Theory of electrophoresis of reacting systems as applied to the sulfhydryl oxidation of creatine kinase

In an attempt to explain the unusual electrophoretic behavior of fish muscle creatine kinase, a phenomenological theory of transport of reacting systems has been formulated for the electrophoresis of a sulfhydryl protein undergoing oxidation in the presence of a gradient of molecular oxygen. The model assumes slow O2-oxidation of sulfhydryl groups followed by rather rapidly reversible sulfhydryl-disulfide interchange with concomitant change in the electrophoretic mobility of the protein. The computed electrophoretic patterns for this model exhibit a sharp, unimodal ascending boundary but a bimodal descending boundary in which the proportions of the two peaks are time dependent. There is a striking similarity between the theoretical patterns and their experimental counterparts (M.D. Doherty, D.A. Bergman, V.M. Re-Miller, and D.J. Winzor, 1980, Arch. Biochem. Biophys. 202, 558-564); and hence support for consideration of the electrophoresis of fish muscle creatine kinase in these terms.     

Anomalous electrophoretic behavior of creatine kinase: A thiol-dependent isomeric system with migration subject to kinetic control

Moving boundary electrophoresis of creatine kinase in 0.1 I diethylbarbiturate buffer, pH 8.9, has yielded anomalous migration behavior that indicates intereonversion between two coexisting states of the enzyme at a rate comparable with the rate at which the two enzymic forms tend to separate by differential migration. Whereas a single, symmetrical boundary is observed in the ascending limb, distinct bimodality of the descending pattern is evident in electrophoresis of enzyme isolated from skeletal muscle of either rabbit or fish (Mugil cephalus): pronounced changes in the nature of this bimodality with time are observed in the case of fish muscle creatine kinase. The abnormal migration behavior is eliminated by inclusion of dithiothreitol in the electrophoresis medium or by covalent modification of the enzyme with 5,5′-dithiobis(2-nitrobenzoic acid). Velocity and equilibrium sedimentation studies have been used to identify the macromolecular event as an isomerization, and studies of sulfhydryl content to implicate either reversible sulfhydryl oxidation or thiol-disulfide interchange in the isomerization mechanism.     

Nonidentity of sulfhydryl oxidase and gama-glutamyltransferase in bovine milk

Sulfhydryl oxidase (glutathione-oxidizing activity) is closely associated with γ-glutamyltransferase (γ-glutamyl transpeptidase) in skim milk membranes. Similar close association of the two enzymatic activities in kidney membranes has led to the recent proposal that glutathione-oxidizing activity can be attributed to the action of γ-glutamyltransferase, itself, in generating cysteinylglycine which, in turn, catalyzes sulfhydryl group oxidation (O. W. Griffith and S. S. Tate, 1980, J. Biol. Chem.255, 5011–5014). However, a previously published procedure for the isolation of highly purified sulfhydryl oxidase from skim milk membranes (V. G. Janolino and H. E. Swaisgood, 1975, J. Biol. Chem.250, 2532–2538) leads to the effective separation of the two activities. Quantitative chromatographic analyses of GSH, GSSG, and Glu levels revealed that the highly purified sulfhydryl oxidase preparation catalyzes the direct oxidation of GSH to GSSG without detectable cleavage of the γ-glutamyl peptide bond. These results were confirmed by monitoring the time course of substrate disappearance and product formation using high-performance liquid chromatography. Conversely, a supernatant fraction enriched in γ-glutamyltransferase activity displayed no sulfhydryl group-oxidizing activity. 6-Diazo-5-oxo-l-norleucine selectively inhibited the transferase in crude preparations containing both sulfhydryl oxidase and γ-glutamyltransferase. It is concluded that sulfhydryl oxidase and γ-glutamyltransferase activities are distinct and separable.     

Histotopography and ultrastructure of the thin limbs of the loop of Henle in the hamster

