLF) was adsorbed to polystyrene or fixed to erythrocytes, a more efficient interaction with collagen was displayed by C1q than by CLF. If collagen represents the solid phase, the binding of CLF is stronger than that of C1q. Inhibition studies indicate that the interaction between C1q and collagens takes place via the collagen-like part of C1q. Intermolecular attraction due to polar amino acid residues seems to be of major importance for this interaction.