Disulfide Bond-reducing Agents Research Articles

Racial Disparity in Antibody Against A rare High Prevalence Antigen; (Anti-Inb)

Abstract Introduction/Objective Introduction: Ina antigen is very rare except in Iranian and Arab populations (10.6% and 11.8%, respectively). In 1973, Badkere et al. identified this antigen in about 3% of Indians from Bombay. Inb is a very high-prevalence antigen. It is prevalent in 99.9% of Caucasians and blacks, 96% of Indians and South Asians, and 90% of Iranians and Arabs. The Indian blood group system also has two other very high prevalence antigens: INFI and INJA. Indian blood group resides in CD44 glycoprotein that binds to hyaluronate of extracellular matrix. CD44 is an adhesion molecule in lymphocytes, monocytes, and other tumor cells, but its function in RBC is unknown. Ina and Inb are sensitive to proteolytic enzyme treatments and destroyed by disulfide bond-reducing agents 2-aminoethyl isothiouronium bromide (AET) and dithiothreitol (DTT). Methods/Case Report Case study: An 11-year-old female of South-East Indian descent with a history of intractable epilepsy was admitted for surgical treatment for epilepsy. Her antibody screen was positive, and her plasma reacted with all cells in PEG AHG except for the auto control. Ficin and DTT-treated cells weakened the reactivity. Suspecting a high-frequency antibody, the patient's plasma was tested against a series of cells in the rare donor cell inventory, including O Bombay cell and Inb-cell considering the patient's ethnicity. With Inb-negative cells, the plasma tested negative. All major alloantibodies from this rare donor cell were ruled out except for anti-C, anti-E, anti-K, and anti-S due to the availability of the rare cell. Results (if a Case Study enter NA) NA Conclusion Antigen Inb has a high prevalence in all populations. The rare phenotype In(b–) has been observed in approximately 2 of 700 Indian blood donors. A patient may be advised to undergo an autologous transfusion. Fortunately, the patient's procedures were uncomplicated, so a blood transfusion was unnecessary. Awareness on the part of blood bank staff regarding the variability of the blood group phenotype and the unpredictability of Anti-Inb may aid in improving patient care. To prevent further exposure, it is recommended that patients with rare antibodies carry an Antibody ID card indicating the rare antibody they possess.Inb antigen blood donors are under represented in the general donors pool. It is recommended to develop a systematic recruitment for Inb antigen donors

Benign High Prevalence Antibody Yta

Abstract Introduction/Objective The Yt system, also known as the Cartwright system, discovered in 1964, consists of five antigens encoded by the ACHE gene on chromosome 7. Yta and Ytb are antithetical antigens on the GPI – linked red cell glycoprotein, acetylcholinesterase (AChE). About 8-10% of individuals of European ancestry are Yt(b+); Yta is highly prevalent in all populations. The other three high-prevalence antigens, YTEG, YTLI, and YTOT, were added to this system in 2018. Yta is not affected by trypsin but is destroyed by α-chymotrypsin treatment of reagent red cells. Yta and Ytb are sensitive to disulfide bond-reducing agents 2-aminoethylisothiouronium bromide (AET) and dithiothreitol (DTT). Yt antibodies are mostly IgG and require Indirect Antiglobulin Test (IAT) for detection. Yt antibodies are not clinically significant, although anti-Yta may cause accelerated destruction of Yt(a+) transfused red cells. Monocyte Monolayer Assay (MMA) is often used to determine whether the anti-Yta is predicted to cause destruction of transfused Yt(a+) red cells. Methods/Case Report A 50 year old female of Hispanic/Latino descent with a BRCA2 mutationand a history of scoliosis and severe migraineswas admitted for robotic-assisted bilateral salpingo-oophorectomy, cystoscopy, and dilation and curettage. A type and screen was ordered preoperatively, and her antibody screen was positive. In her profile, the origin of anti-Yta was identified from an outside facility since 2008, but written documentation of anti-Yta identification could not be located. On antibody identification, the patient’s plasma was reactive with all cells (1+ AHG in PEG) except the auto control. Rare frozen anti-Yta antisera were used for phenotyping the patient for Yta and she was found to be Yta negative. Yta antigens are sensitive or weakened by DTT, so DTT-treated panel screen cells were used to rule out all clinically significant alloantibodies except for the Kell system. Anti-K and anti-k were ruled out by testing the patient’s plasma against SCARF (Serum, Cells, and Rare Fluid Exchange) Yta negative with homozygous K positive and homozygous k positive cells. Anti-Yta was reconfirmed at our facility. Results (if a Case Study enter NA) NA Conclusion Yta-negative blood is so rare that anti-Yta reacts > 99.8% of most populations. In some cases, autologous transfusion may be recommended for a patient. However, anti-Yta is often benign, and antigen-negative blood may not need to be transfused. The patient had uncomplicated procedures, and blood transfusion was not indicated at that time.