Summary In the kidney of the Syrian hamster the descending thin limbs of both the short and long loops of Henle are not spatially separated from each other and descend between the vascular bundles.Ultrastructurally, five different epithelial types are distinguished in the thin limbs of the short and long loops of Henle. Short loops possess only a descending thin limb with a simply organized epithelium (type 1). Long loops comprise an upper and a lower part of the descending thin limb and the ascending thin limb. The upper part of the long descending thin limb is equipped with a complex and highly interdigitating epithelium with shallow junctions (type 2), which gradually transforms into the simple noninterdigitating type-3 epithelium of the lower part. In a minor portion of long descending thin limbs, however, the upper part begins with an even more complexly organized epithelium (type 2a) than type 2. Type-2a epithelium is conspicuously thicker and possesses a more elaborate mode of cellular interdigitation. Along the descent of this tubular part through the inner stripe of the outer medulla, type-2a epithelium transforms into type-2 epithelium. It is suggested that the long descending thin limbs, which start with type-2a epithelium, belong to the longest loops. The type-4 epithelium of the ascending thin limbs is characterized by flat and extensively interdigitating cells with shallow junctions.The unique pattern of the type-2 a epithelium favors the assumption that solute secretion essentially contributes to the increase in concentration of tubular fluid in long descending thin limbs.This investigation was supported by the Deutsche Forschungsgemeinschaft; project Kr 546 Henlesche Schleife     

Characterization of interspecific hybrids betweenOrchis mascula andO. pallens (Orchidaceae) by enzyme electrophoresis

The European orchidsOrchis mascula, O. pallens and their hybrids have been analysed by enzyme electrophoresis on starch gels. The two species differ in the electrophoretic mobilities of four out of eight enzymes tested. Three enzymes, phosphoglucomutase, phosphoglucoisomerase and malic enzyme exhibit typical heterozygote patterns in the hybrid plants demonstrating the presence of both differing parental alleles. Thus, species identification is easy by the electrophoretic analysis of a low number of enzyme loci, and hybrids are detectable even if morphological characters fail.     

Effects of phosphatidylserine on the oxidation of low density lipoprotein

• 1.1. LDL was incubated in the presence of 1 μ M CuSO₄ for 18 hr at 37°C. The content of lipoperoxides was found to be approx. 40 nmol MDA equivalents/mg LDL protein. The addition of 50 μM phosphatidylserine (PS) reduced the content of lipoperoxides to 15% of control values.
• 2.2. The electrophoretic mobility observed for LDL oxidized in the presence of PS approximated the mobility observed for native LDL.
• 3.3. The formation of conjugated dienes was strongly inhibited when LDL was oxidized in the presence of PS.
• 4.4. The addition of 50 μM phosphatidylcholine, phosphatidylglycerol and cardiolipin did not alter the extent of LDL oxidation.
• 5.5. PS did not inhibit the oxidation of LDL mediated by J774 macrophages in the presence of Ham's F-10 culture medium. Under these conditions, PS was found to be an excellent substrate for oxidation.

     

Free radical-mediated oxidation of free amino acids and amino acid residues in proteins

Summary. We summarize here results of studies designed to elucidate basic mechanisms of reactive oxygen (ROS)-mediated oxidation of proteins and free amino acids. These studies have shown that oxidation of proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl groups, sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino groups, and to conversion of some amino acid residues to carbonyl derivatives. Oxidation can lead also to cleavage of the polypeptide chain and to formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives (glycation/glycoxidation) to produce inactive derivatives. Highly specific methods have been developed for the detection and assay of the various kinds of protein modifications. Because the generation of carbonyl derivatives occurs by many different mechanisms, the level of carbonyl groups in proteins is widely used as a marker of oxidative protein damage. The level of oxidized proteins increases with aging and in a number of age-related diseases. However, the accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate oxidized forms of proteins. Thus, the accumulation of oxidized proteins is also dependent upon genetic factors and individual life styles. It is noteworthy that surface-exposed methionine and cysteine residues of proteins are particularly sensitive to oxidation by almost all forms of ROS; however, unlike other kinds of oxidation the oxidation of these sulfur-containing amino acid residues is reversible. It is thus evident that the cyclic oxidation and reduction of the sulfur-containing amino acids may serve as an important antioxidant mechanism, and also that these reversible oxidations may provide an important mechanism for the regulation of some enzyme functions.     

Human galactose-1-phosphate uridylyltrsferase: purification and comparison of the red blood cell and placental enzymes

Galactose-1-phosphate Uridylyltransferase (uridine diphosphoglucose: α-d-galactose-1-phosphate Uridylyltransferase, EC 2.7.7.12) has been purified from human red blood cells and placental tissue. The placental enzyme was obtained as a homogeneous protein with a specific activity of about 100 units/mg of protein by a combination of previously published methods (G. R. Helmer, Jr., and V. P. Williams, 1981,Arch. Biochem. Biophys.210, 573–580) and concanavalin A-Sepharose chromatography. The properties of the two enzyme forms have been examined with respect to subunit size, electrophoretic properties, isozyme distribution, kinetic patterns, and immunological properties.     

