Functional consequences of mitochondrial proteome heterogeneity

Potential functional consequences of the differences in protein distribution between the mitochondria of the rat liver, heart, brain, and kidney, as determined in the companion paper in this issue (Johnson DT, French S, Blair PV, You JS, Bemis KG, Wang M, Harris RA, and Balaban RS. The tissue heterogeneity of the mammalian mitochondrial proteome. Am J Physiol Cell Physiol292: C689–C697, 2006), were analyzed using a canonical metabolic pathway approach as well as a functional domain homology analysis. These data were inserted into the Kyoto Encyclopedia of Genes and Genomes pathway framework to give global and metabolic pathway-specific information on the impact of the differential protein distribution on mitochondrial function. Custom pathway analysis was also performed using pathways limited to the mitochondrion. With the use of this approach, several well-known functional differences between these mitochondrial populations were confirmed. These included GABA metabolism in the brain, urea synthesis in the liver, and the domination of oxidative phosphorylation in the heart. By comparing relative protein amounts of mitochondria across tissues, a greater understanding of functional emphasis is possible as well as the nuclear "programming" required to enhance a given function within the mitochondria. For proteins determined to be mitochondrial and lacking a defined role functional domain BLAST analyses were performed. Several proteins associated with DNA structural modification and a novel CoA transferase were identified. A protein was also identified capable of catalyzing the first three steps of de novo pyrimidine synthesis. This analysis demonstrates that the distribution of nuclear encoded proteins significantly modifies the overall functional emphasis of the mitochondria to meet tissue-specific needs. These studies demonstrate the existence of mitochondrial biochemical functions that at present are poorly defined. oxidative phosphorylation; liquid chromatography; mass spectrometry; electrophoresis; histone; liver; heart; kidney; brain     

Dual trafficking of Slit3 to mitochondria and cell surface demonstrates novel localization for Slit protein

Drosophila slitis a secreted protein involved in midline patterning. Three vertebrateorthologs of the fly slit gene, Slit1, 2, and3, have been isolated. Each displays overlapping, butdistinct, patterns of expression in the developing vertebrate centralnervous system, implying conservation of function. However,vertebrate Slit genes are also expressed in nonneuronaltissues where their cellular locations and functions areunknown. In this study, we characterized the cellular distribution andprocessing of mammalian Slit3 gene product, theleast evolutionarily conserved of the vertebrate Slit genes,in kidney epithelial cells, using both cellular fractionation andimmunolabeling. Slit3, but not Slit2, was predominantly localizedwithin the mitochondria. This localization was confirmed usingimmunoelectron microscopy in cell lines and in mouse kidney proximaltubule cells. In confluent epithelial monolayers, Slit3 was alsotransported to the cell surface. However, we found no evidence of Slit3proteolytic processing similar to that seen for Slit2. We demonstratedthat Slit3 contains an NH₂-terminal mitochondriallocalization signal that can direct a reporter green fluorescentprotein to the mitochondria. The equivalent region from Slit1 cannotelicit mitochondrial targeting. We conclude that Slit3 protein istargeted to and localized at two distinct sites within epithelialcells: the mitochondria, and then, in more confluent cells, the cellsurface. Targeting to both locations is driven by specificNH₂-terminal sequences. This is the first examination ofSlit protein localization in nonneuronal cells, and this study impliesthat Slit3 has potentially unique functions not shared by other Slit proteins.

     

Isolation and Purification of Mitochondrial Mn-Superoxide Dismutase from the Gymnosperm Pinus sylvestris L.

