The 1.4-MDa apoptosome is a critical intermediate in apoptosome maturation

Previously, we demonstrated that both 150 mM KCl and alkaline pH inhibit cytochrome c-mediated activation of procaspase-3 in a unique manner. To determine the mechanism of inhibition, we analyzed the effect of KCl and alkaline pH on the formation of apoptosomes (a large complex consisting of cytochrome c, Apaf-1, and procaspase-9/caspase-9) in vitro. Our results suggest that an initial 700-kDa apoptosome matures through a 1.4-MDa intermediate before a 700-kDa apoptosome is reformed and procaspase-3 is activated. We further demonstrate that 150 mM KCl interferes with the conversion of the initial 700-kDa apoptosome to the 1.4-MDa intermediate, while alkaline pH "traps" the apoptosome in the 1.4-MDa intermediate. Analysis of the cleaved state of procaspase-9 and procaspase-3 suggests that the 1.4-MDa intermediate may be required for cleavage of procaspase-9. Consistent with these results, in vivo data suggest that blocking acidification during the induction of apoptosis inhibits activation of procaspase-3. On the basis of these results, we propose a model of apoptosome maturation. caspase; pH; potassium; apoptosis     

Cytochrome c activates K+ channels before inducing apoptosis

Cellshrinkage is an early prerequisite for apoptosis. Theapoptotic volume decrease is due primarily to loss ofcytoplasmic ions. Increased outward K⁺ currents have indeedbeen implicated in the early stage of apoptosis in many celltypes. We found that cytoplasmic dialysis of cytochrome c(cyt-c), a mitochondria-dependent apoptotic inducer,increases K⁺ currents before inducing nuclear condensationand breakage in pulmonary vascular smooth muscle cells. Thecyt-c-mediated increase in K⁺ currents tookplace rapidly and was not affected by treatment with a specificinhibitor of caspase-9. Cytoplasmic dialysis of recombinant (active)caspase-9 negligibly affected the K⁺ currents. Furthermore,treatment of the cells with staurosporine (ST), an apoptosisinducer that mediates translocation of cyt-c frommitochondria to the cytosol, also increased K⁺ currents,caused cell shrinkage, and induced apoptosis (determined byapoptotic nuclear morphology and TdT-UTP nick end labeling assay).The staurosporine-induced increase in K⁺ currents concurredto the volume decrease but preceded the activation of apoptosis(nuclear condensation and breakage). These results suggest that thecyt-c-induced activation of K⁺ channels and theresultant K⁺ loss play an important role in initiating theapoptotic volume decrease when cells undergo apoptosis.

     

Nitrite-reducing activity of modified cytochrome c-553 from the red alga Porphyra yezoensis Ueda

Crystalline cytochrome c-553 was obtained from Porphyra yezoensisUeda. The cytochrome in areduced form was modified to show anitrite-reducing activity after appropriate treatment with heat,hydrogen peroxide, or photooxidation using methylene blue asthe electron acceptor, but the reducing activity was far lowerthan that of the nitrite reductase isolated from this alga.The modified cytochrome c-553 was autooxidizable and showedan absorption spectrum resembling that of cytochrome c-553 inthe oxidized form except for slight shifts of the absorptionmaximumin the -band region toward shorter wavelengths. ¹ Present address: Department of Biological Sciences, Universityof Tsukuba, Sakura-Mura, Ibaraki, 300-31 Japan. ² Present address: Department of Fisheries, College of Agricultureand Veterinary Medicine, Nihon University, Shimouma, Setagaya-ku,Tokyo, 154 Japan. (Received June 10, 1975; )     

Properties of the Cytochrome c Oxidase Activity of Cytochrome b561 from Photoanaerobically Grown Rhodopseudomonas sphaeroides

Cytochrome b₅₆₁ from Rhodopseudomonas sphaeroides had cytochromec (c2) oxidase activity and a pH optimum at 6.0 for this activity.The activity was affected by the ionic strength of the reactionmixture. The apparent K_m and maximal velocity (V_max) valuesin the absence of addea salts were 14 µM and 120 nmoloxidized per min per mg protein for horse heart cytochrome c.Reduced horse heart cytochrome c was reoxidized in first-orderkinetics by this cytochrome b₅₆₁. The specific activity was0.7 s^–1 per mg protein at 20°C at the concentrationof 30 µMM cytochrome c. Activity was inhibited by KCN and NaN₃, but not by antimycin.The addition of a low concentration of KCN to the cytochromeb₅₆₁ produced a change in the absorption spectrum, evidencethat KCN interacts with the heme moiety of cytochrome b₅₆₁.Results of this and preceeding studies show that the cytochromeoxidase (cytochrome "o") described earlier (Sasaki et al. 1970)is cytochrome b₅₆₁. (Received May 16, 1983; Accepted September 8, 1983)     