Identification of fifteen neighboring protein pairs in the Escherichia coli 50 S ribosomal subunit crosslinked with 2-iminothiolane.

The 50 S ribosomal subunit of Escherichia coli was allowed to react with 2-iminothiolane under conditions in which amidine-linked sulfhydryl derivatives were formed between lysine ?-amino groups in ribosomal proteins and the heterocyclic thioimidate. Crosslinking between sulfhydryl groups close enough to form intermolecular disulfide bonds was promoted by oxidation of the modified ribosomal subunits. Disulfide-linked dimers were partially purified by extraction of the oxidized subunits with lithium chloride and electrophoresis of the salt-extracted fractions in polyacrylamide/urea gels at pH 5.5. Crosslinked protein dimers were separated by polyacrylamide/sodium dodecyl sulfate diagonal gel electrophoresis. Fifteen protein dimers were identified. Many of them involve proteins implicated in functional sites of the 50 S subunit and in ribosome assembly. The crosslinking results show the proximity of many of these proteins at these active centers, and extend the neighborhood by demonstrating the presence of additional proteins.     

Rabbit muscle myogen. Interactions with phosphate as the source of non-enantiography in moving-boundary electrophoresis.

Rabbit muscle myogen has been subjected to moving-boundary electrophoresis and velocity sedimentation in 0.0187 M-potassium phosphate buffer, pH7.7, I = 0.05. The ascending and descending and descending electrophoretic patterns are sufficiently non-enantiographic to suggest the existence of rapid, reversible interactions in the myogen solutions. However, no evidence of pronounced macromolecular association was obtained in velocity-sedimentation experiments. The source of the non-enantiography in electrophoresis has been traced to interactions of phosphate with components of myogen, which should therefore be considered as a mixutre, rather than a complex, of glycolytic enzymes.     

Escherichia coli dihydrofolate reductase: isolation and characterization of two isozymes.

A combination of affinity column chromatography and preparative gel electrophoresis has been used to purify to homogeneity the two isozymes of dihydrofolate reductase from a trimethoprim-resistant strain of Escherichia coli B (RT 500). These enzyme forms are noninterconvertible and are present in crude cell lysates, but other electrophoretic species can be generated durng purification if sulfhydryl-protecting agents, such as dithiothreitol, are not present. The two isozymes, numbered form 1 and form 2 with respect to their decreasing electrophoretic mobilities, have similar molecular weights (18 500), molecular radii (21 A), and apparent Km values for reduced nico inamide adenin- dinucleotide (NADH) and NADH phosphate (NADPH). Both forms contain 2 mol of sulfhydryl/mol of enzyme which can be oxidized to intramolecular disulfide bonds. However, forms 1 and 2 differ physically in their electrophoretic mobility and isoelectric point and kinetically in their pH-activity profile, specific activity, Km for dihydrofolate, and their affinity toward a number of inhibitors.     

Kinetic and functional characterization of a membrane-bound NAD(P)H dehydrogenase located in the chloroplasts of Pleurochloris meiringensis (Xanthophyceae)

Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q₀, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. K _m-values for both pyridine nucleotides were in the molar range (K _m[NADH]=9.8 M, K _m[NADPH]=3.2 M calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5–8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP⁺ reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP⁺ reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.Abbreviations Coenzyme Q₀-2,3-dimethoxy-5-methyl-1,4-benzoquinone - FNR ferredoxin: NADP⁺ reductase - MD menadione - MV methylviologen - NDH NAD(P)H dehydrogenase - PQ plastoquinone - PQ₁₀ decylplastoquinone - SDH succinate dehydrogenase - UQ₁₀ decylubiquinone (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone)     

Functional mimics of catechol oxidase by mononuclear copper complexes of sterically demanding [NNO] ligands

Several mononuclear copper complexes 1(a-b) and 2(a-b) supported over sterically demanding [NNO] ligands namely, N-(aryl)-2-[(pyridin-2-ylmethyl)amino]acetamide [aryl = 2,6-diethylphenyl (1) and mesityl (2)], exhibit catecholase-like activity in performing the aerial oxidation of 3,5-di-t-butylcatehol (3,5-DTBC) to 3,5-di-t-butyl-catequinone (3,5-DTBQ) under ambient conditions. The 1(a-b) and 2(a-b) complexes were directly synthesized from the reaction of the respective ligands 1-2 with CuX₂·nH₂O (X = Cl, NO₃, n = 2, 3) in 55-85% yield. Mechanistic insights on the catalytic cycle as obtained by density functional theory studies for a representative complex 1a suggest that an intramolecular hydrogen transfer, from a catechol-OH moiety to a copper bound superoxo moiety, form the rate-determining step of the oxidation process, displaying an activation barrier of 18.3 kcal/mol (ΔG^‡) [6.9 kcal/mol in Δ(PE + ZPE)^‡ scale].     