Manganese superoxide dismutase (Mn-SOD; EC 1.15.1.1 [EC] ) was purifiedfrom germinating seeds of Scots pine (Pinus sylvestris L.) 3days after the start of imbibition. The purification scheduleincluded (NH₄)₂SO₄ fractionation, anion-exchange and hydrophobic-interactionchromatographies and chromatofocusing. Purified Mn-SOD had anapparent specific activity of 4,130 McCord-Fridovich units (mgprotein)^–1. The molecular mass of the holoenzyme was estimatedto be 91 kDa by size-exclusion chromatography, and a molecularmass of 23 kDa was determined by SDS-PAGE. However, isoelectricfocusing demonstrated that the purified enzyme consisted ofthree similarly migrating isoforms, with isoelectric pointsof approximately 6.5. NH₂-terminal amino acid sequencing ofpurified Mn-SOD revealed no differences among the three isoforms.The comparison of the first 32 NH₂-terminal amino acids withsequences of NH₂-terminal amino acids of Mn-SODs from angiospermsreflected the phylogenetic distances between Scots pine, whichis a gymnosperm, and angiospermic species. Cell fractionationsuggested the mitochondrial localization of Mn-SODs and no evidencefor glyoxysomal localization was found. Mn-SOD activity wasabsent from dry seeds. It was detectable at a considerable levelafter imbibition for 24 h, and it was again absent from 3-week-oldseedlings. (Received February 8, 1994; Accepted May 24, 1994)     

Histone-induced damage of a mammalian epithelium: the role of protein and membrane structure

In a previousreport [T. J. Kleine, A. Gladfelter, P. N. Lewis, and S. A. Lewis.Am. J. Physiol. 268 (Cell Physiol. 37):C1114-C1125, 1995], we found that the cationic DNA-binding proteinshistones H4, H1, and H5 caused a voltage-dependent increase in thetransepithelial conductance in rabbit urinary bladder epithelium. Inthis study, results from lipid bilayer experiments suggest thathistones H5-H1 and H4 form variably sized conductive units. Purifiedfragments of histones H4 and H5 were used to determine the role ofhistone tertiary structure in inducing conductance. Isolated COOH- and NH₂-terminal tails of histone H4,which are random coils, were inactive, whereas the central -helicaldomain induced a conductance increase. Although the activities of thecentral fragment and intact histone H4 were in many ways similar, thedose-response relationships suggest that the isolated central domainwas much less potent than intact histone H4. This suggests than theNH₂- and COOH-terminal tails arealso important for histone H4 activity. For histone H5, the isolatedglobular central domain was inactive. Thus the random-coilNH₂- and COOH-terminal tails areimportant for H5 activity as well. These results indicate that histonemolecules interact directly with membrane phospholipids to form achannel and that protein tertiary structure and the degree of positive charge play an important role in this activity.

     

Evidence from knockout mice against physiologically significant aquaporin 8-facilitated ammonia transport

Aquaporin (AQP)8-facilitated transport of NH₃ has been suggested recently by increased NH₃ permeability in Xenopus oocytes and yeast expressing human or rat AQP8. We tested the proposed roles of AQP8-facilitated NH₃ transport in mammalian physiology by comparative phenotype studies in wild-type vs. AQP8-null mice. AQP8-facilitated NH₃ transport was confirmed in mammalian cell cultures expressing rat or mouse AQP8, in which the fluorescence of a pH-sensing yellow fluorescent protein was measured in response to ammonia (NH₃/NH₄⁺) gradients. Relative AQP8 single-channel NH₃-to-water permeability was 0.03. AQP8-facilitated NH₃ and water permeability in a native tissue was confirmed in membrane vesicles isolated from testes of wild-type vs. AQP8-null mice, in which BCECF was used as an intravesicular pH indicator. A series of in vivo studies were done in mice, including 1) serum ammonia measurements before and after ammonia infusion, 2) renal ammonia clearance, 3) colonic ammonia absorption, and 4) liver ammonia accumulation and renal ammonia excretion after acute and chronic ammonia loading. Except for a small reduction in hepatic ammonia accumulation and increase in ammonia excretion in AQP8-null mice loaded with large amounts of ammonia, there were no significant differences in wild-type vs. AQP8-null mice. Our results support the conclusion that AQP8 can facilitate NH₃ transport but provide evidence against physiologically significant AQP8-facilitated NH₃ transport in mice. water transport; transgenic mouse; liver     

