Biochemistry and physiological role of methionine sulfoxide residues in proteins

The cytochrome system in eggs and embryos of the sea urchin, Hemicentrotus pulcherrimus, was investigated. Difference spectra of the mitochondrial fraction demonstrated the presence of a complete cytochrome system in unfertilized eggs. Cytochrome levels and the activities of respiratory enzymes were measured in crude extracts of eggs both before and after fertilization. Unfertilized eggs contained cytochromes aa₃, b, and c + c₁ in a ratio of 1.0:1.8:0.7. Gastrulae contained almost the same amount of cytochromes aa₃and b as unfertilized eggs. However, the amount of cytochrome c + c₁ in gastrulae was 1.5 times greater than that in unfertilized eggs. The activity of cytochrome oxidase remained unchanged during development. No cytochrome oxidase inhibitor was found in unfertilized eggs. Both antimycin A-sensitive and insensitive NADH-cytochrome c reductase activities increased during development. The activity of succinate-cytochrome c reductase increased during early development, reached a temporary plateau, and then declined at the pluteus stage. These results are discussed in relation to the increase of respiration during early development.     

Quinolinic acid, an endogenous metabolite with neurotoxic properties, alters the physical state of membrane proteins in human erythrocytes

Quinolinic acid (2,3-pyridinedicarboxylic acid), an endogenous, tryptophan metabolite, is neurotoxic when injected into rat striatum (1). To begin to investigate the molecular interactions of quinolinic acid with membranes, electron spin resonance studies of the effects of this neurotoxin on the physical state of lipids, proteins, and cell-surface sialic acid in human erythrocyte ghosts have been performed. Quinolinic acid induced a highly significant alteration in the physical state of membrane proteins (P less than 0.01) while that of sialic acid and membrane lipids was unaffected. These results are similar to those induced by ibotenic acid, an exogenous neurotoxin, and are discussed with reference to possible molecular characteristics of the interaction of these neurotoxins with membrane proteins.     

Electron spin resonance studies of the effects of naturally-occurring excitotoxic amino acid analogues on the physical state of membrane proteins in human erythrocytes

The interaction of the neurotoxic natural products, kainic and ibotenic acids, both of which are also excitatory neurotransmitters and amino acid analogues of glutamic acid, along with the latter compound, with human erythrocyte membranes has been investigated by electron spin resonance methods. Only ibotenic acid caused a statistically significant alteration in the physical state of membrane proteins (P = 0.01) while none of these excitotoxins measurably affected motion of membrane lipids. In order to further investigate some of the molecular characteristics of ibotenic acid that may have contributed to its effect on the conformation of membrane proteins, similar spin labeling studies were performed employing the decarboxylation product and parent ring compound of this excitotoxin, muscimol and isoxazole, respectively. No effect of either of these latter compounds was observed suggesting that the carboxylic acid group of ibotenic acid is essential for its interaction with membrane proteins. These results are discussed in relation to the known different neurotoxic and physiological effects of kainic and ibotenic acids and muscimol.     

Effects of phytohaemagglutinin, wheat-germ agglutinin, and concanavalin-A on the physical state of sialic acid and membrane proteins in human erythrocyte ghosts: a spin label study

Parallel experiments employing sialic acid- and protein specific spin labels have been performed to monitor the effects on the physical state of this carbohydrate and membrane proteins of human erythrocytes induced by the binding of three lectins, $\text{Phaseolus vulgaris}$ phytohaemagglutinin (PHA), wheat germ agglutinin (WGA), and Concanavalin-A (Con-A). PHA and WGA, both of which are known to bind at different sites on the principal sialoglycoprotein of human erythrocytes, glycophorin, had markedly different effects: compared to control values, PHA decreased the apparent correlation time of the sialic acid specific spin probe by 10% while this parameter was decreased by 33% by WGA. The protein specific spin label also monitored differential effects of these lectins: the relevant electron spin resonance parameter (the W/S ratio) was reduced 33% by PHA and increased by WGA over 17% from that of control values. Con-A, which is known to bind to the principal transmembrane protein, Band 3, had no effect on sialic acid or membrane proteins as assessed by the two spin labels employed. These results suggest that (1) the effects of binding of these different lectins, two of which bind to the same cell surface receptor, can be discriminated by use of spin labeling methods; (2) binding events occuring at the cell surface have distinct and pronounced effects on the physical state of proteins within the membrane; (3) the different results with PHA and WGA both of which bind to glycophorin are indicative of multiple and complex interactions of this glycoprotein with the membrane proteins in the erythrocyte; and (4) that the spin labelling technique has the potential to investigate the relationships between cell-surface binding events to membrane structural-functional interactions.     

