Cross-linked Subunits Research Articles

Abstract 3236: Development of a novel cross-linking strategy to identify distinct proteasome subtypes

Abstract Introduction: This research was conducted to develop a practical method to identify distinct proteasome subtypes. Approval of the proteasome inhibitors bortezomib and carfilzomib for clinical use has validated the proteasome as an anticancer target. These inhibitors target two well-recognized proteasome subtypes, the constitutive proteasome and the immunoproteasome, which comprise distinct sets of catalytic subunits. Additional proteasome subtypes containing mixtures of constitutive and immunoproteasome catalytic subunits have more recently been identified. These intermediate proteasome subtypes display unique proteolytic activity profiles and play important roles in the production of certain tumor antigens. However, much regarding their regulation, functions, and relevance as drug targets remains unknown, largely due to limitations in current experimental methods used to differentiate between proteasome subtypes. We have therefore developed bifunctional activity-based probes capable of cross-linking different catalytic subunit pairs within individual proteasome complexes. These probes allow a straightforward readout of proteasome catalytic subunit composition and facilitate functional studies of distinct proteasome subtypes. Experimental Procedures: Bifunctional proteasome probes were synthesized by coupling pairs of proteasome inhibitors via linkers of varying lengths and compositions. Positions for inhibitor derivatization were selected based on computational modeling, and linkers were chosen based on distances between the active sites of each targeted subunit pair. The ability of these compounds to cross-link distinct catalytic subunit pairs in both purified proteasomes and in cancer cell lysates was visualized by western blotting analysis. Competition assays with subunit-selective proteasome inhibitors were employed to confirm the identities of the cross-linked subunit pairs. Data Summary: The structures of the bifunctional proteasome probes were confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Western blotting results demonstrated the ability of these compounds to cross-link multiple pairs of proteasome catalytic subunits, allowing the composition of distinct proteasome subtypes present within cancer cells to be evaluated. Conclusions: We have successfully developed a set of bifunctional proteasome probes for analysis of proteasome catalytic subunit composition. These probes will be used to elucidate the unique functions of distinct proteasome subtypes present within cancer cells. Citation Format: Kimberly C. Carmony, Do-Min Lee, Lalit Kumar Sharma, Jieun Park, Kyung-Bo Kim, Wooin Lee. Development of a novel cross-linking strategy to identify distinct proteasome subtypes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3236. doi:10.1158/1538-7445.AM2014-3236

Differential thermal stability of human, bovine and ovine Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) quaternary structures

Quaternary structure of human, bovine and ovine Follicle-Stimulating Hormones (hFSH, bFSH and oFSH) and Luteinizing Hormone was assessed in sandwich ELISAs using monoclonal anti-oFSHβ or anti-oLHβ antibodies, respectively, for capture and a biotinylated anti-hFSHα (α4 epitope) for detection. Neither free subunit gave any signal in this assay so that it was possible to measure the residual heterodimeric fraction after thermal treatment of the gonadotropins under study. The hormones were subjected to 5-min heating between 37 and 90°C before rapid cooling in melting ice before ELISA. The data show half-dissociation of natural and recombinant human and ovine FSH preparations between 68 and 74°C whereas bovine FSH preparations exhibited lower stability in these conditions with half-dissociation between 61 and 64°C. Moreover, whereas all human and bovine as well as most ovine FSH preparations were fully dissociated at temperatures above 80°C, one natural oFSH and one recombinant hLH preparations contained an important fraction that resisted dissociation even at 93°C and retained in vitro bioactivity. This suggests the existence of gonadotropin αβ heterodimer with covalently linked subunits. Similarly, about 20% of the recombinant hLH preparation was also found withstand heat denaturation and also probably to have cross-linked subunits. The origin and chemical nature of these inter-subunit bonds remain to be determined.

Abstract 2231: Development of bifunctional cross-linking agents to identify intermediate proteasomes.