Proton Transport and pH Control in Sinapis alba Root Hairs: A Study carried out with Double-Barrelled pH Micro-electrodes

Continuous measurements of cytoplasmic pH (pH_c) in Sinapis roothairs have been carried out with double-barrelled pH-micro-electrodesin order to gain information on translocation of protons acrossthe plasmalemma and cytoplasmic pH control. (i) The cytoplasmicpH of Sinapis (7–33 ? 0–12, standard conditions)changes no more than 0.1 pH_c, per pH_o-unit, regardless of whethercyanide is present or not. (ii) Weak acids rapidly acidify pH_cand hyperpolarize, while weak bases alkalize pH_c and depolarizethe cells, (iii) 1.0 mol M,³ NaCN acidifies the cytoplasm by0.4 to 0.7 pH-units, but alkalizes the vacuole. (iv) 20 mmolm^–3 CCCP has no significant effect on pH_c, if added atpH 9.6 or 7.2, but acidifies pH_c by 1.3 units at pH 4.3. Inthe presence of CCCP, cyanide acidifies the cytoplasm, (v) Chloridetransiently acidifies pH_c, while K⁺, Na⁺, and have no significant effects, (vi) Cytoplasmic buffer capacityforms a bell-shaped curve versus pH_c with an optimum of about50 mol m^–3 H⁺pH_c-unit. The modes of proton re-entry and the effects of active and passiveproton transport on cellular pH control are critically discussed.It is suggested that the proton leak, consisting of H⁺-cotransport(e.g. H⁺/Cl^–) rather than H⁺-uniport, is no threat topH_c. The proton export pump, although itself reacting to changesin pH_c, influences pH_c only to a minor extent. It is concludedthat buffer capacity and membrane transport play moderate rolesin pH_c control in Sinapis, while the interlocked H⁺-producingand -consuming reactions of cellular metabolism are the mainregulating factors. This makes pH control in Sinapis quite differentfrom bacterial and animal cells. Key words: Cytoplasmic pH, double-barrelled pH micro-electrode, pH control, proton transport, Sinapis     

Effect of Temperature and External pH on the Cytoplasmic pH of Chara corallina

Cytoplasmic pH (pH_c) in Chara corallina was measured (from [¹⁴C]stribution)as a function of external pH (pH₀)and temperature. With pH₀near 7, pH_c at 25?C is 7.80; pH_cincreases by 0.005 pH units?C^–1 temperature decrease, i.e. pH_c at 5 ?C is 7.90. WithpH? near 5.5, the increase in pH_c with decreasing temperatureis 0.015 units ?C^–1 between 25 and 15?C, but 0.005 units?C^–1 between 15 and 5?C. This implies a more precise regulationof pH_c with variations in pH_o at 5 or 15 ?C compared with 25?C. The observed dp H_c/dT is generally smaller than the –0.017units ?C^–1 needed to maintain a constant H⁺/OH^–1,or a constant fractional ionization of histidine in protein,with variation in temperature. It is closer to that needed tomaintain the fractional ionization of phosphorylated compoundsor of CO₂–HCO₃^– The value of dpH_c/dT has importantimplications for several regulatory aspects of cell metabolism.These include (all as a function of temperature) the rates ofenzyme reactions, the _H+ at the plasmalemma(and hence the energy available for cotransport processes),and the mechanism for pH_c regulation by the control of bidirectionalH⁺ fluxes at the plasmalemma.     

COMPONENTS OF THE ELECTRON-TRANSFERRING SYSTEM IN ACETOBACTER SUBOXYDANS AND RECONSTRUCTION OF THE LACTATE OXIDATION SYSTEM