Conformational modulation of slow skeletal muscle troponin T by an NH(2)-terminal metal-binding extension

TroponinT (TnT) is an essential element in the thin filamentCa²⁺-regulatory system controlling striated musclecontraction. Alternative RNA splicing generates developmental andmuscle type-specific TnT isoforms differing in the hypervariableNH₂-terminal region. Using avian fast skeletal muscle TnTcontaining a metal-binding segment, we have demonstrated a role of theNH₂-terminal domain in modulating the conformation of TnT(Wang J and Jin JP. Biochemistry 37: 14519-14528,1998). To further investigate the structure-function relationship ofTnT, the present study constructed and characterized a recombinantprotein in which the metal-binding peptide present in avian fastskeletal muscle TnT was fused to the NH₂ terminus of mouseslow skeletal muscle TnT. Metal ion or monoclonal antibody binding tothe NH₂-terminal extension induced conformational changes in other domains of the model TnT molecule. This was shown by thealtered affinity to a monoclonal antibody against the COOH-terminal region and a polyclonal antiserum recognizing multiple epitopes. Protein binding assays showed that metal binding to theNH₂-terminal extension had effects on the interaction ofTnT with troponin I, troponin C, and most significantly, tropomyosin.The data indicate that the NH₂-terminal Tx [4-7repeats of a sequence motif His-(Glu/Ala)-Glu-Ala-His] extension confers a specific conformational modulation in the slowskeletal muscle TnT.

     

Quantitative proteomic comparison of rat mitochondria from muscle, heart, and liver

Mitochondria, through oxidative phosphorylation, are the primary source of energy production in all tissues under aerobic conditions. Although critical to life, energy production is not the only function of mitochondria, and the composition of this organelle is tailored to meet the specific needs of each cell type. As an organelle, the mitochondrion has been a popular subject for proteomic analysis, but quantitative proteomic methods have yet to be applied to tease apart subtle differences among mitochondria from different tissues or muscle types. Here we used mass spectrometry-based proteomics to analyze mitochondrial proteins extracted from rat skeletal muscle, heart, and liver tissues. Based on 689 proteins identified with high confidence, mitochondria from the different tissues are qualitatively quite similar. However, striking differences emerged from the quantitative comparison of protein abundance between the tissues. Furthermore we applied similar methods to analyze mitochondrial matrix and intermembrane space proteins extracted from the same mitochondrial source, providing evidence for the submitochondrial localization of a number of proteins in skeletal muscle and liver. Several proteins not previously thought to reside in mitochondria were identified, and their presence in this organelle was confirmed by protein correlation profiling. Hierarchical clustering of microarray expression data provided further evidence that some of the novel mitochondrial candidates identified in the proteomic survey might be associated with mitochondria. These data reveal several important distinctions between mitochondrial and submitochondrial proteomes from skeletal muscle, heart, and liver tissue sources. Indeed approximately one-third of the proteins identified in the soluble fractions are associated predominantly to one of the three tissues, indicating a tissue-dependent regulation of mitochondrial proteins. Furthermore a small percentage of the mitochondrial proteome is unique to each tissue.     