A complex of acidic ribosomal proteins. Evidence of a four-to-one complex of proteins in the Bacillus stearothermophilus ribosome

Two acidic proteins from the 50 S subunit of Bacillus stearothermophilus ribosomes, namely B-L13 (homologous to Escherichia coli protein $\text{L}\text{7}\text{L}\text{12}$) and B-L8, form a complex. Radioactive B-L13, added to ribosomes before dissociation, does not appear in the complex after electrophoresis, so the (B-L13 · B-L8) complex must exist in the ribosome before dissociation. Digestion of B. stearothermophilus ribosomes with polyacrylamide-bound trypsin causes the appearance of new B-L8 and B-L13 spots on two-dimensional polyacrylamide gel electrophoresis, in a pattern which suggests that single molecules of B-L13 are being sequentially cleaved from a four-to-one complex of B-L13 and B-L8.     

Evidence for the participation of two soluble noncatalytic proteins in hepatic microsomal cholesterol synthesis

The capacity of liver soluble fraction to stimulate hepatic microsomal conversion of squalene to cholesterol is lost on treatment with trypsin. Heat treatment of the soluble fraction results in a selective loss of its capacity to stimulate conversion of squalene to cholesterol; the ability to stimulate conversion of lanosterol and desmosterol to cholesterol is however retained. It is proposed that the liver soluble fraction contains at least two noncatalytic proteins, one heat-labile and the other heat-stable, which participate in microsomal cholesterol synthesis. The heat-labile protein mediates the conversion of squalene to lanosterol while the heat-stable protein is needed for the conversion of lanosterol and other sterol precursors to cholesterol.     

Retinoic acid: an endogenous compound of human blood. Unequivocal demonstration of endogenous retinoic acid in normal physiological conditions.

The occurrence of retinoic acid (vitamin A acid) as a normal constituent of the vitamin A reserve from the body is demonstrated. Improved methodologies based on selected peak monitoring (spm) and high-performance liquid chromatography (hplc) are used for the detection of retinoic acid in EDTA-plasma. After extensive cleanup by double-phase extraction and chromatography on Sephadex LH-20, retinoic acid is determined as its methyl derivative by spm. A different approach using double-phase extraction combined with a preextraction step and hplc is used to confirm the findings of the spm experiments. Both techniques proved the presence of retinoic acid in human plasma at a concentration of 1 to 3 ng/ml.     

Lowry protein determination on membrane preparations: need for standardization by amino acid analysis

The protein content of three membrane protein preparations has been determined by the Lowry method with bovine serum albumin as a standard and also by quantitative amino acid analysis as an absolute method. The results differ considerably, the Lowry method giving 29–42% higher values. This implies that many published data for such proteins, based on Lowry protein determinations with bovine serum albumin as the generally applied standard, are in error. Suggestions are made on how to standardize the Lowry method so that reliable values can be obtained for membrane protein.     

Pig red blood cell hexokinase: Regulatory characteristics and possible physiological role

The regulatory properties of pig erythrocyte hexokinase III have been studied. Among mammalian erythrocyte hexokinases, the pig enzyme shows the highest affinity for glucose and a positive cooperative effect with n_H = 1.5 at all the MgATP concentrations studied (for 0.5 to 5 mm). Glucose at high concentrations is also an inhibitor of hexokinase III. Similarly, the apparent affinity constant for MgATP is independent of glucose concentration. Uncomplexed ATP and Mg are both competitive inhibitors with respect to MgATP. Glucose 6-phosphate, known as a stronger inhibitor of all mammalian erythrocyte hexokinases, is a poor inhibitor for the pig enzyme (K_i = 120 μm). Furthermore, this inhibition is not relieved by orthophosphate as with other mammalian red blood cell hexokinases. A variety of red blood cell-phosphorylated compounds were tested and found to be inhibitors of pig hexokinase III. Of these, glucose 1,6-diphosphate and 2,3-diphosphoglycerate displayed inhibition constants in the range of their intracellular concentrations. In an attempt to investigate the role of hexokinase type III in pig erythrocytes some metabolic properties of this cell have been studied. The adult pig erythrocyte is able to utilize 0.27 μmol of glucose/h/ml red blood cells (RBC) compared with values of 0.56–2.85 μmol/h/ml RBC for the other mammalian species. This reduced capacity to metabolize glucose results from a relatively poor ability of the cell membrane to transport glucose. In fact, all the glycolytic enzymes were present and a low intracellular glucose concentration was measured (0.5 mm against a plasma level of 5 mm). Furthermore, transport and utilization were concentration-dependent processes. Inosine, proposed as the major energy substrate of the pig erythrocyte, at physiological concentrations is not as efficient as glucose in maintaining reduced glutathione levels under oxidative stress. Furthermore, newborn pig erythrocytes (fully permeable to glucose) possess hexokinase type II as the predominant glucose-phosphorylating activity. This fact and the information derived from the study of the regulatory characteristics of hexokinase III and from metabolic studies on intact pig erythrocytes permit the hypothesis that the presence of this peculiar hexokinase isozyme (type III) enables the adult pig erythrocyte to metabolize low but appreciable amounts of glucose.     