1. Cytochromes a₁⁵⁹⁰, b⁵⁶⁰, c₁⁵⁵⁴ and c₁⁵⁵² were isolated andpurifiedfrom a strain of Acetobacter suboxydans. The proceduresusedwere described in detail.
2. The main cytochrome band at550-560 mµ in intact cellssplitted at liquid air temperatureinto two bands, 551 mµ(strong) and 559 mµ (weak).
3. Optical and physiological properties of the four cytochromeswere investigated. Lactic dehydrogenase activity was found tobe associated with cytochrome c₁⁵⁵⁴. The two c₁-type cytochromes,especially cytochrome c₁⁵⁵⁴, persisted in their reduced formafter the purification through many steps.
4. By some combinationsof isolated components reconstruction ofthe oxygen uptake systemcould be realized.
5. The oxygen-consuming activity of purifiedoxidase preparationswas accelerated by a-tocopherol but notby Emasoll 4130 andTween 80.
6. Some discussions were made onthe nature of terminal oxidase,the role of cytochrome c₁⁵⁵²in the electron-transport system,and persistence of reducedstate of c₁-type cytochromes.
7. A possible scheme of the electron-transferringsystem of Acetobactersuboxydans was presented.

(Received May 16, 1960; )     

The Osmotic Pressure of Concentrated Solutions of Polyethylene Glycol 6000, and its Variation with Temperature

The vapour pressures of aqueous solutions of polyethylene glycol6000 have been measured (by equilibration with sucrose solutions)up to the saturation point at 25 °C (1.45 g g^–1 water).The reduced-osmotic-pressure (/c), when plotted versus concentration(c), rapidly and linearly increased up to a concentration ofabout 0.8 g g^–1 (crossing the similar plot for sucrose).Above this concentration, the reduced-osmotic-pressure rosemore slowly, but still more rapidly than sucrose. The maximumosmotic pressure achieved at saturation was nearly 18 MPa. Usingthe virial equation: /c= RT/M + RTA₂c, the calculated secondvirial coefficient (A₂) for the linear part is 4.5 x 10^–3mol g^–1, a value slightly greater than most literaturevalues at 25 °C. Data are cited showing that A₂ varies linearlyfrom 5–6 x 10^x3 at 0 °C, to zero at 80–90 °C     

Peroxynitrite induces apoptosis of HL-60 cells by activation of a caspase-3 family protease

Apoptosis is an active process critical for the homeostasis oforganisms. Enzymes of the caspase family are responsible for executingthis process. We have previously shown that peroxynitrite (ONOO), a biologicalproduct generated from the interaction of nitric oxide and superoxide,induces apoptosis of HL-60 cells. The aim of this study was toelucidate the mechanisms involved in the execution process ofperoxynitrite-induced apoptosis. Proteolytic cleavage ofpoly(ADP-ribose) polymerase, an indication of caspase-3 family proteaseactivation and an early biochemical event accompanying apoptosis, wasobserved in a time-dependent manner during peroxynitrite-induced apoptosis of HL-60 cells. Activation of caspase-3 duringperoxynitrite-induced apoptosis was substantiated by monitoringproteolysis of the caspase-3 proenzyme and by measuring caspase-3activity with a fluorogenic substrate. Furthermore, pretreatment ofHL-60 cells withN-acetyl-Asp-Glu-Val-Asp-aldehyde, aspecific inhibitor of caspase-3, but notN-acetyl-Tyr-Val-Ala-Asp-aldehyde, aspecific inhibitor of caspase-1, decreased peroxynitrite-induced apoptosis. These results suggest that the activation of a caspase-3 family protease is essential for initiating the execution process ofperoxynitrite-induced apoptosis of HL-60 cells.

     

Purification and properties of soluble cytochrome b-560 from spinach leaves

A b-type cytochrome having an -band at 560 nm was isolated fromspinach leaves (Spinacia oleracea). A method is described forpreparing this cytochrome, cytochrome b-560 (spinach), in apurified state. The cytochrome has, in its reduced state, absorption bands at560 nm (), 530 nm (ß) and 427 nm (); and in the oxidizedstate at 562 nm (), 529 nm (ß) and 417 nm (). Thepyridine ferro-haemochrome prepared from cytochrome b-560 hadan -band at 556.5 nm, indicating the protohaem-nature of theprosthetic group. The cytochrome has an oxidation-reduction potential (E'⁰) of+0.13V at pH 7.0, as measured using the ferri-ferro oxalate system. The cytochrome is rapidly reduced on illumination with red orfar-red light in the presence of spinach chloroplasts and isoxidized at a slower rate in the dark. This photoreduction isinhibited by 1x10^–6 M 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU). The molecular weight of the cytochrome is 30,000 asestimated by the dextran gel filtration method. (Received December 3, 1971; )     

Identification of the pH sensor and activation by chemical modification of the ClC-2G Cl- channel