Abstract Introduction: This research was conducted to develop bifunctional cross-linking agents that can be used to identify intermediate proteasomes. Two well-characterized proteasome subtypes, the constitutive proteasome and immunoproteasome, differ with respect to their distinct sets of catalytic subunits. Additionally, intermediate proteasome subtypes comprised of non-standard mixtures of catalytic subunits have more recently been discovered. These intermediate proteasomes differ from constitutive and immunoproteasomes in their proteolytic activity profiles, suggesting that they have unique cellular functions. In agreement with this, intermediate proteasomes in cancer cell lines were shown to play important roles in the production of several tumor antigens. However, largely due to limitations of methods currently available to distinguish between subtypes, the detailed functions of intermediate proteasomes remain poorly defined. Bifunctional agents capable of cross-linking two different catalytic subunits within a single proteasome complex will facilitate a simple approach to identify distinct proteasome subtypes present within cells. These compounds will serve as important chemical probes for investigating the functions of individual proteasome subtypes in cancer cells. Experimental Procedures: Bifunctional cross-linking agents were synthesized by coupling two broad-spectrum or subunit-selective proteasome inhibitors through hydrocarbon or polyethylene glycol linkers. Positions for derivatization of these inhibitors were selected based on computational modeling, and linker lengths were varied to increase cross-linking efficiency. The ability of the resulting compounds to cross-link two proteasome catalytic subunits in cancer cell lysates was assessed via western blotting. Competition assays with subunit-selective proteasome inhibitors were conducted to verify the identities of the cross-linked subunit pairs. Data Summary: The structures of the bifunctional cross-linking agents were confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Western blotting results revealed the ability of several of these bifunctional agents to cross-link two catalytic subunits within individual proteasome complexes, facilitating compositional analysis of distinct proteasome subtypes. Conclusions: We have successfully developed bifunctional agents capable of cross-linking two catalytic subunits within individual proteasome complexes. These compounds will be utilized in functional studies of intermediate proteasomes present within cancer cells. Citation Format: Kimberly C. Carmony, Lalit Kumar Sharma, Kyung-Bo Kim. Development of bifunctional cross-linking agents to identify intermediate proteasomes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2231. doi:10.1158/1538-7445.AM2013-2231

A Role for Collagen IV Cross-links in Conferring Immune Privilege to the Goodpasture Autoantigen: STRUCTURAL BASIS FOR THE CRYPTICITY OF B CELL EPITOPES

The detailed structural basis for the cryptic nature (crypticity) of a B cell epitope harbored by an autoantigen is unknown. Because the immune system may be ignorant of the existence of such "cryptic" epitopes, their exposure could be an important feature in autoimmunity. Here we investigated the structural basis for the crypticity of the epitopes of the Goodpasture autoantigen, the alpha3alpha4alpha5 noncollagenous-1 (NC1) hexamer, a globular domain that connects two triple-helical molecules of the alpha3alpha4alpha5 collagen IV network. The NC1 hexamer occurs in two isoforms as follows: the M-isoform composed of monomer subunits in which the epitopes are accessible to autoantibodies, and the D-isoform composed of both monomer and dimer subunits in which the epitopes are cryptic. The D-isoform was characterized with respect to quaternary structure, as revealed by mass spectrometry of dimer subunits, homology modeling, and molecular dynamics simulation. The results revealed that the D-isoform contains two kinds of cross-links as follows: S-hydroxylysyl-methionine and S-lysyl-methionine cross-links, which stabilize the alpha3alpha5-heterodimers and alpha4alpha4-homodimers, respectively. Construction and analysis of a three-dimensional model of the D-isoform of the alpha3alpha4alpha5 NC1 hexamer revealed that crypticity is a consequence of the following: (a) sequestration of key residues between neighboring subunits that are stabilized by domain-swapping interactions, and (b) by cross-linking of subunits at the trimer-trimer interface, which stabilizes the structural integrity of the NC1 hexamer and protects against binding of autoantibodies. The sequestrated epitopes and cross-linked subunits represent a novel structural mechanism for conferring immune privilege at the level of quaternary structure. Perturbation of the quaternary structure may be a key factor in the etiology of Goodpasture disease.