All human Na(+)-K(+)-ATPase alpha-subunit isoforms have a similar affinity for cardiac glycosides

Three-subunit isoforms of the sodium pump, which is the receptor forcardiac glycosides, are expressed in human heart. The aim of this studywas to determine whether these isoforms have distinct affinities forthe cardiac glycoside ouabain. Equilibrium ouabain binding to membranesfrom a panel of different human tissues and cell lines derived fromhuman tissues was compared by an F statistic to determinewhether a single population of binding sites or two populations ofsites with different affinities would better fit the data. For alltissues, the single-site model fit the data as well as the two-sitemodel. The mean equilibrium dissociation constant(K_d) for all samples calculated using thesingle-site model was 18 ± 6 nM (mean ± SD). No differencein K_d was found between nonfailing and failinghuman heart samples, although the maximum number of binding sites infailing heart was only ~50% of the number of sites in nonfailingheart. Measurement of association rate constants and dissociation rateconstants confirmed that the binding affinities of the different human-isoforms are similar to each other, although calculatedK_d values were lower than those determined byequilibrium binding. These results indicate both that the affinity ofall human -subunit isoforms for ouabain is similar and that theincreased sensitivity of failing human heart to cardiac glycosides isprobably due to a reduction in the number of pumps in the heart ratherthan to a selective inhibition of a subset of pumps with differentaffinities for the drugs.

     

Stress Proteins in Mammalian Hibernation

Many organisms whose body temperatures (T_b) vary when they areexposed to a wide range of environmental temperatures exhibitdifferential expression of stress (heat shock) proteins, presumablyto minimize protein damage during thermal stress. In contrast,we know relatively little about natural variation in stressproteins in homeotherms (i.e., birds and mammals), perhaps dueto the relatively constant T_b that is the hallmark of this vertebrategroup. The significant changes in T_b and metabolism characteristicof mammalian hibernators suggest they could provide insightinto the ecological relevance of stress proteins in mammals.Here we examined differential expression of stress proteinsin several tissues of active and torpid 13-lined ground squirrels.There were few significant differences in expression of inducibleHsp70 protein in liver, kidney, heart, intestine, and skeletalmuscle between active and torpid squirrels, and neither fastingin active squirrels nor torpor bout length in hibernators significantlyaltered Hsp70 abundance in these tissues. However, Hsp70 proteinwas lower in brown adipose tissue (BAT) of torpid compared withactive squirrels. In contrast, abundance of GRP75, the mitochondria]form of Hsp70, was greater in liver, skeletal muscle and intestineof torpid compared with active squirrels, with the greatestchange in intestine. Because GRP75 has been shown to be inducedby non-thermal stressors including glucose deprivation and oxidativestress, these results suggest that the ecological significanceof stress proteins for hibernators may be more closely associatedwith the metabolic demands of heterothermy rather than thermalstress per se.     

Adenine nucleotide-creatine-phosphate module in myocardial metabolic system explains fast phase of dynamic regulation of oxidative phosphorylation

Computational models of a large metabolic system can be assembled from modules that represent a biological function emerging from interaction of a small subset of molecules. A "skeleton model" is tested here for a module that regulates the first phase of dynamic adaptation of oxidative phosphorylation (OxPhos) to demand in heart muscle cells. The model contains only diffusion, mitochondrial outer membrane (MOM) permeation, and two isoforms of creatine kinase (CK), in cytosol and mitochondrial intermembrane space (IMS), respectively. The communication with two neighboring modules occurs via stimulation of mitochondrial ATP production by ADP and P_i from the IMS and via time-varying cytosolic ATP hydrolysis during contraction. Assuming normal cytosolic diffusion and high MOM permeability for ADP, the response time of OxPhos (t_mito; generalized time constant) to steps in cardiac pacing rate is predicted to be 2.4 s. In contrast, with low MOM permeability, t_mito is predicted to be 15 s. An optimized MOM permeability of 21 µm/s gives t_mito = 3.7 s, in agreement with experiments on rabbit heart with blocked glycolytic ATP synthesis. The model correctly predicts a lower t_mito if CK activity is reduced by 98%. Among others, the following predictions result from the model analysis: 1) CK activity buffers large ADP oscillations; 2) ATP production is pulsatile in beating heart, although it adapts slowly to demand with "time constant" 14 heartbeats; 3) if the muscle isoform of CK is overexpressed, OxPhos reacts slower to changing workload; and 4) if mitochondrial CK is overexpressed, OxPhos reacts faster. systems biology; computational model; creatine kinase; phosphocreatine shuttle; regulatory module; mitochondrial membrane permeability; oxygen consumption     

