Fc fragment of human immunoglobulin G: Reduction and carboxymethylation of inter-chain disulfide bonds enhances complement activating capacity

The complement activating capacity of the Fc fragment of human immunoglobulin G increases after selective reduction and carboxymethylation of the inter-chain disulfide bonds. Differences in the circular dichroism spectra of intact and modified fragment suggest that minor conformational changes have occurred. Our results are discussed in relation to the molecular requirements for complement activation.     

The chemical structure of a fragment of Micrococcus lysodeikticus cell-wall.

A fragment of Micrococcus lysodeikticus cell-wall obtained by cetylpyridinium recipitation from the nondialyzable portion of the degradation products of egg-white lysozyme was studied by the periodate oxidation and methylation procedures. The fragment consists of a polysaccharide chain composed of about 40 repeating (1 leads to 4)-O-(2-acetamido-2-deoxy-beta-D-mannopyranosyluronic acid)-(1 leads to 6)-O-(alpha-D-glucopyranosyl) residues with D-glucopyranosyl residues at both ends. The alpha-D-glucopyranose residue at the reducing end is linked to a phosphate group that is also linked to C-6 of a 2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl residue of a peptidoglycan chain composed of four repeating (1 leads to 4)-O-[2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl] residues. The peptidoglycan chain has, as nonreducing group, a 2-acetamido-2-deoxy-beta-D-glucopyranosyl group, and, as reducing residue, a 2-acetamido-3-O-(D-1-carboxytheyl)-2-deoxy-beta-D-glucose residue.     

The reaction of 4,4'-bis-dimethylaminodiphenylcarbinol with the sulfhydryl group. A new reagent for sulfhydryl analysis

4,4′-bis-Dimethylaminodiphenylcarbinol (BDC-OH) dissociates in aqueous buffers at pH values below neutrality to form a resonance-stabilized carbonium-immonium ion (BDC⁺) which exhibits an absorbance maximum at 606 nm. In the presence of 4.0 M guanidine hydrochloride, BDC⁺ has an apparent molar absorption coefficient of 70,800 M^?1cm^?1 and an absorbance maximum of 612 nm. Sulfhydryl groups react with the cation to form S-(4,4′-bis-dimethylaminodiphenylmethyl-) derivatives with a concomitant quantitative loss of the 612-nm absorbance. This quantitative interaction has been exploited in the development of a new and convenient technique for the quantitative determination of sulfhydryl groups in proteins. Results of sulfhydryl determinations on simple thiols and five proteins are presented, along with comparison data obtained via other sulfhydryl techniques.     

Purification and partial characterization of hatching protease of the sea urchin, Strongylocentrotus purpuratus.

The supernatant above hatched sea urchin (Strongylocentrotus purpuratus) blastulae contains crude hatching protease, which is heterogeneous in molecular weight, solubility, charge, and density. It requires urea treatment (6 m, 22 °C, 6 h) to dissociate from the enzyme the heterogeneous population of fragments it has generated in digesting its substrate, the fertilization envelope. It can then be purified 340-fold by diethylaminoethyl-cellulose, ammonium sulfate, and Sephadex G-100. The resulting preparation, homogeneous by the criteria of gel exclusion chromatography, sodium dodecyl sulfate gel electrophoresis, and thermal inactivation, has the following properties: specific activity = 1.44 U mg^?1 (1.44 μmol min^?1 mg^?1); k_cat = 0.72 s^-1; molecular weight = 29,000; energy of activation = 12.9 kcal mol^?1 on dimethylated casein;K_m = 0.93 mgml^?1 dimethylated casein. The pure enzyme is optimally active at pH 7 to 9, 0.5 m NaCl, 10 mm Ca²⁺, and 42 °C. Purification renders the enzyme less stable to freezing and thawing and increases the rate of its thermal inactivation at 37 °C by 100-fold.     

Calcium and pancreatic β-cell function: 3. Validity of the La3+-wash technique for discriminating between superficial and intracellular 45Ca

Exposure to La³⁺ has been proposed as a tool for discriminating between superficially and intracellularly located ⁴⁵Ca. Two different pools of glucose-stimulated ⁴⁵Ca were identified in β-cell-rich pancreatic islets microdissected from ob/ob mice using a standard procedure of washing with 2 mM La³⁺ at 37°C. The results were not critically dependent on the concentration of La³⁺ within a range of 1–20 mM. However, the distribution of ⁴⁵Ca was markedly influenced by the temperature of the washing medium. Reduction of the temperature to 1°C abolished the glucose effect on the La³⁺-displaceable ⁴⁵Ca and considerably increased the amounts of ⁴⁵Ca not displaceable with La³⁺. Separate studies in a perifusion system revealed that La³⁺ in itself was less effective than temperature reduction in depressing the efflux of ⁴⁵Ca from the isolated islets. The data indicate that washing in cold La³⁺ solution gives a better estimate of the intracellular islet calcium than the original procedure. It is suggested that the glucose-stimulated part of the ⁴⁵Ca, which is displaced by washing with La²⁺ at 37°C, is derived from the interior of the β-cells.     