Production and characterization of a novel aminopeptidase A from Lactococcus lactis

Aminopeptidase A (Pep A) is a metal-dependent enzyme that specifically hydrolyze peptides with the N-terminal amino acids glutamic acid (Glu) and aspartic acid (Asp). A possible application of PepA is the hydrolysis of Glu/Asp-rich food proteins such as wheat gluten and casein, increasing the flavor and solubility of food protein. In the present study, the gene encoding a Pep A from Lactococcus lactis ssp. lactis IL1403 was synthesized and introduced into Pichia pastoris GS115 (His4). Lc-Pep A was successfully expressed and secreted to the culture medium, followed by identification and purification to homogeneity. Characteristics study demonstrated that Lc-Pep A could specifically hydrolyze the substrates Glu-pNA and Asp-pNA with similar catalytic activity, and this was further confirmed by the kinetics parameters measured. Additionally, Lc-Pep A showed a broad thermostability and pH stability with an optimum temperature of 60 ℃ and an optimum pH of 8.0. The enzyme activity of Lc-Pep A was activated by metal ions Co2+, Mn2+, and Zn2+ but was strongly inhibited by Ni2+and Cu2+. The routine proteinase inhibitor had no effect on the activity of Lc-Pep A. However, Lc-Pep A was strongly inhibited by the metallopeptidase inhibitor, EDTA, and disulfide bond-reducing agents. The study may facilitate production and application of Lc-Pep A.

Putative extracellular α-class carbonic anhydrase, EcaA, of Synechococcus elongatus PCC 7942 is an active enzyme: a sequel to an old story.

Carbonic anhydrase (CA) EcaA of Synechococcus elongatus PCC 7942 was previously characterized as a putative extracellular α-class CA, however, its activity was never verified. Here we show that EcaA possesses specific CA activity, which is inhibited by ethoxyzolamide. An active EcaA was expressed in heterologous bacterial system, which supports the formation of disulfide bonds, as a full-length protein (EcaA+L) and as a mature protein that lacks a leader peptide (EcaA-L). EcaA-L exhibited higher specific activity compared to EcaA+L. The recombinant EcaA, expressed in a bacterial system that does not support optimal disulfide bond formation, exhibited extremely low activity. This activity, however, could be enhanced by the thiol-oxidizing agent, diamide; while a disulfide bond-reducing agent, dithiothreitol, further inactivated the enzyme. Intact E. coli cells that overexpress EcaA+L possess a small amount of processed protein, EcaA-L, whereas the bulk of the full-length protein resides in the cytosol. This may indicate poor recognition of the EcaA leader peptide by protein export systems. S. elongatus possessed a relatively low level of ecaA mRNA, which varied insignificantly in response to changes in CO2 supply. However, the presence of protein in the cells is not obvious. This points to the physiological insignificance of EcaA in S. elongatus, at least under the applied experimental conditions.

A Novel Glutamyl (Aspartyl)-Specific Aminopeptidase A from Lactobacillus delbrueckii with Promising Properties for Application.

Lactic acid bacteria (LAB) are auxotrophic for a number of amino acids. Thus, LAB have one of the strongest proteolytic systems to acquit their amino acid requirements. One of the intracellular exopeptidases present in LAB is the glutamyl (aspartyl) specific aminopeptidase (PepA; EC 3.4.11.7). Most of the PepA enzymes characterized yet, belonged to Lactococcus lactis sp., but no PepA from a Lactobacillus sp. has been characterized so far. In this study, we cloned a putative pepA gene from Lb. delbrueckii ssp. lactis DSM 20072 and characterized it after purification. For comparison, we also cloned, purified and characterized PepA from Lc. lactis ssp. lactis DSM 20481. Due to the low homology between both enzymes (30%), differences between the biochemical characteristics were very likely. This was confirmed, for example, by the more acidic optimum pH value of 6.0 for Lb-PepA compared to pH 8.0 for Lc-PepA. In addition, although the optimum temperature is quite similar for both enzymes (Lb-PepA: 60°C; Lc-PepA: 65°C), the temperature stability after three days, 20°C below the optimum temperature, was higher for Lb-PepA (60% residual activity) than for Lc-PepA (2% residual activity). EDTA inhibited both enzymes and the strongest activation was found for CoCl2, indicating that both enzymes are metallopeptidases. In contrast to Lc-PepA, disulfide bond-reducing agents such as dithiothreitol did not inhibit Lb-PepA. Finally, Lb-PepA was not product-inhibited by L-Glu, whereas Lc-PepA showed an inhibition.