Interactions of rotor subunits in the chloroplast ATP synthase modulated by nucleotides and by Mg 2+

ATP synthases – rotary nano machines – consist of two major parts, F O and F 1, connected by two stalks: the central and the peripheral stalk. In spinach chloroplasts, the central stalk (subunits γ, ε) forms with the cylinder of subunits III the rotor and transmits proton motive force from F O to F 1, inducing conformational changes of the catalytic centers in F 1. The ε subunit is an important regulator affecting adjacent subunits as well as the activity of the whole protein complex. Using a combination of chemical cross-linking and mass spectrometry, we monitored interactions of subunit ε in spinach chloroplast ATP synthase with III and γ. Onto identification of interacting residues in subunits ε and III, one cross-link defined the distance between ε-Cys6 and III-Lys48 to be 9.4 Å at minimum. ε-Cys6 was competitively cross-linked with subunit γ. Altered cross-linking yields revealed the impact of nucleotides and Mg 2+ on cross-linking of subunit ε. The presence of nucleotides apparently induced a displacement of the N-terminus of subunit ε, which separated ε-Cys6 from both, III-Lys48 and subunit γ, and thus decreasing the yield of the cross-linked subunits ε and γ as well as ε and III. However, increasing concentrations of the cofactor Mg 2+ favoured cross-linking of ε-Cys6 with subunit γ instead of III-Lys48 indicating an approximation of subunits γ and ε and a separation from III-Lys48.

Effect of Kiwifruit Juice on Beef Collagen

The objective of this study is to clarify the difference in susceptibility to protease digestion by kiwifruit juice between collagen domains under different conditions. In addition, the effect of pre-treatment with kiwifruit juice on collagen in meat during cooking processes was examined. Kiwifruit juice can degrade denatured collagen, but it can not cleave the triple helical domain of collagen. Thus, kiwifruit juice does not have collagenase activity. On the other hand, the cross-linked subunits of acid-soluble collagen were converted to monomeric subunits by kiwifruit juice treatment at acidic pH, suggesting that the globular domains, in which cross-links preferentially occur, can be degraded by kiwifruit juice. The pre-treatment with kiwifruit juice significantly decreased the shear force of connective tissue in comparison with other pre-treatments without protease activity, but inversely increased the liberation of collagen-related peptides in the outer solution by heating processes at 50 and 70 degrees C or by a shorter heating time at 100 degrees C. This can be explained by the protease-mediated degradation of globular domains. However, this effect was not observed with a prolonged heating period at 100 degrees C, and the liberation of collagen-related peptides by pre-treatment with kiwifruit juice at 100 degrees C was less than that at 70 degrees C for all heating periods. Thus, it can be suggested that the pre-treatment with kiwifruit juice might be useful in meat softening under vacuum-cooking and grilling, but not under stewing.

Acrolein Modifies and Inhibits Cytosolic Aspartate Aminotransferase

Acrolein is a reactive lipid peroxidation byproduct, which is found in ischemic tissue. We examined the effects of acrolein on cytosolic aspartate aminotransferase (cAAT), which is an enzyme that was previously shown to be inhibited by glycating agents. cAAT is thought to protect against ischemic injury. We observed that acrolein cross-linked cAAT subunits as evidenced by the presence of high molecular weight bands following SDS-PAGE. Acrolein-modified cAAT resisted thermal denaturation when compared with native cAAT. We also observed a decrease in intrinsic fluorescence (290 nm, ex; 380 nm, em). These observations are consistent with an acrolein-induced change in conformation that is more rigid and compact than native cAAT, suggesting that intramolecular cross-links occurred. Acrolein also inhibited activity, and the inhibition of enzyme activity correlated with the acrolein-induced formation of cAAT cross-links.