Rabbit and human ClC-2GCl channels are voltagesensitive and activated by protein kinase A and low extracellular pH.The objective of the present study was to investigate the mechanism involved in acid activation of the ClC-2GCl channel and to determinewhich amino acid residues play a role in this acid activation. Channelopen probability(P_o) at ±80 mV holding potentials increased fourfold in a concentration-dependent manner with extracellular H⁺concentration (that is, extracellular pH,pH_trans), with anapparent acidic dissociation constant of pH 4.95 ± 0.27. 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide-catalyzed amidation of the channel with glycine methyl ester increasedP_o threefold atpH_trans 7.4, at which the channelnormally exhibits lowP_o. Withextracellular pH reduction (protonation) or amidation, increasedP_o was due to asignificant increase in open time constants and a significant decreasein closed time constants of the channel gating, and this effect wasinsensitive to applied voltage. With the use of site-directedmutagenesis, the extracellular region EELE (amino acids416-419) was identified as the pH sensor and amino acid Glu-419was found to play the key or predominant role in activation of theClC-2G Cl channel byextracellular acid.

     

Proton-Chloride Symport in Barley Roots

An increase in the acidity of the incubation medium from pH6 5 to pH 4.0 increased Cl- flux into ATP-depleted Hordeum vulgareL roots more than three times This pH-dependent Cl^– fluxwas inhibited by p-chloromercuriphenyl sulphonic acid. The effectof pH on Cl- influx was eliminated when the pH gradient wasdissipated by addition of salts of permeable weak acids, andin K-loaded roots in the presence of a protonophore togetherwith valinomycin The results support the assumption that a H⁺-Cl^–symportsystem is present in barley root cells Hordeum vulgare L., barley, excised roots, ion transport, proton-chloride symport     

Nitrobacter agilis Cytochrome c-550: Isolation, Physicochemical and Enzymatic Properties, and Primary Structure

Nitrobacter agilis cytochrome c-550 was purified to an electrophoreticallyhomogeneous state, and some of its properties were determined.The cytochrome showed an absorption peak at 410 nm in the oxidizedform, and peaks at 416, 521 and 550 nm in the reduced form.Its isoelectric point was 8.1 at 5?C. Analysis of the aminoacid composition showed that the cytochrome molecule was composedof 108 amino acid residues, 16 of which were lysine residues. The cytochrome reacted rapidly with N. agilis cytochrome c oxidaseand yeast cytochrome c peroxidase and more slowly with Pseudomonasaeruginosa nitrite reductase and bovine cytochrome c oxidase.The reactivities with these redox enzymes suggested that thecytochrome might be an evolutionary stage between bacterialand eukaryotic cytochromes c. The primary structure of the cytochrome from the N-terminusto the 85th residue was determined. The N-terminal sequencewas homologous to the corresponding portion of the primary structureof horse cytochrome c. ¹ Present adress: Department of Chemistry, Faculty of Science,Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo,152, Japan. (Received December 3, 1981; Accepted January 28, 1982)     

Nitrate Uptake and Reduction of Aseptically Cultivated Spruce Seedlings, Picea abies (L.) Karst

Spruce (Picea abies (L.) Karst.) seedlings were asepticallycultivated and the effects of different N-nutrition on net uptakeand reduction of nitrate were investigated. The characteristicsof nitrate uptake were calculated, K_s as 0?2 mol m^–3 andV_max as 18 µmol g^–1 d^–1. Low pH, and Al³⁺ in the medium caused adecrease in nitrate uptake rate. An in vivo assay was set upwhich allowed the measurement of NRA in both roots and needlesof spruce seedlings. The in vivo nitrate reductase activitywas repressed by ammonium and stimulated by nitrate. Nitratereduction was similar to nitrate uptake, negatively affectedby low pH and ammonium. Therefore, a limited N-supply to spruceseemed to occur when pH was low in the rhizosphere combinedwith the presence of Al³⁺ and . Key words: Spruce, nitrate uptake, nitrate reduction     

STUDIES ON COMPONENTS OF RESPIRATORY SYSTEM OF BEGONIA LEAVES

Investigations were made of the properties of diaphorase, cytochromec reductases, cytochrome c oxidase, and other components ofelectron transfer system in various fractions of leaf homogenateof Begonia semperflorens.