Bidirectional effects of hydrogen sulfide via ATP-sensitive K+ channels and transient receptor potential A1 channels in RIN14B cells

Hydrogen sulfide (H2S) reportedly acts as a gasotransmitter because it mediates various cellular responses through several ion channels including ATP-sensitive K+ (KATP) channels and transient receptor potential (TRP) A1 channels. H2S can activate both KATP and TRPA1 channels at a similar concentration range. In a single cell expressing both channels, however, it remains unknown what happens when both channels are simultaneously activated by H2S. In this study, we examined the effects of H2S on RIN14B cells that express both KATP and TRPA1 channels. RIN14B cells showed several intracellular Ca2+ concentration ([Ca2+]i) responses to NaHS (300µM), an H2S donor, i.e., inhibition of spontaneous Ca2+ oscillations (37%), inhibition followed by [Ca2+]i increase (24%), and a rapid increase in [Ca2+]i (25%). KATP channel blockers, glibenclamide or tolbutamide, abolished any inhibitory effects of NaHS and enhanced NaHS-mediated [Ca2+]i increases, which were inhibited by extracellular Ca2+ removal, HC030031 (a TRPA1 antagonist), and disulfide bond-reducing agents. NaHS induced 5-hydroxytryptamine (5-HT) release from RIN14B cells, which was also inhibited by TRPA1 antagonists. These results indicate that H2S has both inhibitory and excitatory effects by opening KATP and TRPA1 channels, respectively, in RIN14B cells, suggesting potential bidirectional modulation of secretory functions.

Mutations in Orai1 transmembrane segment 1 cause STIM1-independent activation of Orai1 channels at glycine 98 and channel closure at arginine 91

Stim and Orai proteins comprise the molecular machinery of Ca(2+) release-activated Ca(2+) (CRAC) channels. As an approach toward understanding the gating of Orai1 channels, we investigated effects of selected mutations at two conserved sites in the first transmembrane segment (TM1): arginine 91 located near the cytosolic end of TM1 and glycine 98 near the middle of TM1. Orai1 R91C, when coexpressed with STIM1, was activated normally by Ca(2+)-store depletion. Treatment with diamide, a thiol-oxidizing agent, induced formation of disulfide bonds between R91C residues in adjacent Orai1 subunits and rapidly blocked STIM1-operated Ca(2+) current. Diamide-induced blocking was reversed by disulfide bond-reducing agents. These results indicate that R91 forms a very narrow part of the conducting pore at the cytosolic side. Alanine replacement at G98 prevented STIM1-induced channel activity. Interestingly, mutation to aspartate (G98D) or proline (G98P) caused constitutive channel activation in a STIM1-independent manner. Both Orai1 G98 mutants formed a nonselective Ca(2+)-permeable conductance that was relatively resistant to block by Gd(3+). The double mutant R91W/G98D was also constitutively active, overcoming the normal inhibition of channel activity by tryptophan at the 91 position found in some patients with severe combined immunodeficiency (SCID), and the double mutant R91C/G98D was resistant to diamide block. These data suggest that the channel pore is widened and ion selectivity is altered by mutations at the G98 site that may perturb α-helical structure. We propose distinct functional roles for G98 as a gating hinge and R91 as part of the physical gate at the narrow inner mouth of the channel.