Photoaffinity Labeling and Photoaffinity Cross-Linking of Phosphofructokinase-1 from Saccharomyces cerevisiae by 8-Azidoadeninenucleotides

Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of four α- and four β-subunits, each of them carrying catalytic and regulatory bindings sites for MgATP. In this paper, various photoaffinity labels, such as 8-azidoadenosine 5′-triphosphate, 8-azido-1,N6-ethenoadenosine 5′-triphosphate, and 8-N3-3′(2′)-O-biotinyl-8-azidoadenosine 5′-triphosphate have been used to study their interaction with the enzyme in the dark and during irradiation. All nucleotidetriphosphates function as phosphate donor forming fructose 1,6-bisphosphate from fructose 6-phosphate. However, the kinetic analysis revealed distinctly differences between them. Photolabeling causes a decrease in enzyme activity to a similar extent, and ATP acts as competitive effector to inactivation. Three bifunctional diazidodiadeninedinucleotides (8-diN3AP4A, monoϵ-8-diN3AP4A, and diϵ-8-diN3AP4A) were applied for studying the spatial arrangement of the nucleotide binding sites. No cross-linking of the subunits was obtained by irradiation of the enzyme with 8-diN3AP4A. Photolabeling with diϵ-8-diN3AP4A resulted in the formation of two α-β cross-links with different mobilities in the SDS–polyacrylamide gel electrophoresis, while monoϵ-8-diN3AP4A yielded only one α-β cross-link. Because an interfacial location of the catalytic sites between two subunits is less likely, we suggest that the formation of cross-linked subunits may be the result of specific interactions of the bifunctional photolabels with regulatory sites at the interface of both subunits.

Dibromopropanone Cross-linking of the Phosphopantetheine and Active-site Cysteine Thiols of the Animal Fatty Acid Synthase Can Occur Both Inter- and Intrasubunit: REEVALUATION OF THE SIDE-BY-SIDE, ANTIPARALLEL SUBUNIT MODEL

The objective of this study was to test a new model for the homodimeric animal FAS which implies that the condensation reaction can be catalyzed by the amino-terminal beta-ketoacyl synthase domain in cooperation with the penultimate carboxyl-terminal acyl carrier protein domain of either subunit. Treatment of animal fatty acid synthase dimers with dibromopropanone generates three new molecular species with decreased electrophoretic mobilities; none of these species are formed by fatty acid synthase mutant dimers lacking either the active-site cysteine of the beta-ketoacyl synthase domain (C161A) or the phosphopantetheine thiol of the acyl carrier protein domain (S2151A). A double affinity-labeling strategy was used to isolate dimers that carried one or both mutations on one or both subunits; the heterodimers were treated with dibromopropanone and analyzed by a combination of sodium dodecyl sulfate/polyacrylamide gel electrophoresis, Western blotting, gel filtration, and matrix-assisted laser desorption mass spectrometry. Thus the two slowest moving of these species, which accounted for 45 and 15% of the total, were identified as doubly and singly cross-linked dimers, respectively, whereas the fastest moving species, which accounted for 35% of the total, was identified as originating from internally cross-linked subunits. These results show that the two polypeptides of the fatty acid synthase are oriented such that head-to-tail contacts are formed both between and within subunits, and provide the first structural evidence in support of the new model.

Myosin filament ATPase is enhanced by intramolecularly cross-linked actin

Reaction of rabbit skeletal muscle F-actin with the lysine-directed photolabile cross-linker, N-5-azido-2-nitrobenzoyloxy succinimide was limited to Lysine-328 and Lysine-326, with Lysine-328 being labelled to a greater extent. Photolysis of the modified actin enhanced the actin-activated MgATPase activity of filamentous scallop myosin 3-4-fold more than unmodified actin, without affecting calcium sensitivity. Unphotolysed modified actin behaved as untreated actin, indicating that photolysis was essential for the effect. The actin-activated ATPase of filamentous rabbit myosin was similarly increased by photolysed N-5-azido-2-nitrobenzoyloxy succinimide-modified actin. After photolysis in either the monomeric (G-) or filamentous (F-) form, N-5-azido-2-nitrobenzoyloxy succinimide-modified actin moved as a monomeric (42 kDa) species on SDS gels, and depolymerized and polymerized readily, demonstrating that any cross-linking event produced by photolysis must be intramolecular. In contrast to the substantial increase in actin-activated ATPase activity observed when photolysed ANB-NOS-modified actin was added to filamentous myosin, the enhancement was not observed with the soluble HMM and S-1 fragments of myosin. Photolysed modified actin showed only poor movement on a rabbit HMM-coated surface in vitro motility assays. These results can be explained if the internally cross-linked G-actin subunits which comprise only a fraction of the actin population, either weaken the actin-actin contacts or have an increased affinity for myosin.