The Influence of Nitrate and Ammonium Nutrition on the Growth of Wheat (Triticum aestivum) and Maize (Zea mays) Plants

The effects of NO^-₃ and NH⁺₄ nutrition on hydroponically grownwheat (Triticum aestivum L.) and maize (Zea mays L.) were assessedfrom measurements of growth, gas exchange and xylem sap nitrogencontents. Biomass accumulation and shoot moisture contents ofwheat and maize were lower with NH⁺₄ than with NO^-₃ nutrition.The shoot:root ratios of wheat plants were increased with NH⁺₄compared to NO^-₃ nutrition, while those of maize were unaffectedby the nitrogen source. Differences between NO^-₃ and NH⁺₄-fedplant biomasses were apparent soon after introduction of thenitrogen into the root medium of both wheat and maize, and thesedifferences were compounded during growth. Photosynthetic rates of 4 mM N-fed wheat were unaffected bythe form of nitrogen supplied whereas those of 12 mM NH⁺₄-fedwheat plants were reduced to 85% of those 12 mM NO^-₃-fed wheatplants. In maize supplied with 4 and 12 mM NH⁺₄ the photosyntheticrates were 87 and 82% respectively of those of NO^-₃-fed plants.Reduced photosynthetic rates of NH⁺₄ compared to NO^-₃-fed wheatand maize plants may thus partially explain reduced biomassaccumulation in plants supplied with NH⁺₄ compared to NO^-₃ nutrition.Differences in the partitioning of biomass between the shootsand roots of NO^-₃-and NH⁺₄-fed plants may also, however, arisefrom xylem translocation of carbon from the root to the shootin the form of amino compounds. The organic nitrogen contentof xylem sap was found to be considerably higher in NH⁺₄- thanin NO^-₃-fed plants. This may result in depletion of root carbohydrateresources through translocation of amino compounds to the shootin NH⁺₄-fed wheat plants. The concentration of carbon associatedwith organic nitrogen in the xylem sap of maize was considerablyhigher than that in wheat. This may indicate that the shootand root components of maize share a common carbon pool andthus differences induced by different forms of inorganic nitrogenare manifested as altered overall growth rather than changesin the shoot:root ratios.Copyright 1993, 1999 Academic Press Triticum aestivum, wheat, Zea mays, maize, nitrogen, growth, photosynthesis, amino acids, xylem     

Ammonium release by zooplankton in suspensions of heat-killed algae and an evaluation of the flow-cell method

The maximum excretion rate of NH₄ (39 nmol mg dry wt^–1h^–1) was directly measured for Daphnia pulex by measuringNH₄ accumulation in bottles containing D. pulex and dense, satiatingsuspensions of heat-killed algae. Ammonium release rates inthe algal suspensions were compared to those of individual animalsremoved from the suspension and placed in flow cells. Ammoniumrelease rate, R (nmol mg dry wt^–1 h^–1). in the flowcell decreased very rapidly with time, t (min), after removalaccording to the relation R = 26 + 25e^–0.16t. Ammoniumexcretion obtained by the flow cell method after extrapolationto time zero was not significantly different from that obtainedin the bottles. The considerable experiment-to-experiment variationin NH₄ excretion was in large part correlated (r² = 0.73) withthe feeding rate on the algae.     

beta-Adrenergic receptor-stimulated apoptosis in adult cardiac myocytes involves MMP-2-mediated disruption of beta1 integrin signaling and mitochondrial pathway