Resolving the native conformation of Escherichia coli OmpA

The native conformation of the 325-residue outer membrane protein A (OmpA) of Escherichia coli has been a matter of contention. A narrow-pore, two-domain structure has vied with a large-pore, single-domain structure. Our recent studies show that Ser163 and Ser167 of the N-terminal domain (1-170) are modified in the cytoplasm by covalent attachment of oligo-(R)-3-hydroxybutyrates (cOHBs), and further show that these modifications are essential for the N-terminal domain to be incorporated into planar lipid bilayers as narrow pores (≈ 80 pS, 1 m KCl, 22 °C). Here, we examined the potential effect(s) of periplasmic modifications on pore structure by comparing OmpA isolated from outer membranes (M-OmpA) with OmpA isolated from cytoplasmic inclusion bodies (I-OmpA). Chemical and Western blot analysis and 1H-NMR showed that segment 264-325 in M-OmpA, but not in I-OmpA, is modified by cOHBs. Moreover, a disulfide bond is formed between Cys290 and Cys302 by the periplasmic enzyme DsbA. Planar lipid bilayer studies indicated that narrow pores formed by M-OmpA undergo a temperature-induced transition into stable large pores (≈ 450 pS, 1 M KCl, 22 °C) [energy of activation (Ea) = 33.2 kcal·mol(-1)], but this transition does not occur with I-OmpA or with M-OmpA that has been exposed to disulfide bond-reducing agents. The results suggest that the narrow pore is a folding intermediate, and demonstrate the decisive roles of cOHB-modification, disulfide bond formation and temperature in folding OmpA into its native large-pore configuration.

MER1, a novel organic arsenic derivative, has potent PML-RARα- independent cytotoxic activity against leukemia cells

Arsenic trioxide (ATO) is an inorganic arsenic derivative that is highly effective against PML-RARalpha-positive leukemia but much less against other hematological malignancies. We synthesized an organic arsenic derivative (OAD), S-dimethylarsino-thiosuccinic acid (MER1), which offers a superior toxicity profile and comparable in vitro activity relative to ATO. In Swiss Webster mice, maximally-tolerated cumulative dose of MER1 when given i.v. for 5 days was 100 mg/kg/d. We demonstrated that MER1 induced apoptosis and dose- and time-dependent inhibition of survival and growth in a panel of myeloid leukemia cell lines. Unlike ATO, this activity was independent of PML-RARalpha status and was not associated with induction of myeloid maturation. In NB4 and HL60 cells, MER1 and ATO induced caspase activation and dissipation of mitochondrial transmembrane potential. At the same time, MER1 induced generation of reactive oxygen species (ROS) and cell cycle arrest in G2/M phase and proved to be more potent than ATO at inducing apoptosis. ROS generation and intracellular glutathione levels were key modulators of MER1-induced cytotoxicity as evidenced by abrogation of apoptosis in myeloid leukemia cell lines pretreated with the disulfide bond-reducing agent dithiothreitol or the radical scavenger N-acetyl-L-cysteine. Collectively, these data indicate that MER1 induces apoptosis in PML-RARalpha-positive and -negative myeloid leukemia cells by enhancing oxidative stress. This agent, therefore, combines low in vivo toxicity with formidable in vitro pro-apoptotic ROS-mediated activity, and may represent a novel OAD suitable for clinical development against a variety of hematological malignancies.

Oligomerization of G-protein-coupled Receptors Shown by Selective Co-immunoprecipitation

Recent studies have shown that G-protein-coupled receptors (GPCRs) can assemble as high molecular weight homo- and hetero-oligomeric complexes. This can result in altered receptor-ligand binding, signaling, or intracellular trafficking. We have co-transfected HEK-293 cells with differentially epitope-tagged GPCRs from different subfamilies and determined whether oligomeric complexes were formed by co-immunoprecipitation and immunoblot analysis. This gave the surprising result that the 5HT(1A) receptor was capable of forming hetero-oligomers with all GPCRs tested including the 5HT(1B), 5HT(1D), EDG(1), EDG(3), GPR(26), and GABA(B2) receptors. The testing of other GPCR combinations showed similar results with hetero-oligomer formation occurring for the 5HT(1D) with the 5HT(1B) and EDG(1) receptor. Control studies showed that these complexes were present in co-transfected cells before the time of lysis and that the hetero-oligomers were comprised of GPCRs at discrete stoichiometries. These findings suggest that GPCRs have a natural tendency to form oligomers when co-transfected into cells. Future studies should therefore investigate the presence and physiological role of GPCR hetero-oligomers in cells in which they are endogenously expressed.

Free thiol groups are essential for infectivity of human cytomegalovirus.