1. All the fractions tested showed the existence of cytochromec oxidase, succinic- and reduced diphosphopyridine nucleotide-cytochromec reductases, and diaphorase. Activities of these enzymes werefound to be associated mainly with the particulate fractions.The particulate fractions showed, in particular, a capacityof reducing oxidized cytochrome c with fumarate, malate, -ketoglutarate,ß-hydroxy-butyrate, and citrate.
2. Optimum pH foroxidation of cytochrome c by the particulatefractions was foundto be 5.5, while that for reduction was7.2.
3. The activityof cytochrome c reductase was partially suppressedby malonate.Partial inhibition of cytochrome c oxidase wascaused by azideand cyanide, the inhibitory effects observedbeing strongerwith particulate fractions than with solublefractions.

(Received August 11, 1962; )     

Intra- and extracellular measurement of reactive oxygen species produced during heat stress in diaphragm muscle

Skeletalmuscles are exposed to increased temperatures during intense exercise,particularly in high environmental temperatures. We hypothesized thatheat may directly stimulate the reactive oxygen species (ROS) formationin diaphragm (one kind of skeletal muscle) and thus potentially play arole in contractile and metabolic activity. Laser scan confocalmicroscopy was used to study the conversion of hydroethidine (a probefor intracellular ROS) to ethidium (ET) in mouse diaphragm. During a30-min period, heat (42°C) increased ET fluorescence by 24 ± 4%, whereas in control (37°C), fluorescence decreased by 8 ± 1% compared with baseline (P < 0.001). The superoxidescavenger Tiron (10 mM) abolished the rise in intracellularfluorescence, whereas extracellular superoxide dismutase (SOD; 5,000 U/ml) had no significant effect. Reduction of oxidized cytochromec was used to detect extracellular ROS in rat diaphragm.After 45 min, 53 ± 7 nmol cytochrome c · g drywt¹ · ml¹ were reduced in heatcompared with 22 ± 13 nmol · g¹ · ml¹ in controls(P < 0.001). SOD decreased cytochrome creduction in heat to control levels. The results suggest that heatstress stimulates intracellular and extracellular superoxideproduction, which may contribute to the physiological responses tosevere exercise or the pathology of heat shock.

     

A probable lack of function of c-type cytochrome in the photosynthetic system of the blue-green alga Anabaena variabilis

Quantitative study of the cytochrome c acting in the photosyntheticsystem of the blue-green alga Anabaena variabilis (M-2) wasdone with membrane fragments and intact cells. Membrane fragments highly active in the NADP⁺-Hill reaction(above 200 µmoles/mg chl.a;-hr) retained photoresponsivecytochrome c equal only one-tenth that of P700, while the plastocyanincontent was almost equal to that of P700. The cytochrome contentin intact cells was a little larger than that in membrane fragmentson the chlorophyll a basis. However, the values relative toP700 (1/9) and plastocyanin (1/10) were identical with thosein membrane fragments. The content was also far smaller thanthat of reaction center II's (1/6). If the cytochrome mediatesall electrons from reaction center II, the cytochrome oxidation-reductionshould have a rate constant of 2.4?102 sec^–1 which isone order above of the rate constant of the cytochrome reduction(2.3 to 3.5?10¹sec^–1). These quantitative relationshipsindicate that in Anabaena variabilis (M-2), c-type cytochrome,either cytochrome f or algal cytochrome c, cannot function inthe main electron flow between two reaction centers. (Received September 8, 1978; )     

Role of Na+-K+-Cl- cotransport and Na+/Ca2+ exchange in mitochondrial dysfunction in astrocytes following in vitro ischemia

Na⁺-K⁺-Cl^– cotransporter isoform 1 (NKCC1) and reverse mode operation of the Na⁺/Ca²⁺ exchanger (NCX) contribute to intracellular Na⁺ and Ca²⁺ overload in astrocytes following oxygen-glucose deprivation (OGD) and reoxygenation (REOX). Here, we further investigated whether NKCC1 and NCX play a role in mitochondrial Ca²⁺ (Ca_m²⁺) overload and dysfunction. OGD/REOX caused a doubling of mitochondrial-releasable Ca²⁺ (P < 0.05). When NKCC1 was inhibited with bumetanide, the mitochondrial-releasable Ca²⁺ was reduced by 42% (P < 0.05). Genetic ablation of NKCC1 also reduced Ca_m²⁺ accumulation. Moreover, OGD/REOX in NKCC1^+/+ astrocytes caused dissipation of the mitochondrial membrane potential (_m) to 42 ± 3% of controls. In contrast, when NKCC1 was inhibited with bumetanide, depolarization of _m was attenuated significantly (66 ± 10% of controls, P < 0.05). Cells were also subjected to severe in vitro hypoxia by superfusion with a hypoxic, acidic, ion-shifted Ringer buffer (HAIR). HAIR/REOX triggered a secondary, sustained rise in intracellular Ca²⁺ that was attenuated by reversal NCX inhibitor KB-R7943. The hypoxia-mediated increase in Ca_m²⁺ was accompanied by loss of _m and cytochrome c release in NKCC1^+/+ astrocytes. Bumetanide or genetic ablation of NKCC1 attenuated mitochondrial dysfunction and astrocyte death following ischemia. Our study suggests that NKCC1 acting in concert with NCX causes a perturbation of Ca_m²⁺ homeostasis and mitochondrial dysfunction and cell death following in vitro ischemia. intracellular calcium ion; mitochondrial membrane potential; sodium ion influx; bumetanide; cytochrome c; glial cell death     