Surface and internal galactosyl receptors are heterooligomers and retain this structure after ligand internalization or receptor modulation

We have developed a specific chemical affinity reagent for the hepatic galactosyl receptor (GalR) by derivatizing asialoorosomucoid (ASOR) with the homobifunctional N-hydroxysuccinimide (NHS) ester cross-linker disuccinimidyl suberate [Herzig, M. C. S., & Weigel, P. H. (1989) Biochemistry 28, 600]. NHS-ASOR cross-links with 30-50% efficiency to the three GalR subunits, designated rat hepatic lectins (RHL) 1, 2, and 3. Here, we examined the subunit structure of both surface and internal receptors of two functionally distinct GalR subpopulations, designated state 1 or state 2 GalR. Freshly isolated cells, referred to as state 1 cells, kept at 4 degrees C express only active state 1 GalR on their surface. When these cells are equilibrated at 37 degrees C, they then express both state 1 GalR and state 2 GalR on their surface. These cells are referred to as state 1,2 cells. After incubation at 4 degrees C with NHS-125I-ASOR, surface or internal GalR of state 1 cells or of state 1,2 cells incorporated 125I-ASOR into all three RHL subunits. As analyzed by autoradiography of SDS-PAGE, radiolabeling was identical for all conditions and was in a ratio of 1:1:1 for RHL 1:2:3. Native GalR structure was also examined by first cross-linking nonradiolabeled NHS-ASOR at 4 degrees C to surface or internal receptors of state 1 or state 1,2 hepatocytes. These cells were then washed with EGTA, extracted with Triton X-100, immunoprecipitated with anti-orosomucoid antibody, and subjected to Western blot analysis. Antisera specific for RHL 1 or RHL 2/3 detected cross-linked complexes of Mr congruent to 85K or congruent to 90K-115K, respectively, as well as un-cross-linked native subunits. In all four cases, the ratio of free to cross-linked subunits was greater than or equal to 5:1 for RHL 1 and less than or equal to 0.5:1 for RHL 2/3. Internalized GalR had the same ratio of free to cross-linked subunits as noninternalized GalR. Depletion of ATP either before or after cross-linking GalR to NHS/ASOR also did not alter the ratio of free cross-linked RHL subunits. We conclude that the surface and internal GalR of the two functionally distinct GalR populations have the same heterooligomeric subunit composition and that this GalR structure persists following endocytosis or ATP depletion.

Synthesis and characterization of N-hydroxysuccinimide ester chemical affinity derivatives of asialoorosomucoid that covalently crosslink to galactosyl receptors on isolated rat hepatocytes