A critical physiological role of zinc in the structure and function of biomembranes

Of the large number of recognized zinc metalloenzymes, the activities of only a few are significantly decreased in severely zinc deficient animals. On the other hand, physiological pathology is manifested rapidly after dietary zinc deprivation. This shows that zinc exerts physiological and biochemical roles other than as a component of the known zinc metalloenzymes. The research results reviewed here suggest that zinc plays an important role in the maintenance of membrane structure and function.     

Immunochemical analysis of the membrane proteins of rat liver and Zajdela hepatoma mitochondria

The contents of mitochondrial inner membrane protein complexes were compared in normal liver and in Zajdela hepatoma mitochondria by the immunotransfer technique. Antibodies against core proteins 1 and 2, cytochrome c1, the iron-sulfur protein of Complex III, subunits I and II of cytochrome oxidase, and the alpha and beta subunits of the F1-ATPase were used. In addition, antibodies against a primary dehydrogenase, beta-hydroxybutyrate dehydrogenase, as well as the outer membrane pore protein were used. The results indicate that the components of the cytochrome chain and porin are greatly enriched in hepatoma mitochondria compared to normal rat liver mitochondria. This enrichment was also reflected in the rates of respiration in tumor mitochondria using a variety of substrates. Enrichment of porin may partially account for increased hexokinase binding to tumor mitochondria. In contrast to the respiratory chain components, the F1-ATPase and F0 (measured by DCCD binding) were not increased in tumor mitochondria. Thus, Zajdela hepatoma mitochondria components are nonstoichiometric, being enriched in oxidative capacity but relatively deficient in ATP synthesizing capacity. Finally, beta-hydroxybutyrate dehydrogenase, which is often decreased in hepatoma mitochondria, was shown here by immunological methods to be decreased by only 40%, whereas enzyme activity was less than 5% of that in normal rat liver.     

Identification of vitelline membrane proteins in Drosophila melanogaster

In Drosophila melanogaster, proteins involved in vitelline membrane production are secreted by ovarian follicle cells during stages 9 and 10 of oogenesis. We have used SDS-PAGE and two-dimensional electrophoresis to identify six major size classes of radiolabeled components in purified vitelline membrane preparations. Analyses of in vivo labeled proteins from egg chambers of different developmental stages and stage 10 follicle cells show that components of five of these size classes are synthesized by follicle cells during the period of vitelline membrane deposition. Immunological analysis of eggshell antigens utilizing complex antisera raised to purified eggshell fragments has confirmed the identity of components of three size classes.     

Light-dependent chemical modification of thylakoid membrane proteins with carboxyl-directed reagents

Spinach chloroplast thylakoid membranes were chemically modified with membrane penetrating reagents reactive toward protein carboxyl groups, a carbodiimide and the nucleophiles [¹⁴C]glycine ethyl ester or [³H]serotonin. The reagents, being weak bases, were accumulated within the inner aqueous space in the light, due to the low pH inside. Both the accumulation and the low pH stimulating effect on the carbodiimide activation step contributed to a greater labeling in the light compared to dark, and uncouplers inhibited most of the light-dependent increase. Hence, it is likely that the proteins showing the light-dependent, uncoupler-sensitive labeling have those parts located within the inner aqueous space or within the membrane itself. While many membrane proteins which separated on sodium dodecyl sulfate-polyacrylamide gels (12.5–25% gradient) showed some increased labeling in the light, the most conspicuous were the four polypeptides of the chlorophyll $\text{a}\text{b}$ light-harvesting complex. The light-harvesting complex was purified from dark- and light-treated, labeled membranes. The resultant preparation showed about a sixfold, light-dependent, uncoupler-sensitive labeling increase compared to dark conditions. Polypeptides near 6 and 8 kdalton showed light-dependent, uncoupler-resistent increases in carboxyl group modification, which could be due to localized acidic conditions near sites of proton release.     

Inhibition of β-lactamase-I by 6-β-sulfonamidopenicillanic acid sulfones: Evidence for conformational change accompanying the inhibition process

A number of 6-β-sulfonamidopenicillanic acid sulfones were examined for their ability to inhibit Bacillus cereus$\text{569}\text{H}$ β-lactamase I. Among these, 6-β-trifluoromethane sulfonamidopenicillanic acid sulfone was found to be the most potent inhibitor, effecting rapid and irreversible inactivation of the enzyme. Optical rotatory dispersion and differential scanning calorimetry were employed to probe the possible conformational changes accompanying the inactivation of B. cereus$\text{569}\text{H}$ β-lactamase 1 by 6-β-trifluoromethane sulfonamidopenicillanic acid sulfone. Optical rotatory dispersion measurements indicated the presence of approximately 29 and 17% helical structure in the native and inactivated enzyme, respectively. Differential scanning calorimetry determinations revealed that the inactivated enzyme was less thermostable than the native β-lactamase. The temperatures of maximum heat absorption were 48.4(±0.5) and 57.4(±0.1)°C for the inactivated and the native enzyme, respectively. Extensive conformational changes accompanying the interaction of the enzyme with the inhibitor may be responsible for the irreversible loss in the catalytic activity.     

Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a positively charged membrane filter

Zeta-bind, a positively charged nylon membrane, was tested as an immobilizing matrix for the electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels. It was found that Zeta-bind has a considerably greater capacity than does nitrocellulose for protein binding. Because of this property, more efficient elution of proteins from gels can be used (by omitting methanol from transfer buffers). The procedure described is more amenable to quantitation than usual nitrocellulose-based transfer. Antibody or lectin overlay techniques are also more sensitive on Zeta-bind than on nitrocellulose.     

Evidence for the spontaneous formation of interspecies hybrid molecules of human, rat and bovine serum albumins

Utilizing electrophoretic and gel filtration techniques it was shown that a bovine C-terminal peptic fragment [residues 307-582] spontaneously forms interspecies hybrid molecules with three complementary N-terminal fragments derived from human [residues 1-308; 49-308] and rat [residues 1-308] albumins. The fragments associate with 1:1 stoichiometry to produce an albumin-like complex which has a molecular weight and electrophoretic mobility similar to intact albumin. These data demonstrate, for the first time, that albumin fragments derived from different species associate in a complementary fashion and provide direct evidence that the tertiary structure may be highly conserved.     

Triton X-114 phase fractionation of membrane proteins of the cyanobacterium Anacystis nidulans R2

The thylakoid polypeptides of the cyanobacterium Anacystis nidulans R2 were analyzed by Triton X-114 phase fractionation [C. Bordier (1981) J. Biol. Chem.256, 1604–1607, as adapted for photosynthetic membranes by T. M. Bricker and L. A. Sherman (1982) FEBS Lett.149, 197–202]. In this procedure, polypeptides with extensive hydrophobic regions (i.e., intrinsic proteins) form mixed micelles with Triton X-114, and are separated from extrinsic proteins by temperature-mediated precipitation of the mixed Triton X-114-intrinsic protein micelles. The polypeptide pattern after phase fractionation was highly complementary, with 62 of the observed 110 polypeptide components partitioning into the Triton X-114-enriched fraction. Identified polypeptides fractionating into the Triton X-114 phase included the apoproteins for Photosystems I and II, cytochromes f and b₆, and the herbicide-binding protein. Identified polypeptides fractioning into the Triton X-114-depleted (aqueous) phase included the large and small subunits of RuBp carboxylase, cytochromes c₅₅₀ and c₅₅₄, and ferredoxin. Enzymatic radioiodination of the photosynthetic membranes followed by Triton X-114 phase fractionation allowed direct identification of intrinsic polypeptide components which possess surface-exposed regions susceptible to radioiodination. The most prominent of these polypeptides was a 34-kDa component which was associated with photosystem II. This phase partitioning procedure has been particularly helpful in the clarification of the identity of the membrane-associated cytochromes, and of photosystem II components. When coupled with surface-probing techniques, this procedure is very useful in identifying intrinsic proteins which possess surface-exposed domains. Phase fractionation, in conjunction with the isolation of specific membrane components and complexes, has allowed the identification of many of the important intrinsic thylakoid membrane proteins of A. nidulans R2.     

Construction and analytical applications of liquid membrane electrode for trinitrobenzenesulfonic acid (TNBS)

A new liquid membrane electrode which responds to trinitrobenzenesulfonate (TNBS^?) ion is described. The electrode exhibits very good experimental characteristics: (i) The working pH range is 2.0–12.5; (ii) its response is consistent with the Nernst equation in the range 1.10^?1–5.10^?5m; (iii) its response time is 5 s; and (iv) its selectivity for TNBS^? against 14 tested ions is very high. Ions such as $\text{ClO}{}_4{}^{\mathrm{?}}$, $\text{IO}{}_4{}^{\mathrm{?}}$, I^?, and biphthalates interfere with potentiometric selectivity coefficients in the range 1.0 × 10^?2–9.0 × 10^?2. The electrode is suitable for direct potentiometry, potentiometric titrations, and kinetic potentiometric methods. Instructions for the application of the electrode for the potentiometric assay of aminoacids are given. The application of the electrode in the potentiometric precipitation titration of methylene blue with TNBS, and its use in the kinetic potentiometric determination of aminoacids is also described. The electrode has very good slope stability (60 mV/decade change of activity) for a period ofat least 2 months.