The membrane-impermeable thiol blocker 5'5-dithiobis 2- nitrobenzoic acid (DTNB) blocked infectivity of human cytomegalovirus (CMV) although the virus still bound to cells. DTNB-treated CMV regained 65% of its infectivity after incubation with the disulfide bond-reducing agent dithiothreitol. These observations suggest that free thiol groups on CMV are required for infectivity and may participate in disulfide bond formation during virus entry.

Purification and characterization of a low-molecular-weight phospholipase A2 from developing seeds of elm.

Phospholipase A2 (PLA2) was purified about 180,000 times compared with the starting soluble-protein extract from developing elm (Ulmus glabra) seeds. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified fraction showed a single protein band with a mobility that corresponded to 15 kD, from which activity could be recovered. When analyzed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry, the enzyme had a deduced mass of 13,900 D. A 53-amino acid-long N-terminal sequence was determined and aligned with other sequences, giving 62% identity to the deduced amino acid sequence of some rice (Oryza sativa) expressed sequence tag clones. The purified enzyme had an alkaline pH optimum and required Ca2+ for activity. It was unusually stable with regard to heat, acidity, and organic solvents but was sensitive to disulfide bond-reducing agents. The enzyme is a true PLA2, neither hydrolyzing the sn-1 position of phosphatidylcholine nor having any activity toward lysophosphatidylcholine or diacylglycerol. The biochemical data and amino acid sequence alignments indicate that the enzyme is related to the well-characterized family of animal secretory PLA2s and, to our knowledge, is the first plant enzyme of this type to be described.

The Removal of the Sperm Perinuclear Theca and Its Association with the Bovine Oocyte Surface during Fertilization

The perinuclear theca (PT) is a unique cytoskeletal structure whose anterior part is intercalated between the inner acrosomal membrane and the nuclear envelope of the mammalian sperm head and is important for spermiogenesis and stabilization of sperm structures (Oko and Maravei,Biol. Reprod.50, 1000–1014, 1994; Oko and Maravei,Microsc. Res. Tech.32, 520–532, 1995). Using immunofluorescence labeling of inseminated bovine oocytes and serial sectioning–ultrastructural analysis, we demonstrate that the PT is removed from the sperm nucleus following the loss of the sperm plasma membrane and the interaction of oocyte cortex with the PT. These events precede the development of the male pronucleus. The removal of the PT involves the elongated oocyte microvilli, rich in actin microfilaments, since it can be blocked by the microfilament-disrupting drug cytochalasin B. Reduction of disulfide bonds, which is a major factor supporting the disassembly of the sperm nucleus and accessory structures during mammalian fertilization, seems to exert little effect on the PTin vitro,as evidenced by the treatment of isolated bull sperm with the disulfide bond-reducing agent dithiothreitol.In vivo,intact bull sperm microinjected into mature oocytes do not undergo disassembly of the PT. Consequently, the decondensation of the sperm nucleus does not occur. These data suggest that the binding of the PT to the oocyte microvillar region and its removal from the sperm nucleus constitute an early step in mammalian fertilization, which is required for the conversion of the sperm nucleus into a male pronucleus.

Depletion of glutathione during bovine oocyte maturation reversibly blocks the decondensation of the male pronucleus and pronuclear apposition during fertilization.

Oocyte-produced glutathione (the tripeptide gamma-glutamyl-cysteinyl-glycine; GSH) has been implicated in the reduction of disulfide bonds in the sperm nucleus during fertilization and thus in the development of the male pronucleus (PN). In this study, we show that the depletion of endogenous glutathione by 10 mM buthionine sulfoximine (BSO; specific inhibitor of GSH synthesis) during bovine oocyte maturation (24 h in vitro; represents prophase I to metaphase II transition in this species) blocks the formation of a male PN in > 85% of treated oocytes (vs. 6.8% in controls) and prevents the assembly of the sperm aster microtubules in approximately 35%. Consequently, the pronuclear migration and apposition do not occur. Ultrastructural observations suggest that the effect of BSO on pronuclear apposition might be due to incomplete disassembly of the sperm tail connecting piece, which normally leads to the release of the sperm centriole and to the reconstitution of the zygotic centrosome during fertilization. The sperm nucleus decondensation and migration blocks were reversed by the treatment of the GSH-depleted oocytes with 1-10 mM dithiothreitol (a disulfide bond-reducing agent) applied 8 h after insemination: 82% of these oocytes exhibited a normal male PN and pronuclear apposition 20 h after insemination. The pool of glutathione seems to be generated during oocyte maturation since > 80% of oocytes that were matured in the absence of BSO displayed a normal male PN, as apposed to a female PN, when inseminated and cultured in the presence of 10 mM BSO. These data suggest that the reduction of disulfide bonds in the sperm after incorporation is important for the formation of the male PN, as well as for the disassembly of the sperm tail connecting piece and pronuclear apposition. The lack of disulfide-reducing power in the GSH-depleted oocytes can be reversed by treatment with disulfide bond-reducing agents.