Metal ion induced reaction specificity in vitamin B6 model systems: The effects of Zn2+ and Cu2+ on the 5-deoxypyridoxal catalyzed reactions of α-phenyl-α-aminomalonic acid

In the absence of metal ions, the reaction of 5-deoxypyridoxal (III) with α-phenyl-α-aminomalonic acid, (II; R=C₆H₅) leads to the formation of two 5-deoxypyridoxal-derived products, 5-deoxypyridoxamine (VII) and a dimer-like product (VIII) formed from the condensation of a molecule of III and a molecule of VII (J. W. Thanassi, Biochemistry12, 5109 (1973)). The addition of excess Zn²⁺ ions or limiting Cu²⁺ ions leads to the formation of VII only. Addition of excess Cu²⁺ ions results in an entirely different 5-deoxypyridoxal-derived product, VI, which is a peptide of 5-deoxypyridoxic acid and d,l-α-phenylglycine (V), and which is formed in an oxidative reaction. Mechanisms for the formation of these compounds are discussed in relation to reaction selectivity in vitamin B₆ catalysis.     

X-Ray crystal and molecular structure of 1,2,3,5-tetra-O-acetyl-4-deoxy-4-C-[(S)-ethylphosphinyl]-α-d-ribofuranose,and conformations of aldopentofuranose analogs having phosphorus in the hemiacetal ring

X-Ray crystallographic analysis was performed on the compound to which had been assigned the structure 1,2,3,5-tetra-O-acetyl-4-deoxy-4-C-[(S)-ethylphosphinyl]-α-d-ribofuranose. The results showed that the compound has the proposed configuration, the five-membered ring is in the ³T₂ conformation with a tendency towards the E₂ form, the substituents on C-1, C-4, and P-5 are linked bisectionally, and the acetoxyl groups on C-2 and C-3 are respectively attached axially and equatorially. Based on the X-ray crystallographic and ¹H-n.m.r.-spectral data, favored conformations of P-in-ring analogs of aldopentofuranose peracetates in solution are discussed.     

600 MHz H nuclear magnetic resonance studies of the kringle 4 fragment of human plasminogen

Kringle 4, a approximately 10,000-Da domain in the heavy chain of human plasminogen, has been isolated intact and studied by H NMR spectroscopy at 600 MHz. The spectroscopic data indicates that kringle 4 possesses a globular and flexible structure which exhibits relatively fast amide-hydrogen exchange. About 17 NH groups show retarded exchange, with half-lives of approximately 7 h in 2H2O at pH* 6.45, 25 degrees C, which indicates that regions of the kringle are buried and shielded from direct interaction with the solvent. Analysis of the methyl region spectrum accounts for all singlets and doublets in terms of the amino acid composition; resonances from the C- and N-termini residues could be identified from the magnitude of their J couplings and their response to pH titration. It is shown that elastase digestion of plasminogen generates two species of kringle 4, one that terminates with Ala85 and another that extends to Val87. The heterogeneity can be resolved by chromatography on CM-Sephadex. The interaction of kringle 4 with BASA (p-benzylaminesulfonic acid), an antifibrinolytic drug presumed to bind to the plasminogen lysine-binding sites, has been investigated through the effects of added ligand on the kringle spectrum. The kringle lysine-binding site would appear to be integrated by a cluster of interacting His and aromatic residues since many of these resonances follow a definite saturation curve pattern upon BASA titration. In contrast, only minor changes are detected in the aliphatic methyl spectra. The association constant for the BASA-kringle 4 interaction is estimated to be Ka approximately 74 mM-1, which should be compared with Ka approximately 145 mM-1 previously measured for kringle 1 under identical conditions. It is proposed that residues in the proximity of the Cys80-Cys1 disulfide bridge are proximal to, or form part of, the lysine-binding site.     