Stimulation of -adrenergic receptors (-AR) induces apoptosis in adult rat ventricular myocytes (ARVMs) via the JNK-dependent activation of mitochondrial death pathway. Recently, we have shown that inhibition of matrix metalloproteinase-2 (MMP-2) inhibits -AR-stimulated apoptosis and that the apoptotic effects of MMP-2 are possibly mediated via its interaction with ₁ integrins. Herein we tested the hypothesis that MMP-2 impairs ₁ integrin-mediated survival signals, such as activation of focal adhesion kinase (FAK), and activates the JNK-dependent mitochondrial death pathway. Inhibition of MMP-2 using SB3CT, a selective gelatinase inhibitor, significantly increased FAK phosphorylation (Tyr-397 and Tyr-576). TIMP-2, tissue inhibitor of MMP-2, produced a similar increase in FAK phosphorylation, whereas treatment of ARVMs with purified active MMP-2 significantly inhibited FAK phosphorylation. Inhibition of MMP-2 using SB3CT inhibited -AR-stimulated activation of JNKs and levels of cytosolic cytochrome c. Treatment of ARVMs with purified MMP-2 increased cytosolic cytochrome c release. Furthermore, inhibition of MMP-2 using SB3CT and TIMP-2 attenuated -AR-stimulated decreases in mitochondrial membrane potential. Overexpression of ₁ integrins using adenoviruses expressing the human _1A-integrin decreased -AR-stimulated cytochrome c release and apoptosis. Overexpression of ₁ integrins also inhibited apoptosis induced by purified active MMP-2. These data suggest that MMP-2 interferes with the ₁ integrin survival signals and activates JNK-dependent mitochondrial death pathway leading to apoptosis. matrix metalloproteinases; focal adhesion kinase; c-Jun NH₂-terminal kinase; cytochrome c     

Changes in the composition of exocellular proteins of suspension-cultured Lupinus albus cells in response to fungal elicitors or CuCl2

Among many aspects of plant defence responses to pathogenicinfection are changes in the composition of the exocellularmatrix. To study potential defences in white lupin (Lupinusalbus L.), suspension-cultured cells were treated for 24 h withone of three different elicitors: CuCl₂, and two fungal elicitorpreparations from purified cell walls of yeast and ColletotrichumIindemuthianum. Two subsets of exocellular proteins: ionicallyboundwall proteins and proteins secreted into culture medium, wereisolated, and their patterns compared following electrophoreticseparation. Only a few proteins were observed in culture filtrateswith dominating bands at 27, 33, and 42 kDa. About 30 proteinswere observed in cell wall extracts. Changes in protein intensitiesevoked by elicitor treatments depended on the type of elicitorused, the age and composition of lupin cell culture, and concentrationof applied fungal elicitor. Based on these observations, tenproteins were chosen for N-terminal sequencing, and sequences5–30 amino acids long for nine proteins were obtained.Three of the major proteins sequenced were identified as acidicexocellular chitinase, polygalacturonaseinhibiting protein,and germin/oxalate oxidase. Key words: Lupinus aibus, defence response, exocellular proteins, elicitation, N-terminal amino acid sequencing, suspension culture     

Peptides inhibit selectin-mediated cell adhesion in vitro, and neutrophil influx into inflammatory sites in vivo

The selectins are cell adhesion molecules whose carbohydrate-bindingdomain (C-type lectin) is thought to be involved in leukocyteadhesion to activated vascular endothelium in the inflammatoryprocess. A series of peptides, based on a conserved region (⁴⁸YYWIGIRK⁵⁵-NH₂)of the lectin domain of E-, L- and P-selectins, were analysedfor their ability to block selectin-mediated cell adhesion invitro, and neutrophil infiltration into sites of inflammationin vivo. The peptides inhibited the adhesion of myeloid cellsto recombinant forms of E- and P-selectin. The adhesion of myeloidcells to human endothelial cells, stimulated to express E-selectin,was also inhibited by the peptides. Finally, the peptides blockedthe adhesion of lymphocytes, expressing L-selectin, to highendothelial venules in lymph nodes which contain the ligandfor L-selectin. A clear structure-activity relationship wasestablished when peptides of different amino acid chain lengthswere tested in these assays. Peptides lacking tyrosine residues(e.g. WIGIR-NH₂) at their amino terminus were poor inhibitorsof selectin-mediated cell adhesion in vitro. The peptides thatwere found to be inhibitors of cell adhesion in vitro were alsofound to inhibit (up to 70%) neutrophil infiltration into sitesof inflammation in a thioglycollate-induced peritonitis mousemodel system. They also significantly reduced (>50%) themigration of neutrophils into cytokine-treated skin. These resultsstrongly suggest that compounds based on these tyrosine-containing,selectin-derived peptides could be used as anti-inflammatorytherapeutic agents. inflammation neutrophils peptides selectins     