Mitochondrial sheath movement and detachment in mammalian, but not nonmammalian, sperm induced by disulfide bond reduction.

The successful completion of the fertilization process requires the properly choreographed unsheathing of the tightly packaged sperm once it has been fully incorporated into the egg's cytoplasm. The nuclear and accessory structures of mammalian sperm become stabilized by disulfide bonds (S-S) during epididymal maturation. This stabilization is reversed during fertilization by the reduction of S-S cross-linking, but little is known about the effect of S-S reduction on individual disulfide-hardened structures such as the sperm's connecting piece, fibrous sheath, and mitochondria. Here, we demonstrate the action of the S-S-reducing environment on the mitochondrial sheath of mammalian sperm, visualized by the vital fluorescent probe MitoTracker and by electron microscopy. In both human and bull sperm, mitochondria form a compact helix (mitochondrial sheath) wrapped around the midpiece and connecting piece that can be fluorescently labelled by a short incubation with 100 nM MitoTracker. Exposure of bull sperm to 0.1-10 mM dithiothreitol (DTT; a disulfide bond-reducing agent) induced a time and dose-dependent sliding of the mitochondrial sheath down the axoneme, accompanied by the excision of the sperm tail and decondensation of the sperm nucleus. Increasing the concentration of DTT to 100 mM accelerated mitochondrial movement, causing a completed stripping of sperm mitochondria and partial disassembly of the connecting piece. Likewise, human sperm responded to DTT treatment by the sliding or removal of the mitochondrial sheath and decondensation of the sperm chromatin. These events were not observed in the sperm of lower vertebrates and invertebrates (Xenopus laevis and Lytechinus pictus, respectively) exposed to an excess of DTT. Thus the sensitivity of sperm mitochondria to the S-S reducing environment seems to be an exclusive feature of mammalian sperm. The movement of sperm mitochondria induced by S-S reduction may be an initial critical step in the disassembly of the mammalian sperm tail during fertilization.

Role of disulfide bonds in maintaining the structural integrity of the sheath of Leptothrix discophora SP-6.

Isolated sheaths of Leptothrix discophora SP-6 (ATCC 51168) were tested for susceptibility to degradation by a variety of chemical denaturants and lytic enzymes and found to be resistant to many reagents and enzyme treatments. However, disulfide bond-reducing agents such as dithiothreitol (DTT), beta-mercaptoethanol, sodium cyanide, and sodium sulfite degraded the sheath, especially at elevated pH (pH 9) and temperature (50 degrees C). DTT and beta-mercaptoethanol caused more rapid degradation of the sheath than cyanide or sulfite. Treatment of the sheath with 1 N NaOH resulted in rapid breakdown, while treatment with 1 N HCl resulted in slow but significant hydrolysis. Transmission electron microscopy showed that the 6.5-nm fibrils previously shown to be an integral structural element of the sheath fabric (D. Emerson and W. C. Ghiorse, J. Bacteriol. 175:7808-7818, 1993) were progressively dissociated into random masses during DTT-induced degradation. Quantitation of disulfide bonds with DTT showed that the sheaths contained approximately 2.2 mumol of disulfides per mg of sheath protein. Reaction with 5,5'-dithio-bis-(2-nitrobenzoic acid) showed that sheaths also contained approximately 0.8 mumol of free sulfhydryls per mg of protein. A sulfhydryl-specific fluorescent probe (fluorescein 5-maleimide) showed that the free sulfhydryls in sheathed cell filaments were evenly distributed throughout the sheath. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography of [14C]iodoacetamide-labeled sheaths and DTT-dissociated sheath fibril suspensions showed that the majority of 14C-labeled sulfhydryls in the sheaths did not enter the gel. However, low-molecular-mass silver-staining bands (14 to 45 kDa) did appear in the gels after iodoacetic acid or iodoacetamide alkylation of the dissociated fibrils. These bands did not stain with Coomassie blue. Their migration in gels was slightly affected by digestion with pronase. The fibrils contained 20 to 25% protein. These results confirm that the sheath fibrils consist of high molecular-weight heteropolysaccharide-protein complexes. We hypothesize that proteins in the fibril complexes provide interfibril cross-linking to maintain the structural integrity of the sheath.