We have developed chemical affinity reagents for the hepatic galactosyl receptor. Asialoorosomucoid (ASOR) was derivatized with five homobifunctional N-hydroxysuccinimide (NHS) ester cross-linkers. NHS/ASOR derivatives were synthesized, purified, and applied within 10 min to isolated rat hepatocytes at 4 degrees C. Specific binding of these 125I-labeled derivatives was approximately 90% in the presence of either EGTA or excess ASOR. Specific cross-linking assessed by the resistance of specifically bound NHS/125I-ASOR to release by EGTA, was 50-75% of the specifically bound ligand. The extent of specific cross-linking correlated with the average number of NHS groups per ASOR and was controlled by varying the molar ratio of cross-linker to ASOR during the synthesis. Cross-linking proceeded rapidly at 4 degrees C as a first-order process (k = 0.25 min-1, t1/2 = 2.8 min). After being cross-linked with any of the NHS/125I-ASOR derivatives, cells were washed with EGTA, solubilized in Triton X-100, and analyzed by SDS-PAGE and autoradiography. Major bands were observed at Mr congruent to 84K, 93K, and 105K corresponding to the expected size of 1:1 adducts between NHS/ASOR (Mr congruent to 41.3K) and the three subunits of the receptor, Mr congruent to 43K, 50K, and 60K. The three subunits, rat hepatic lectin (RHL) 1, 2, and 3, were labeled in the ratio of about 1.0:1.2:1.0, respectively. After cross-linking, a polyclonal goat antibody to the receptor immunoprecipitated up to 100% of the specifically cross-linked NHS/125I-ASOR. Preimmune IgG immunoprecipitated less than 1% of the radiolabeled ligand. Cell surface receptors were cross-linked to NHS-ASOR, extracted with Triton X-100, immunoprecipitated with anti-orosomucoid-Sepharose, and subjected to Western blot analysis. By use of anti-sera specific for RHL 1 or RHL 2/3 (from K. Drickamer), cross-linked complexes of Mr congruent to 85K or approximately 90-115K, respectively, were detected as were un-cross-linked native subunits. The ratio of free to cross-linked subunits was approximately 10:1 for RHL 1 and approximately 0.5:1 for RHL 2/3. We conclude that all three receptor subunits can cross-link to ligand. We propose a model in which the native receptor is a heterohexamer composed of four subunits of RHL 1 and two subunits of RHL 2 and/or RHL 3.

Properties of carboxymethylated cross-linked hemoglobin A.

The selective carboxymethylation of the N-terminal amino groups of hemoglobin A with glyoxylic acid and sodium cyanoborohydride has been studied as a function of the state of ligation of hemoglobin. The N-terminal residues have been established as the primary sites of reaction by peptide mapping of the tryptic digest of each chain and subsequent amino acid analysis of the modified peptides. With oxyhemoglobin, the desired derivatives with a carboxymethyl group at the N-terminal of either or both chains amounted to 55% [Di Donato, A., Fantl, W. J., Acharya, A. S., & Manning, J. M. (1983) J. Biol. Chem. 258, 11890-11895]. In the present study it is shown that with deoxyhemoglobin the amount of the desired derivative is increased to 75%. The oxygen equilibrium curve of hemoglobin A carboxymethylated on its four N-terminal residues [0.5 mM as tetramer in 50 mM [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane (Bis-Tris), pH 7.5, 37 degrees C] had a P50 value of 30 mmHg (Hill coefficient n = 2.8, alkaline Bohr value = 0.4) compared to a P50 of 9 mmHg for unmodified hemoglobin under the same conditions (n = 2.5, alkaline Bohr value = 0.5). In carboxymethylated oxyhemoglobin A, cross-linked with the mild agent glycolaldehyde for 3.5 h, there was 85% of Mr 64,000 species and 15% of Mr 128,000 or higher species. For the former, the extent of cross-linking between two subunits was 19%. For the latter, there was 29% of two cross-linked subunits and 13% of three cross-linked subunits. Termination of cross-linking, which may be desirable in some circumstances, can be successfully achieved with isonicotinic acid hydrazide.(ABSTRACT TRUNCATED AT 250 WORDS)

Column centrifugation generates an intersubunit disulfide bridge in Escherichia coli F1-ATPase.

Passage of F1-ATPase through a centrifuge column [Penefsky, H. S. (1979) Methods Enzymol. 56, 527-530] caused formation of a product with a relative molecular mass of 72,000 as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The product was identified as cross-linked alpha and delta subunits by using Western blots and subunit-specific monoclonal antibodies. The cross-link was reversed by 50 mM dithiothreitol implying that it was a disulfide bridge. Formation of the cross-link was inhibited by 2 mM EDTA and was stimulated in some buffers by the addition of 10 microM CuCl2. Time course experiments indicated that the majority of the cross-link formed while the enzyme was passing through the column. Thus the cross-link induced by column centrifugation arose from the rapid, heavy-metal-ion-catalysed oxidation of two sulfhydryl groups, one on the alpha subunit and one on the delta subunit, to a disulfide. These results demonstrate that care must be exercised when running proteins through centrifuge columns as potentially deleterious disulfide formation can result. An anti-beta monoclonal antibody was capable of immunoprecipitating the entire enzyme including the cross-linked subunits, implying that the cross-linked alpha and delta subunits were still a part of F1. The formation of the cross-link affected neither the hydrolytic activity of the enzyme nor its susceptibility to inhibition by epsilon subunit. The cross-linked enzyme was unable to bind to F1-depleted membranes in experiments in which soluble F1 and membranes were separated by centrifugation. Column centrifugation did not generate the cross-link on membrane-bound enzyme. These results indicate that the alpha-delta cross-link results in a loss of binding affinity between F1 and F0.