S-acylation regulates Kv1.5 channel surface expression

The number of ion channels expressed on the cell surface shapes the complex electrical response of excitable cells. An imbalance in the ratio of inward and outward conducting channels is unfavorable and often detrimental. For example, over- or underexpression of voltage-gated K⁺ (Kv) channels can be cytotoxic and in some cases lead to disease. In this study, we demonstrated a novel role for S-acylation in Kv1.5 cell surface expression. In transfected fibroblasts, biochemical evidence showed that Kv1.5 is posttranslationally modified on both the NH₂ and COOH termini via hydroxylamine-sensitive thioester bonds. Pharmacological inhibition of S-acylation, but not myristoylation, significantly decreased Kv1.5 expression and resulted in accumulation of channel protein in intracellular compartments and targeting for degradation. Channel protein degradation was rescued by treatment with proteasome inhibitors. Time course experiments revealed that S-acylation occurred in the biosynthetic pathway of nascent channel protein and showed that newly synthesized Kv1.5 protein, but not protein expressed on the cell surface, is sensitive to inhibitors of thioacylation. Sensitivity to inhibitors of S-acylation was governed by COOH-terminal, but not NH₂-terminal, cysteines. Surprisingly, although intracellular cysteines were required for S-acylation, mutation of these residues resulted in an increase in Kv1.5 cell surface channel expression, suggesting that screening of free cysteines by fatty acylation is an important regulatory step in the quality control pathway. Together, these results show that S-acylation can regulate steady-state expression of Kv1.5. quality control; potassium; channels; palmitoylation; posttranslational     

Effect of Hydrogen Peroxide on Degradation of Cell Wall Associated Proteins in Growing Bean Hypocotyls

The possible involvement of active oxygen species and an apoplasticendopeptidase (EP) in the digestion of cell wall proteins wasstudied in extracellular fluid (EF) from hypocotyls of Phaseolusvulgaris at different stages of elongation. EF proteins underwentsignificant changes in polypeptide pattern during hypocotylgrowth, which were characterized by increases in 35, 39, 40and 50 kDa peptides and appearance of 61, 70 and 75 kDa peptidesat the exponential growth phase. EFs also contain endopeptidase[Gómez et al. (1994) Agriscientia 11:3]. Autolysis experimentswithout or with purified EP revealed that many cell wall polypeptidesare liable to degradation by the protease. Besides, EF polypeptidesincreased their susceptibility to EP during hypocotyl elongation.The 50 and 40 kDa polypeptydes were poorly degraded when extractedfrom hypocotyls in active growth, but greatly hydrolyzed whenextracted from fully elongated tissues, suggesting that in thecourse of growth proteins underwent modifications that renderedthem more prone to proteolytic attack. These modifications seemedto involve active oxygen species, as indicated by: (a) H₂O₂level rised when protein susceptibility to EP increased; and(b) EF proteins from growing hypocotyls (comparatively lesssusceptible to EP) treated with H₂O₂ were rapidly degraded bythe protease. (Received April 27, 1995; Accepted July 31, 1995)     

Isozymes of Superoxide Dismutase in Chlamydomonas and Purification of One of the Major Isozymes Containing Fe