Identification and initial topological analysis of the Rickettsia prowazekii ATP/ADP translocase

The Rickettsia prowazekii ATP/ADP translocase was identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis using antibodies raised against a synthetic peptide corresponding in sequence to the carboxyl-terminal 17 amino acids of the carrier. Both the translocase of R. prowazekii and that expressed by Escherichia coli transformants containing the rickettsial gene had an apparent molecular mass of 36,500 Da by SDS-PAGE analysis, a mass considerably less than that deduced from the sequence of the gene. The SDS-solubilized translocase aggregated upon heating at 100 degrees C in the presence of disulfide bond-reducing agents. Similar concentrations of disulfide bond-reducing agents inhibited the exchange transport of adenine nucleotides by both R. prowazekii and translocase-expressing E. coli. These data suggested that an intramolecular disulfide bond in the translocase was essential for transport activity. The antipeptide antibodies used for identification of the translocase bound preferentially to inside-out membrane vesicles of translocase-expressing E. coli relative to right-side-out spheroplasts, thus indicating that the carboxyl terminus of the carrier is located on the cytoplasmic side of the bacterial inner membrane. Protease studies were unable to localize the carboxyl terminus because of the resistance of this region of the native translocase to proteolytic cleavage. These data in conjunction with hydrophobicity analysis were used to construct an initial topological model of the translocase within the cell membrane.

Activation/inactivation of rat tissue pyroglutamate aminopeptidase by disulfide bond-reducing agents

The effects of dithiothreitol on pyroglutamate aminopeptidase activity in rat serum, spinal cord, brain, liver, kidney, and adrenal extracts were investigated in vitro . Addition of dithiothreitol to the reaction mixture resulted in noncompetitive inhibition of serum (Ki=2.1±0.3 mM) and brain (Ki=3.2 ± 0.28 mM) enzymes and stimulation of liver, spinal cord, kidney and adrenal enzymes with an increase in V max but not K m. The findings of these studies suggest that the properties of Pyroglutamate aminopeptidases vary from tissue to tissue.

Nature of the vasoactive substance in CSF from patients with subarachnoid hemorrhage.

The purpose of this experiment was to study the nature of vasoactive substance in cerebrospinal fluid (CSF) from patients with aneurysmal subarachnoid hemorrhage. The authors have examined the effects of disulfide bond-reducing agents, a sulfhydryl group oxidizing agent, and specific antagonists of so-called "classical" neurotransmitters on the in vitro isometric contraction of canine basilar arterial strips induced by bloody human CSF. The disulfide bond-reducing agents dithiothreitol (10(-4) M) and dithioerythritol (10(-4) M) suppressed the contraction induced by bloody CSF by an average of 40.3% and 61.2%, respectively. This suppression was achieved under conditions that did not alter KCl-induced contraction. The sulfhydryl group oxidizing agent, 5,5'-dithiobis-(2-nitrobenzoic acid), 10(-4) M, reversed the inhibitory effect of dithioerythritol on the contractile response to bloody CSF. No significant suppression of any response in any preparation was observed with methysergide (10(-7) M), mepyramine (10(-7) M), phenoxybenzamine (10(-5) M), propranolol (10(-6) M), or atropine (10(-6) M). These results indicate that disulfide bonds in the arterial smooth-muscle cells are involved in the contractile responses of canine basilar artery to bloody CSF. Prostaglandins, hemoglobin, and lipid hydroperoxides may all be spasmogens in bloody CSF, while serotonin, histamine, norepinephrine, and acetylcholine are probably not involved.

Individual Characteristics of Chemically Modified Human Hairs Revealed by Scanning Electron Microscopy

Abstract Hair that is treated with several different chemical reagents including those that are proteolytic, denaturing, or disulfide bond-reducing agents, undergoes structural alterations both internally and externally as revealed by scanning electron microscopic analysis. Some of these agents produce varying degrees of morphologic alterations in hairs obtained from different individuals. It would seem that this technique can be useful in the discrimination of human hairs from different individuals, since the chemically induced topological changes on the hair shaft apparently exhibit a high degree of intraindividual consistency.