Activity of penicillin-binding protein 3 from Escherichia coli.

The activity of penicillin-binding protein 3 of Escherichia coli has been studied both in vivo and in ether-permeabilized cells. The peptidoglycan transpeptidase activity of penicillin-binding protein 3 appears to use either nascent or exogenously added UDP-N-acetylmuramyl tripeptide-derived substrates as acceptors. By means of a defilamentation system which elicited the activity of penicillin-binding protein 3 in vivo, the structure of peptidoglycan made by this enzyme has been elucidated. This peptidoglycan, very probably of septal location, contained increased amounts of cross-linked peptidoglycan as well as a higher ratio of tripeptide-containing cross-linked subunits.

Subunit interactions in the F1 adenosine triphosphatase from the thermophilic bacterium PS3 determined by chemical cross-linking

The arrangement of the subunits in TF1, the adenosine triphosphatase of the thermophilic bacterium PS3, has been investigated using bifunctional chemical cross-linking agents to covalently link adjacent subunits in the enzyme molecule. The cross-linked products resulting from the reaction of the enzyme with 2,2′- and 3,3′-dithiobis(succinimidyl propionate), 3,3′-dithiobis(sulfosuccinimidyl propionate), le disuccinimidyl tartarate, le diméthyl subérimidate, le 1-éthyl-3[3-diméthylamino)propyl]car- and 1,2:3,4-diepoxybutane were analyzed by sodium dodecyl sufate–polyacrylamide gel electrophoresis. Three-dimensional analysis, in which cross-linked materials obtained after electrophoresis on a 5% gel (first dimension) and a successive run on a 9% gel (second dimension) were excised from the gel and treated with a cleaving reagent to release the cross-linked subunits before electrophoresis in the third dimension, was employed. The following cross-linked dimers were identified: αα, αβ, αγ, βγ, αδ, and γε. Two trimers, α2δ and γαδ, were recognized. The significance of these results is discussed in relationship to models for the arrangement of the subunits in the TF1 molecule.

Electrophoretic determination of sulfhydryl groups and its application to complex protein samples, in vitro protein synthesis mixtures, and cross-linked proteins.

Without prior fractionation, the number of sulfhydryl groups of individual polypeptides in a protein mixture can be determined, provided their molecular weights and approximate isoelectric points are known. Urea-denatured protein samples are reacted with iodoacetamide and iodoacetate in a modified version of Creighton's procedure. After separation by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and isoelectric focusing, the number of sulfhydryl groups is determined by counting the protein bands which have additional negative charges. This method requires little material and provides an additional parameter, besides the molecular weight and isoelectric point, for the identification and characterization of a protein. The sensitivity may be enhanced for nonradioactive proteins by using 14C-labeled iodoacetamide and iodoacetate. The procedure has been applied to prokaryotic in vitro protein synthesis mixtures, bacterial membrane protein, and trypsin-cleaved or chemically cross-linked subunits of the F1 ATPase from Escherichia coli.

The molecular morphology of bovine heart mitochondrial NADH—-ubiquinone reductase. Native disulfide-linked subunits and rotenone-induced conformational changes.