Transient Cytoplasmic pH Changes in Correlation with Opening of Potassium Channels in Eremosphaera

Steigner, W. Khler, K., Simonis, W. and Urbach, W. 1988. Transientcytoplasmic pH changes in correlation with opening of potassiumchannels in Eremosphaera.—J. exp. Bot. 39: 23–36. The role of the cytoplasmic pH (pH_c) of Eremosphaera viridisin the signal transduction chain after light-off from the chloroplaststo the K⁺ channels in the plasmalemma of this unicellular algawas investigated. The temporary opening of K⁺ channels is indicatedby a transient hypcrpolarization (TP). To record rapid changesof pH_c, continuous measurements with pH sensitive micro-electrodeswere carried out. (i) Under normal conditions pH_c in the light(7·56 ±0·2) did not differ from pHc inthe dark (7·62 ±0·2). (ii) The vacuolepH ranged between 4·8 and 5·2. (iii) After light-offa rapid transient acidification of pHc O19±0·07occurred and a TP was released, (iv) In every case, the startof the transient acidification after light-off preceded thehyperpolarization by about 3s. (v) Light-on caused a rapid transientalkalinization but never a TP. (vi) Change to acid externalmedium (3.2) transiently acidified the cytoplasm and was ableto release a TP. (vii) After addition of NH₄Cl, pH_c again showeda rapid transient acidification and the release of a TP. The origin of the protons appearing in the cytoplasm after light-offis discussed critically with respect to the buffer capacity.Either direct or indirect translocation is a possible mechanismfor the movement of H⁺ from the chloroplasts into the cytoplasm.The intracellular acidification and its relation to the openingof potassium channels in the plasmalemma leads us to suggestthat a sudden change of pH_c is a potent internal signal factorin Eremosphaera viridis. Key words: Cytoplasmic pH, transient potential, K⁺–channels, Eremosphaera viridis     

Cloning and characterization of a sialidase from the murine histocompatibility-2 complex: low levels of mRNA and a single amino acid mutation are responsible for reduced sialidase activity in mice carrying the Neula allele

The Neu1 locus, in the S region of the murine histocompatibility-2complex, regulates the sialic acid content of several liverlysosomal enzymes. Three alleles, Neu1^a, Neu1^b, and Neu1^c, havebeen described on the basis of differential sialylation of theenzyme liver acid phosphatase. The Neu1^a allele occurs in asmall number of mouse strains, e.g., SM/J and is associatedwith sialidase deficiency. We recently described G9, a sialidasegene in the human major histocompatibility complex (Milner etal. (1997) J. Biol. Chem., 272, 4549–4558), and we nowreport the characterization of the equivalent gene in mouse.The protein product of the murine G9 gene is 409 amino acidsin length and is 83% identical to its human orthologue. Expressionof the murine G9 protein in insect cells has confirmed thatit is a sialidase, with optimal activity at pH 5. To elucidatethe basis of sialidase deficiency in mouse strains carryingthe Neu1^a allele, we have sequenced the G9 coding regions frommice carrying the three Neu1 alleles and hence defined the aminoacid sequence characteristic of each allotype. Of particularinterest is a Leu-209 to Ile mutation that is unique to theNeu1^a allotype and is associated with reductions in sialidaseactivity of 68% and 88% compared to the Neu1^b and Neu1^c allotypes,respectively, when these three protein variants are expressedin insect cells. Additional factors, such as differential expression,may also influence the activities of the Nen1 allotypes in vivo.We have observed that the level of G9 mRNA is substantiallyreduced in mice carrying the Neu1^a allele compared to the Neu1^b(85–95% reduction) and Neu1^c (70% reduction) alleles. H2 complex MHC Neu1 sialidase