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

The subunit composition of the high molecular weight proteins cylindrin and torin from human erythrocyte ghosts has been studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis on 3 to 30% ‘Gradipore’ polyacrylamide gradient slab gels. Torin has been shown to be a multimer of a single polypeptide of approx. $\text{M}{}_{\mathrm{r}}$ 20 000. Cylindrin appears to contain five polypeptides, three of which predominate, in the $\text{M}{}_{\mathrm{r}}$ range 22 000 to 25 000. The isoelectric points (pI) of cylindrin and torin have been determined as 4.6 and 4.8, respectively. The molecular properties of cylindrin and torin are discussed in relation to the previous studies by the authors and others on these proteins.

In the silkworm, Bombyx mori, a group of structurally related proteins referred to as ‘30K proteins’ comprises the major plasma proteins of the last instar larvae. Four protein components consisting of 30K proteins were purified to homogeneity from the larval hemolymph and designated Component 1, 2, 3 and 4, respectively. Close similarity in amino acid composition was noticed between Components 1 and 3, and between Components 2 and 4. Rabbit antibody prepared against Component 4 crossreacted with Component 2 as well as Component 4 but not with Components 1 or 3. In a cell-free translation system, RNA isolated from the fat body of the last instar larvae directed the synthesis of proteins reactive with anti-Component 4 antibody. Developmental change in the hemolymph concentration of 30K proteins well reflected the level of functional mRNA for these proteins in the fat body, indicating that the biosynthesis of 30K proteins is regulated during development at pre-translational level.

A simple procedure is described for renaturing dodecyl sulfate-unfolded enzymes. The method involves the direct addition of a large molar excess of the non-ionic detergent Triton X-100 to protein-dodecyl sulfate complexes either in solution or as a band on a polyacrylamide gel. The cytoplasmic enzymes lactate dehydrogenase and malate dehydrogenase have been renatured by this protocol. On the other hand, no recovery of activity was found with the mitochondrial isoenzyme of malate dehydrogenase or the mitochondrial enzymes glutamate dehydrogenase or fumarase. Possible implications of the differences in the ability of cytosolic and mitochondrial enzymes to renature under these conditions are discussed in terms of their biosynthesis.

The functional properties of two new abnormal hemoglobins with high oxygen affinity were studied. Hb Hôtel Dieu β99 (G1) Asp → Gly is situated in the α₁β₂ contact. Hb Pitié Salpétrière β34 (B16) Val → Phe is situated in the α₁β₁ contact. Both hemoglobins exhibited similar functional properties with a 10-fold increased oxygen affinity, a decreased cooperativity, a decreased Bohr effect and a normal or slightly decreased 2,3-diphosphoglycerate effect. The structure-function relationship is discussed in the light of these results.

The action of trypsin at 55°C and pH 8.3 on pig IgM anti-Salmonella has been compared with the action of pepsin at 37°C and pH 4.6. Both processes cause the gradual removal of Fab arms and C_μ2 domains to produce eventually an (Fc)₅ fragment. However, during tryptic digestion Fab arms are preferentially removed from the same subunit, whereas peptic digestion causes random removal from any subunit. At intermediate stages of digestion both processes produce partially fragmented molecules which consist of an (Fc)₅ portion still attached to limited numbers of Fab arms. Both processes cause a gradual decrease in the ability of molecules to agglutinate Salmonella, but complement fixation by the complexes declines much more rapidly. A stage is reached where molecules having four Fab arms can still agglutinate but there is no complement fixation. However, the remaining arms on the tryptic molecules are distributed in pairs on the same subunit, whereas those on the peptic molecules are distributed randomly. Hence the number of remaining Fab arms, rather than their distribution, appears to be the critical factor which influences biological activity. A possible explanation for this is discussed.

An uncharged $\text{N-}\text{hydroxysuccinimide ester}$ derivative of agarose, Affi-Gel 10, exhibited excellent capacity for immobilization, at pH 7.5, of proteins having isoelectric points of 6.5–11.0. Under identical conditions, acidic proteins with isoelectric points of 3.3–5.9 did not couple well to this activated gel. Immobilization of acidic proteins increased in the presence of 80 mM CaCl₂, or at a pH equal to or less than the isoelectric point. Affi-Gel 15, a new $\text{N-}\text{hydroxysuccinimide ester}$ derivative of agarose containing a tertiary amine in the spacer arm, coupled acidic proteins efficiently at pH 7.5 but basic proteins coupled poorly. The immobilization of basic proteins to Affi-Gel 15 was increased to useful levels by increasing the ionic strength, or the pH, of the reaction medium. The lectin concanavalin A was efficiently immobilized using either activated gel, and the concanavalin A-agarose derivatives bound 3.9–4.1 mg ovalbumin/ml gel. These studies demonstrate that the charge of the protein relative to the charge of the gel is an important factor affecting the level of protein immobilization to active ester gels.