Electrophoretic analysis of Chlamydomonas reinhardtii extractrevealed at least 4 distinct superoxide dismutase (SOD) activitybands as well as several additional minor bands. Among them,one was deduced to be Fe-type and the other three Mn-type basedon their susceptibility to KCN and H₂O₂. The Fe-SOD, which occupiedabout 40% of the total soluble activity, was purified to homogeneityusing ammonium sulfate fractionation followed by DEAE-cellulose,hydroxyapatite, and Superdex 75 gel-permeation chromatography.The 40-kDa native enzyme was composed of two identical 20-kDasubunits with a low shoulder of absorption at {small tilde}350nm. The NH₂-terminal amino acid sequence determined up to residue29 showed a high homology to those of Fe-SOD from Arabidopsisthaliana, Glycine max, and Nicotiana plumbaginifolia. (Received December 21, 1992; Accepted May 28, 1993)     

Topological analysis of NHE1, the ubiquitous Na+/H+ exchanger using chymotryptic cleavage

Proteases,glycosidases, and impermeant biotin derivatives were used incombination with antibodies to analyze the subcellular distribution andtransmembrane disposition of theNa⁺/H⁺exchanger NHE1. Both native human NHE1 in platelets and epitope-tagged rat NHE1 transfected into antiport-deficient cells were used for thesestudies. The results indicated that1) the entire population ofexchangers is present on the surface membrane of unstimulated platelets, ruling out regulation by recruitment of internal stores ofNHE1; 2) the putative extracellularloops near the NH₂ terminus areexposed to the medium and contain all the N- andO-linked carbohydrates;3) by contrast, the putativeextracellular loops between transmembrane domains 9-10 and11-12 are not readily accessible from the outside and may befolded within the protein, perhaps contributing to an aqueous iontransport pathway; 4) the extreme COOH terminus of the protein was found to be inaccessible toextracellular proteases, antibodies, and other impermeant reagents,consistent with a cytosolic localization; and5) detachment of ~150 amino acidsfrom the NH₂-terminal end of theprotein had little effect on the transport activity of NHE1.

     

Differential modulation of voltage-dependent K+ currents in colonic smooth muscle by oxidants

The effect of oxidants on voltage-dependent K⁺ currents was examined in mouse colonic smooth muscle cells. Exposure to either chloramine-T (Ch-T), an agent known to oxidize both cysteine and methionine residues, or the colon-specific oxidant monochloramine (NH₂Cl) completely suppressed the transient outward K⁺ current (I_to) while simultaneously enhancing the sustained delayed rectifier K⁺ current (I_dr). In contrast, the cysteine-specific oxidants hydrogen peroxide (H₂O₂) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) exhibited partial and slow suppression of I_to by inducing a shift in channel availability of -18 mV without affecting I_dr. After enhancement by NH₂Cl or Ch-T, I_dr was sensitive to 10 mM tetraethylammonium but not to other K⁺ channel blockers, suggesting that it represented activation of the resting I_dr and not a separate K⁺ conductance. Extracellular dithiothreitol (DTT) partially reversed the effect of H₂O₂ and DTNB on I_to but not the actions of NH₂Cl and Ch-T on either I_dr or I_to. Dialysis of myocytes with GSH (5 mM) or DTT (5 mM) prevented suppression of I_to by H₂O₂ and DTNB but did not alter the effects of NH₂Cl or Ch-T on either I_dr or I_to. Ch-T and NH₂Cl completely blocked I_to generated by murine K_v4.1, 4.2, and 4.3 in Xenopus oocytes, an effect not reversible by intracellular DTT. In contrast, intracellular DTT reversed the effect of H₂O₂ and DTNB on the cloned channels. These results suggest that I_to is suppressed via modification of both methionine and cysteine residues, whereas enhancement of I_dr likely results from methionine oxidation alone. colon; colitis; redox; ion channel