Bovine heart mitochondrial NADH----ubiquinone reductase (complex I), contains two disulfide-linked subunits of 75 and 33 kDa as revealed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with beta-mercaptoethanol omitted from preparation of the sample for the first dimension. Two unidentified polypeptides (110-115 and 69 kDa) are also found in disulfide linkage with the two complex I subunits. The 110-115-kDa polypeptide appears to be pyridine dinucleotide transhydrogenase by several criteria including selective precipitation with an antibody raised to the purified transhydrogenase. The two disulfide-linked subunits were also found in a product cross-linked for 2 min with dithiobis (succinimidyl propionate) (DSP) along with five other complex I subunits of 53-57, 42, 24-27, 17-18, and 12.5-15.5 kDa (Gondal, J.A., and Anderson, W.M. (1985) J. Biol. Chem. 260, 5931-5935) indicating that these seven subunits lie within 11-12 A of each other at one or more points in space in the enzyme's interior. Cross-linking of complex I with DSP for 2 min in the presence of 1 microM rotenone yielded a cross-linked product consisting of the two natural disulfide-linked subunits and the 110-115- and 69-kDa polypeptides. This suggests that rotenone induces a conformational change in the enzyme that moves the seven DSP cross-linked subunits away from each other and outside the 11-12 A bridging distance of DSP. This alteration in conformation may be communicated to iron-sulfur center N-2 within the hydrophobic outer shell of the enzyme to prevent electron transfer to its natural electron acceptor, ubiquinone. A model of rotenone action based upon these observations is presented.

4-Aminobutyrate aminotransferase reaction of sulfhydryl residues connected with catalytic activity.

4-Aminobutyrate aminotransferase is inactivated by preincubation with N-(1-pyrene)maleimide (mixing molar ratio 10:1) at pH 7. The reaction with N-(1-pyrene)maleimide was monitored by fluorescence spectroscopy and the degree of labeling of the enzyme determined by absorption spectroscopy. The blocking of 2 cysteinyl residues/enzyme dimer is needed for inactivation of the aminotransferase. The time course of the reaction is significantly affected by the substrate alpha-ketoglutarate, which afforded complete protection against the loss of catalytic activity. Trypsin digestion of pyrene-labeled aminotransferase, followed by gel filtration and "fingerprint" analysis, revealed the presence of only one peptide tagged with the fluorescent probe. The reaction of approximately 1.9 SH residues/dimer with iodosobenzoate resulted in enzyme inactivation together with a formation of an oligomeric species of Mr = 100,000 detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cross-linked subunits are dissociated by addition of 2-mercaptoethanol which also restores full catalytic activity. Altogether, these observations are consistent with the concept that inactivation of 4-aminobutyrate aminotransferase by iodosobenzoate proceeds through disulfide bond formation between vicinal cysteinyl residues of the protein. It is postulated that the critical sulfhydryl groups of the enzyme are situated on opposite sides of the dimeric structure at the subunit interfaces.

Topology, organization, and function of the psi subunit in the F0 sector of the H+-ATPase of Escherichia coli.

The F1F0 H+-ATPase in membranes of Escherichia coli was amplified by heat induction of a lysogenic lambda-unc+ transducing phage. Inverted membrane vesicles were stripped of the F1 sector of the ATPase complex by washing with EDTA. The stripped membranes were treated with dithiobis(succimidylpropionate) to cross-link subunits of the F0 sector of the ATPase complex. After electrophoresis under nonreducing conditions in one dimension, cross-linked subunits were identified by off-diagonal electrophoresis in a second dimension following cleavage of the cross-linked products with beta-mercaptoethanol. A psi-psi dimer was the major cross-linked product identified. In addition, a chi-psi product and chi-psi2 product were identified. These results support the proposed chi-psi2 stoichiometry of subunits in F0. When the F1-stripped membranes were treated with trypsin, the psi subunit was rapidly degraded, whereas psi was protected from degradation when F1 was bound to the membrane. Trypsin-treated, stripped membranes, lacking an intact psi subunit, did not bind the F1 portion of the ATPase with high affinity. However, these trypsin-treated stripped membranes remained as permeable to protons as untreated stripped membranes, and the H+ conductivity was blocked by dicyclohexylcarbodiimide. These results indicate that the portion of the psi subunit exposed on the cytoplasmic face of the inner membrane is involved in the binding of the F1 portion of the ATPase, but is not necessary for H+ conduction mediated by the F0 sector of the complex.