A human brain E-type ATPase (HB6 ecto-apyrase) was subjected to site-directed mutagenesis to assess the functional significance of two highly conserved tryptophan residues (Trp 187 and Trp 459), the only two tryptophans conserved in nearly all E-type ATPases. Mutation of tryptophan 187 to alanine yielded a poorly expressed ecto-apyrase completely devoid of nucleotidase activity. Immunolocalization of the W187A mutant in mammalian COS cells showed a cellular distribution clearly different from that of the wild-type enzyme, with the majority of the immunoreactivity concentrated in the interior of the cell. Unlike the wild-type enzyme, this mutant did not bind the nucleotide analogue Cibacron Blue and was sensitive to proteolytic digestion by chymotrypsin. These results suggest alteration of the tertiary structure, causing the enzyme to be improperly folded and retained within the cell. In contrast, mutation of tryptophan 459 to alanine resulted in an ecto-apyrase with enhanced NTPase activity, but diminished NDPase activity. Immunolocalization of this active mutant ecto-apyrase revealed a cellular pattern similar to that of the wild-type enzyme, distributed along the cell periphery and in cell processes. Coupling this active W459A mutation to a previously described mutation (D219E) resulted in an enzyme which preferentially hydrolyzes nucleoside triphosphates over diphosphates. The D219E/W459A double mutant had an ATPase:ADPase ratio of 11:1 and a UTPase:UDPase ratio of 148:1. In addition, the double mutant is substantially less sensitive to inhibition by azide, a more potent inhibitor of ecto-apyrases than ecto-ATPases. Thus, mutation of only two amino acids of an E-type ATPase essentially converts an ecto-apyrase to an ecto-NTPase.
Indirect immunofluorescence procedures reported thus far are not effective at localizing two antigens in the same preparation when both primary antibodies are raised in the same species. In this case, the secondary antibodies can crossreact with both primary antibodies. We report here a protocol in which mouse monoclonal antibodies (MAb) specifrc for actin and myosin were used sequentially to stain the same frozen section of guinea pig skeletal muscle. The myosin-specific mAb was applied first and was localized with a rabbit antimouse IgG-rhodamine secondary antibody. The sections were then ''blocked with a non-binding mouse MAb and unconjugated goat anti-mouse IgG F(ab) fragments. The actin-specific mAb was then applied and localized with a
Dyads (transverse tubule--junctional sarcoplasmic reticulum complexes) were enriched from rat ventricle microsomes by continuous sucrose gradients. The major vesicle peak at 36% sucrose contained up to 90% of those membranes which possessed dihydropyridine (DHP) binding sites (markers for transverse tubules) and all membranes which possessed ryanodine receptors and the putative junctional foot protein (markers for junctional sarcoplasmic reticulum). In addition, the 36% sucrose peak contained half of the vesicles with muscarine receptors. Vesicles derived from the nonjunctional plasma membrane as defined by a low content of dihydropyridine binding sites per muscarine receptor and from the free sarcoplasmic reticulum as defined by the M(r) 102K Ca2+ ATPase were associated with a diffuse protein band (22-30% sucrose) in the lighter region of the gradient. These organelles were recovered in low yield. Putative dyads were not broken by French press treatment at 8,000 psi and only partially disrupted at 14,000 psi. The monoclonal antibody GE4.90 against skeletal muscle triadin, a protein which links the DHP receptor to the junctional foot protein in skeletal muscle triad junctions, cross-reacted with a protein in rat dyads of the same M(r) as triadin. Western blots of muscle microsomes from preparations which had been treated with 100 mM iodoacetamide throughout the isolation procedure showed that cardiac triadin consisted predominantly of a band of M(r) 95 kD. Higher molecular weight polymers were detectable but low in content, in contrast with the ladder of oligomeric forms in rat psoas muscle microsomes. Cardiac triadin was not dissolved from the microsomes by hypertonic salt or Triton X-100, indicating that it, as well as skeletal muscle triadin, was an integral protein of the junctional SR. The cardiac epitope was localized to the junctional SR by comparison of its distribution with that of organelle markers in both total microsome and in French press disrupted dyad preparations. Immunofluorescence localization of triadin using mAb GE4.90 revealed that intact rat ventricular muscle tissue was stained following a well-defined pattern of bands every sarcomere. This spacing of bands was consistent with the interpretation that triadin was present in the dyadic junctional regions.
We have investigated the effect of cross‐linking on the enzymatic activity and oligomer formation of the chicken stomach ecto‐apyrase. Cross‐linking with the hydrophobic, lysine‐specific dithiobis(succinimidylpropionate) (DSP) caused equal inhibition of ATPase and ADPase activity in both the membrane‐bound and detergent‐solubilized ecto‐apyrase. The inhibitory effect of cross‐linking was reversed upon the addition of the reductant dithiothreitol. Western blots of aliquots of the cross‐linked samples show decreased amounts of the monomeric 80 kDa ecto‐apyrase and the appearance of a 160 kDa dimer under conditions inducing enzyme inhibition. Therefore, the chicken stomach ecto‐apyrase, like the chicken gizzard ecto‐ATPase, is likely a homodimer in vivo. Unlike the related gizzard ecto‐ATPase, however, the native stomach ecto‐apyrase is not stimulated, but rather inhibited by cross‐linking, presumably due to different quaternary structural stability of the two enzymes.
SUMMARYWe have generated a polyclonal antibody (CKG2) against native chicken gizzard ecto-ATPase for immunolocalization and immunoprecipitation. Active ecto-ATPase is immunoprecipitated from solubilized chicken and rat membranes and shown to be localized to the plasma membrane of the chicken smooth muscle cells. This antibody is specific for the ecto-ATPases, since the more abundant chicken stomach ecto-apyrase is not recognized in immunoprecipitation, western blot or immunolocalization analyses. The CKG2 antibody cross-reacts with mammalian (rat) ectoATPase in western blots, with testis being the most abundant source. Interestingly, when the same rat membranes are analyzed by western blot under non-reducing conditions, the 66 kDa ecto-ATPase is not recognized, instead a 200 kDa protein is detected, previously postulated to be an oligomer of ecto-ATPase. However, this 200 kDa cross-reacting protein is not related to the ecto-ATPases, but is instead an immunoglobulin binding protein, comprised of 50 kDa subunits.
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