Sequence of the cysteinyl-containing peptides of 4-aminobutyrate aminotransferase. Identification of sulfhydryl residues involved in intersubunit linkage

Three cysteine-containing tryptic peptides were isolated and sequenced from mitochondrial 4-aminobutyrate aminotransferase using DABIA (4-dimethylaminoazobenzene-4-iodoacetamide) as specific labeling reagent for sulfhydryl groups. The enzyme is a dimer made up of two identical subunits, but four out of the six cysteinyl residues/dimer form disulfide bonds when treated with iodosobenzoate to yield inactive enzyme species. To identify the cysteinyl residues undergoing reversible oxidation/reduction, the S-DABIA-labeling patterns of the fully reduced (active) and fully oxidized (inactive) forms of the enzyme were compared. Tryptic digests of the reduced enzyme contained three labeled peptides. If the enzyme was treated with iodosobenzoate prior to reaction with DABIA and tryptic digestion, only one labeled peptide was detected and identified (peptide I), indicating that the two missing cysteinyl-containing peptides (peptides II, III) have been oxidized. The sulfhydryl groups undergoing oxidation/reduction were found to be intersubunit, based on SDS/polyacrylamide gel electrophoresis results. The loss of catalytic activity of 4-aminobutyrate aminotransferase by oxidation of sulfhydryl residues is related to constraints imposed at the subunit interface by the insertion of disulfide bonds.     

PROTEINS OF WHEAT EMBRYOS IN THE PERIOD OF VERNALIZATION

1. Soluble proteins prepared from non-vernalized and vernalizedwheat embryos were investigated by means of column chromatographyand disc electrophoresis. As vernalization proceeded, new proteinswere detectable as the fastest moving electrophoretic bandsor as the new peaks which were eluted with 0.025 M and 0.05M phosphate buffer (pH 7.0) from DEAE-cellulose. Plumule wasfound to be the part of embryo in which these proteins appear.After vernalization, protein pattern of winter wheat embryowas very similar to that of spring wheat embryo.
2. Histoneswere prepared from non-vernalized and vernalized wheatembryosand their chromatographic patterns were investigated.Winterwheat embryo showed more complicated pattern than didspringwheat embryo. Upon vernalization, histone pattern ofwinterwheat embryo became less complicated and similar to thatofspring wheat embryo.

(Received October 4, 1966; )     

Oxidations of sulfur rich heterocycles - new S-oxides of the parent 1,2,4-trithiolane and its tetramethyl derivative: synthesis and structural investigations

Whereas oxidation of 1,2,4-trithiolane (1) with 1 molar equiv. of m-chloroperbenzoic acid (mCPBA) yielded 1,2,4-trithiolane 4-oxide (3) and a small amount of 1,2,4-trithiolane 1-oxide (2), the reaction with 2.5 molar equiv. of mCPBA afforded exclusively 1,2,4-trithiolane 1,4-dioxide (trans-7). The oxidation of 3,3,5,5-tetramethyl-1,2,4-trithiolane (4) with peroxyacetic acid (1 molar equiv. H₂O₂/AcOH) gave a mixture of regioisomeric 3,3,5,5-tetramethyl-1,2,4-trithiolane 4-oxide (6) as a major product and only traces of 1-oxide 5. Using 2.5 molar equiv. of peroxyacetic acid in reaction with 4 a mixture of both stereoisomers of 3,3,5,5-tetramethyl-1,2,4-trithiolane 1,4-dioxides cis-8 and trans-8 was isolated. Furthermore, 4 was oxidized to 3,3,5,5-tetramethyl-1,2,4-trithiolane 1,1,4,4-tetraoxide (9) using 6 molar equiv. of peroxyacetic acid. The molecular structures of 3, trans-7, trans-8 and 9 were unambiguously established by X-ray structure analysis. Compounds 1-4, trans-7, trans-8 and 9 were investigated by Raman spectroscopy. Ab initio calculations were used to obtain the optimized geometries and the vibrational wavenumbers of the title compounds. The vibrational assignment was accomplished by using the calculated harmonic wavenumbers and their Raman intensities. The calculated values of both structural parameters and the vibrational modes fitted in with experimental data. The spectroscopic changes observed in the spectra were correlated with the structural parameters in order to gain information about the influence of the oxidation on the molecule structure. The experimental data indicated, that in comparison with starting 1,2,4-trithiolanes 1 and 4 their oxidized derivatives showed remarkable shortening of the S-S bonds.     

