Recombinant human insulin.

Insulin is a well-characterized peptide that can be produced by recombinant DNA technology for human therapeutic use. A brief overview of insulin production from both traditional mammalian pancreatic extraction and recombinant bacterial and yeast systems is presented, and detection techniques, including electrophoresis, are reviewed. Analytical systems for insulin separation are principally based on reversed-phase chromatography, which resolves the deamidation product(s) (desamido insulin) of insulin, proinsulin, and insulin. Process-scale separation is a multistep process and includes ion exchange, reversed-phase, and size exclusion chromatography. Advantages and/or disadvantages of various separation approaches, as described by the numerous literature references on insulin purification, are presented.     

The heparin binding domain of S-protein/vitronectin binds to complement components C7, C8, and C9 and perforin from cytolytic T-cells and inhibits their lytic activities

S-Protein/vitronectin is a serum glycoprotein that inhibits the lytic activity of the membrane attack complex of complement, i.e., of the complex including the proteins C5b, C6, C7, C8, and C9n. We show that intact S-protein/vitronectin or its cyanogen bromide generated fragments also inhibit the hemolysis mediated by perforin from cytotoxic T-cells at 45 and 11 microM, respectively. The glycosaminoglycan binding site of S-protein/vitronectin is responsible for the inhibition, since a synthetic peptide corresponding to a part of this highly basic domain (amino acid residues 348-360) inhibits complement- as well as perforin-mediated cytolysis. In the case of C9, the synthetic peptide binds to the acidic residues occurring in its N-terminal cysteine-rich domain (residues 101-111). Antibodies raised against this particular segment react 25-fold better with the polymerized form of C9 as compared with its monomeric form, indicating that this site becomes exposed only upon the hydrophilic-amphiphilic transition of C9. Since the cysteine-rich domain of C9 has been shown to be highly conserved in C6, C7, and C8 as well as in perforin, the inhibition of the lytic activities of these molecules by S-protein/vitronectin or by peptides corresponding to its heparin binding site may be explained by a similar mechanism.     

Effect of substrate and pH on the oxidative half-reaction of phenol hydroxylase

The oxidative half-reaction of phenol hydroxylase has been studied by stopped-flow spectrophotometry. Three flavin-oxygen intermediates can be detected when the substrate is thiophenol, or m-NH2, m-OH, m-CH3, m-Cl, or p-OH phenol. Intermediate I, the flavin C(4a)-hydroperoxide, has an absorbance maximum at 380-390 nm and an extinction coefficient approximately 10,000 M-1 cm-1. Intermediate III, the flavin C(4a)-hydroxide, has an absorbance maximum at 365-375 nm and an extinction coefficient approximately 10,000 M-1 cm-1. Intermediate II has absorbance maxima of 350-390 nm and extinction coefficients of 10,000-16,000 M-1 cm-1 depending on the substrate. A Hammett plot of the logarithm of the rates of the oxygen transfer step, the conversion of intermediate I to intermediate II, gives a straight line with a slope -0.5. Fluoride ion is a product of the enzymatic reaction when 2,3,5,6-tetrafluorophenol is the substrate. These results are consistent with an electrophilic substitution mechanism for oxygen transfer. The conversions of I to II and II to III are acid-catalyzed. A kinetic isotope effect of 8 was measured for the conversion of II to III using deuterated resorcinol as substrate. The conversion of III to oxidized enzyme is base-catalyzed, suggesting that the reaction depends on the removal of the flavin N(5) proton. Product release occurs at the same time as the formation of intermediate III, or rapidly thereafter. The results are interpreted according to the ring-opened model of Entsch et al. (Entsch, B., Ballou, D. P., and Massey, V. (1976) J. Biol. Chem. 251, 2550-2563).     

Manganese-54 accumulation by Chlorella spp., Daphnia magna and yellow perch (Perca flavescens)

Concentration factor and biological half-life of ⁵⁴Mn were determined in three species representing an ecologically and economically important food chain. Green algae (Chlorella spp.), Daphnia magna and yellow perch (Perca flavescens) were exposed to ⁵⁴Mn in water and assayed for ⁵⁴Mn uptake. Steady state concentration factors computed from the laboratory data for algae, Daphnia and perch were 4230, 17 000 and 11, respectively. Respective biological half-lives were 1.6, 1.2 and 8.3 days.     

The stoichiometry of the Ca2+ pump in human erythrocyte vesicles: modulation by Ca2+, Mg2+ and calmodulin

Active Ca2+ uptake and the associated (Ca2+ + Mg2+)-ATPase activity were studied under the same conditions in an inside-out vesicle preparation of human red blood cells made essentially by the procedure of Quist and Roufogalis (Journal of Supramolecular Structure 6, 375-381, 1977). Some preparations were treated with 1 mM EDTA at 30 degrees to further deplete them of endogenous levels of calmodulin. As the Ca2+ taken up by the EDTA-treated inside-out vesicles, as well as the non-EDTA treated vesicles, was maintained after addition of 4.1 mM EGTA, the vesicles were shown to be impermeable to the passive leak of Ca2+ over the time course of the experiments. In the absence of added calmodulin, both active Ca2+ uptake and (Ca2+ + Mg2+)-ATPase were sensitive to free Ca2+ over a four log unit concentration range (0.7 microM to 300 microM Ca2+) at 6.4 mM MgCl2. Below 24 microM Ca2+ the stoichiometry of calcium transported per phosphate liberated was close to 2:1, both in EDTA and non-EDTA treated vesicles. Above 50 microM Ca2+ the stoichiometry approached 1:1. When MgCl2 was reduced from 6.4 mM to 1.0 mM, the stoichiometry remained close to 2:1 over the whole range of Ca2+ concentrations examined. In contrast to the results at 6.4 mM MgCl2, the Ca2+ pump was maximally activated at about 2 microM free Ca2+ and significantly inhibited above this concentration at 1 mM MgCl2. Calmodulin (0.5-2.0 microgram/ml) had little effect on the stoichiometry in any of the conditions examined. The possible significance of a variable stoichiometry of the Ca2+ pump in the red blood cell is discussed.     

Extending the Cellulosome Paradigm: the Modular Clostridium thermocellum Cellulosomal Serpin PinA Is a Broad-Spectrum Inhibitor of Subtilisin-Like Proteases

Clostridium thermocellum encodes a cellulosomal, modular, and thermostable serine protease inhibitor (serpin), PinA. PinA stability but not inhibitory activity is affected by the Fn(III) and Doc(I) domains, and PinA is a broad inhibitor of subtilisin-like proteases and may play a key role in protecting the cellulosome from protease attack.