Tritium labeling of thermolysin, elastase, and ribonuclease by exposure to tritium gas at low pressure

The bacterial metalloendoprotease thermolysin, bovine pancreatic ribonuclease, and porcine pancreatic elastase have been tritiated by exposure to subcurie amounts of tritium gas at pressures below 50 mTorr for periods of 1 to 6 h. Thermolysin, ribonuclease, and elastase have been purified to specific radioactivities of 15, 5, and 1 Ci/mol, respectively. Amino acid analyses of the tritiated enzymes revealed higher relative specific radioactivities for His, Pro, and Phe in all three proteins while Val and Ile were among the residues with the lowest relative specific radioactivities. The recovery of enzyme activity was always greater than 95% and the formation of tritiated decomposition products was not observed. This lowpressure gas exposure process requires less tritium gas and less time than the original method of Wilzbach to achieve equal or higher levels of tritium incorporation. In addition, the enzymes were completely active and did not show the presence of highly radioactive byproducts which have been observed in earlier studies of the Wilzbach labeling of proteins.     

Direct measurement of carbon monoxide bound to different subunits of hemoglobin A in solution and in red cells by infrared spectroscopy

Infrared spectra for carbon monoxide bound to alpha and beta subunits of human hemoglobin A have subunit differences near 1950 cm-1 and indicate that 92% of the alpha subunits exist in one conformer and 5% in a second conformer under conditions where 99% of the beta subunit is in only one conformation. The sum of the separated subunit spectra is equivalent to the alpha 2 beta 2 tetramer spectrum. CO infrared spectra indicate that CO displaces O2 from HbO2 in red cells or in solution preferentially at the beta subunits. The measurement of C-O stretch bands provides a direct method for characterization of ligand binding sites within intact cells.     

Primary structure of the southern bean mosaic virus coat protein: First results

Studies on the southern bean mosaic virus coat protein have established the molecular weight of this protein, its amino acid composition, the nature of its C-terminal amino acid, and the blockage of the N-terminal residue by an acetyl group. After hydrolysis of the protein by trypsin, the hydrolysate was fractionated by ion-exchange chromatography. Among the purified tryptic peptides were isolated the N- and the C-terminal peptides where sequences were determined, principally by mass spectrometry.     

Human complex-forming glycoprotein, heterogeneous in charge: the primary structure around the cysteine residues and characterization of a disulfide bridge

L-929 cell surface membranes have been assayed in vitro and found to contain significant protein kinase activity. A steady-state kinetic analysis indicated that at least two distinct protein kinases were present. Plots of reaction velocity (v) against substrate (ATP) concentration were distinctly biphasic, as were Lineweaver-Burk plots of $\text{1}\text{v}$ versus $\text{1}\text{ATP}$. Michaelis constants of the two enzymes were calculated to be 22 and 173 μm, respectively. Sodium dodecyl sulfate polyacrylamide gel analysis of the phosphorylated membrane proteins provided additional support for the existence of more than one protein kinase. Different endogenous proteins were phosphorylated at 1 μm ATP compared to 1 μm ATP. Further studies of the low K_m (22 μm) enzyme suggested that it is a typical cyclic 3′,5′-AMP-independent protein kinase. Its activity was dependent on the presence of Mg²⁺, but it was not affected by cyclic 3′,5′-AMP, cyclic 3′,5′-GMP, or the heat-stable inhibitor of cyclic 3′,5′-AMP-dependent protein kinases. ATP and GTP, but not other nucleoside triphosphates, could serve as phosphoryl donor and maximum kinase activity was expressed at pH 7.0. Phosvitin and casein were superior to histones as exogenous substrates for the low K_m enzyme.     

A model reaction demonstrating alkylating properties of pyridoxol, involving an o-quinone methide intermediate

The synthesis of several specifically substituted pyridoxine derivatives 5, 6, 7, 8, 9, 10, 11, and 12, is described. A pyridoxol derived o-quinone methide, postulated as the common reaction intermediate, gives rise to the specific pyridoxol substitution encountered in these products. The mechanism is discussed as a model for a new type of pyridoxine transformation, either in known vitamin-B₆-dependent enzymatic reactions or in toxicological reactions induced by pyridoxine.     

Synthesis and characterization of a metal-binding peptide fragment of human carbonic anhydrase B

A 36-amino acid residue peptide containing the presumed metal-binding ligands at the active site of human erythrocyte carbonic anhydrase B was synthesized by the standard solid phase method. The synthetic peptide was purified by ion-exchange chromatography and was homogeneous as judged by cellulose acetate gel electrophoresis. Amino acid analysis, dansylation, C-terminal determination, and four cycles of Edman degradation all gave results consistent with the anticipated sequence. The peptide binds Co(II) with an apparent dissociation constant of about 7 × 10^?5M (uncorrected) but has little, if any, of the catalytic activity of carbonic anhydrase. Possible explanations for the weak binding of the metal ion are discussed along with prospects and strategies for designing polypeptide models of enzymatic catalysts.     