Escherichia coli K-12, which is rich in alkaline phosphatase, exhibits phosphorescence characteristic of tryptophan at room temperature. E. coli mutants which do not have alkaline phosphatase do not show long-lived phosphorescence. The phosphorescence spectrum and lifetime of E. coli K-12 was similar to that of purified alkaline phosphatase from E. coli. These results indicate that the long-lived tryptophan phosphorescence in E. coli is likely to be derived from alkaline phosphatase in situ. The temperature dependence of tryptophan phosphorescence life-time of purified alkaline phosphatase and E. coli K-12 differ; this may imply that alkaline phosphatase in E. coli may be associated with the cell envelope and is therefore protected against structural changes in the protein which result in increased phosphorescence decay rates.

A strategy is reported which allows the rapid characterization of different hemoglobins D found in Ivory Coast. It involves reverse-phase HPLC for peptide separations and micro-sequencing by the manual solid-phase method of Chang. Identification of hemoglobin Avicenna, hemoglobin Korle Bu and hemoglobin Cocody, a new variant (β 21 (B3) Asp → Asn), is described.

The lactose-blockable lectin activity from fetal calf skeletal muscle has been purified to apparent homogeneity. The purification entails differential centrifugation, ammonium sulfate precipitation, asialofetuin affinity chromatography with a lactose gradient and ion-exchange chromatography on DEAE-cellulose. In the last step, the activity is resolved into a major and minor species, designated ion-exchange-purified lectins I and II, respectively. Both lectin activities are reversibly inhibited by lactose and appear as single bands with identical mobilities on SDSpolyacrylamide gel electrophoresis. Lectin I1 was not obtained in sufficient quantities for further characterization. Lectin I is characterized by a functional requirement for reducing agents and sensitivity to N-ethylmaleimide, which suggests a role for an essential thiol in its activity. Subunit moleculas weight determinations by SDS-polyacrylamide gel electrophoresis (12000 ±1 000) and by gel filtration in 6 M guanidine · HCI (13 000 ± 1 000), when compared with that obtained under native conditions on Bio-Rad P-60 gels (27 000 ± 2 000), suggest a true M_r of 25 000 ± 3 000 for the dimeric molecule. Amino acid composition data, when fitted to this molecular weight, lead to the tentative conclusion that the intact dimer is composed of two very similar but compositionally non-identical chains, designated α and β. While the only detectable N-terminal amino acid is tryptophan, the isoelectric focusing pattern of lectin I supports this heterodimeric structure. In addition, a lactose-insensitive hemagglutinating activity which can be separated from the lactose-blockable activity by affinity chromatography was also observed.

EPR spectra of nitrosyl hemes were used to study the quaternary structure of hemoglobin. Human adult hemoglobin has been titrated with nitric oxide at pH 7.0 and 25°C. After the equilibration of NO among the α and β subunits the samples were frozen for EPR measurements. The spectra were fitted by linear combinations of three standard signals: the first arising from NO-β-hemes and the other two arising from NO-α-hemes of molecules in the high- and low-affinity conformations. The fractional amounts of α subunits exhibiting the high-affinity spectrum fitted the two-state model (Edelstein, S.J. (1974) Biochemistry 13, 4998–5002) with the allosteric constant L = 7 · 10⁶ and relative affinities c_NO^α and c_NO^β approx. 0.01. Hemoglobin has been marked with nitric oxide at one chain using low-saturation amounts of nitric oxide. The EPR spectra were studied as a function of oxygen saturation. Linear combinations of the three standard signals above fitted these spectra. The fractions of molecules exhibiting the high-affinity spectrum fitted the two-state model with L = 7 · 10⁶, c_O2 = 0.0033 and c_NO^α = 0.08, instead of c_NO^α = 0.01. Thus, the two-state model is not adequate to describe the conformational transition of these hybrids. The results present evidence of the non-equivalence between oxygen and nitric oxide as ligands.