Immunocytochemical localization of Na+, K+-ATPase in the rat kidney

Summary To determine if rat kidney Na⁺, K⁺-ATPase can be localized by immunoperoxidase staining after fixation and embedding, we prepared rabbit antiserum to purified lamb kidney medulla Na⁺, K⁺-ATPase. When sodium dodecylsulfate polyacrylamide electrophoretic gels of purified lamb kidney Na⁺, K⁺-ATPase and rat kidney microsomes were treated with antiserum (1200), followed by [¹²⁵I]-Protein A and autoradiography, the rat kidney microsomes showed a prominent radioactive band coincident with the -subunit of the purified lamb kidney enzyme and a fainter radioactive band which corresponded to the -subunit. When the Na⁺, K⁺-ATPase antiserum was used for immunoperoxidase staining of paraffin and plastic sections of rat kidney fixed with Bouin's, glutaraldehyde, or paraformaldehyde, intense immunoreactive staining was present in the distal convoluted tubules, subcapsular collecting tubules, thick ascending limb of the loops of Henle, and papillary collecting ducts. Proximal convoluted tubules stained faintly, and the thin portions of the loops of Henle, straight descending portions of proximal tubules, and outer medullary collecting ducts did not stain. Staining was confined to basolateral surfaces of tubular epithelial cells. No staining was obtained with preimmune serum or primary antiserum absorbed with purified lamb kidney Na⁺, K⁺-ATPase, or with osmium tetroxide postfixation. We conclude that the basolateral membranes of the distal convoluted tubules and ascending thick limb of the loops of Henle are the major sites of immunoreactive Na⁺, K⁺-ATPase concentration in the rat kidney.Supported by Grant AM 17047 from NIH and by the Veterans Administration     

Hemin-mediated oxidation of dithiothreitol reduces oxygen to H2O

Summary Hemin catalyses the oxidation of dithiothreitol. One mole of oxygen is consumed for every 2 moles of dithiothreitol oxidized and the product is shown by spectral studies to be the intramolecular disulphide. The reaction shows a specificity for dithiol and for free heme moieties. Hemin molecules exhibit cooperativity in oxygen reduction. Oxygen radicals do not seem to be involved. H₂O₂ is not required for this oxidation of dithiothreitol and does not appear to be an intermediate in the reduction of O₂ to H₂O. However, an independent minor reaction involving a 2-electron transfer with the formation of H₂O₂ also occurs. These studies on the hemin-catalyzed oxidation of dithiothreitol provide a chemical model for a direct 4-electron reduction of O₂ to H₂O.Abbreviations HMGCoA 3-hydroxy-3-methylglutaryl coenzyme A - DTT dithiothreitol - Tris-HCl tris(hydroxymethyl)-aminomethane hydrochloride - HEPES N-2,hydroxylethypiperazine-N-2-ethane-sulphonic acid     

Self-assembled pentagonal bipyramidal and skew trapezoidal organotin(IV) complexes of substituted benzoic acids: Their antibacterial, antifungal, cytotoxic, insecticidal and urease inhibition activities

Fourteen di- and triorganotin(IV) derivatives with pentagonal bipyramidal and skew trapezoidal geometries have been synthesized and characterized. Dimethylstannyl bis[3-amino-4-chlorophenylcarboxylate] (1), bis[3-amino-4-chlorophenylcarboxylato] tetraethyldistannoxane] (2) and bis[3,5-dinitro-4-chlorophenylcarboxylato] tetra-n-butyldistannoxane (10) are dinuclear and tetranuclear complexes of bidentate ligands. The crystal structure of (1) is dimeric in which each Sn atom is seven coordinated. Study of weak intramolecular Sn?O interactions is very important to decide geometry around tin. Furthermore, it has been investigated that these compounds exhibit fairly good antibacterial, antifungal, cytotoxic, insecticidal and antiurease activities.