Reactivity of singlet molecular oxygen with cholesterol in a phospholipid membrane matrix. A model for oxidative damage of membranes

Photooxidation of cholesterol in liposomes with hematoporphyrin sensitization has been studied. With liposomal samples in which the hematoporphyrin is incorporated in the membrane, the yield of the characteristic singlet oxygen product, 3β-hydroxycholest-6-ene 5α-hydroperoxide, was approximately 6 times greater than that observed in the samples in which the hematoporphyrin was outside the membrane. Small amounts of 3β-hydroxycholest-5-ene 7α- and 7β-hydroperoxides, radical autoxidation products, were formed in both samples. Photolysis of a dispersion of cholesterol in an aqueous solution of hematoporphyrin gave no singlet oxygen products. It is concluded from these results that endogenous singlet oxygen when formed in the phospho-lipid membrane has a sufficiently long lifetime to effect oxygenation of cholesterol; whereas exogenous singlet oxygen generated outside the membrane is quenched by solvent before appreciable diffusion into the membrane can occur.     

Intrinsic perturbing ability of alkanols in lipid bilayers

The proportionality constant between the equipotency concentrations of a series of solutes and the fraction of a solute in the membrane phase is directly related to the solute to lipid mol ratio. Experimental measurements of partition coefficient and of several alkanol-induced effects show that the solute/lipid mol ratlos for a series of alkanols are not constant at their equipotency concentrations. The deviations in the solute/lipid ratios are similar in the various systems, and these deviations seem to depend primarily upon the chain length and branching in alkanols. It is suggested that such intrinsic differences in the perturbing ability of alcohols arise from a specificity of interaction between alkanols and lipid bilayer. We have correlated partition coefficients (in n-octanol, in egg phosphatidylcholine liposomes, and in dipalmitoyl phosphatidylcholine liposomes) for thirteen alkanols to the equipotency concentrations for their ability to modify the order-disorder thermotropic transition in dipalmitoyl phosphatidylcholine, ability to stimulate the hydrolysis of phosphatidylcholine in a bilayer by bee venom phospholipase A₂, and for the activation of the galactoside transport system in Escherichia coli. Significant correlation is found between equipotency concentrations for perturbing the order-disorder transition, the activation of phospholipase A₂-catalyzed hydrolysis and the activation of galactoside transport system.     

Variations on stoichiometry of ribosomal proteins in Escherichia coli

Experiments are described in which the Stoichiometry of the ribosomal proteins before and after ribosome release from mRNA is compared. Polysomes labeled with ³H (or ¹⁴C) and run-off 70 S particles (Subramanian el al., 1969) labeled with ¹⁴C (or ³H) were separately isolated, mixed, and the ribosomal proteins extracted and fractionated by two-dimensional gel electrophoresis. The measurement of the isotopic ratios shows that 47 proteins out of the 53 investigated are present in the same amount in polysomes as in run-off ribosomes indicating that they remain with the ribosome during the release step. Proteins S₁, S₂, S₆, S₂₁, $\text{L}{}_{\mathrm{<mtext>7</mtext>}}\text{L}{}_{\mathrm{<mtext>12</mtext>}}$ (Wittmann et al., 1971), however, show higher amounts in polysomes than in run-off ribosomes. The significance of these results is discussed.     

Molecular conformation of prostaglandin A1 monoclinic cyrstalline polymorph

The molecular conformation of the monoclinic crystalline polymorph of prostaglandin A₁ has been determined by X-ray diffraction techniques. The space group is P2₁ with a = 13.637 (2), b = 7.567 (1), I c = 10.576 (2) Å, β = 107.37 (3)°; D_c = 1.073 g·cm⁻³ for Z = 2. The molecular conformation is characterized by the nearly parallel arrangement of the C₁–C₇ and C₁₃–C₂₀ side chains, with a general flattening of the overall structure when compared with the orthorhombic polymorph. The cyclopentenone moiety assumes a C₈ envelope conformation with C₈ and O₉ displaced +0.29 Å and −0.18 Å from the C₉–C₁₀=C₁₁–C₁₂ plane respectively. Concerted, small variations of the torsion angles, primarily about the C₈–C₁₂, C₁₄–C₁₅ and C₁₆–C₁₇ bonds, bring the monoclinic and orthorhombic conformations into coincidence.