Heme can serve Haemophilus influenzae as a source of both essential porphyrin and iron. In extracellular mammalian body fluids neither free heme nor free iron is available, since they are tightly bound to hemopexin and transferrin, respectively. Since H. influenzae grows in the presence of iron-transferrin and heme-hemopexin and is known to express a saturable receptor for transferrin, we investigated the process by which this pathogen acquired heme from hemopexin for use as an iron source. The ability of human and rabbit hemopexin to donate heme as a source of iron to H. influenzae type b strains was demonstrated by plate bioassays. With a dot enzyme assay with biotinylated hemopexin as ligand, H. influenzae bound heme-hemopexin and apo-hemopexin following growth in iron-restricted, but not in iron-sufficient, medium. Competitive binding studies with heme-hemopexin and apo-hemopexin demonstrated saturability of binding. Neither heme, protoporphyrin IX, hemoglobin, nor transferrin blocked the binding of hemopexin to whole cells, demonstrating the specificity of binding. Treatment of whole H. influenzae cells with trypsin abolished binding. Taken together, these observations suggest that H. influenzae type b expresses an outer membrane protein(s) which acts as a receptor for hemopexin and which is regulated by the availability of iron in the growth medium. In iron-restricted media, H. influenzae 706705 and DL42 did not express the 100-kDa hemopexin-binding protein previously reported (M.S. Hanson, S.E. Pelzel, J. Latimer, U. Muller-Eberhard, and E.J. Hansen, Proc. Natl. Acad. Sci. USA 89:1973-1977, 1992). The putative iron-regulated hemopexin receptor was solubilized from cell envelopes of H. influenzae 706705, DL42, and Eagan with the detergent CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) and isolated by affinity chromatography on heme-hemopexin-Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins bound to the affinity resin revealed three proteins of 29, 38, and 57 kDa, of which the 57- and 29-kDa proteins bound hemopexin after Western blotting (immunoblotting). A monoclonal antibody to the 57-kDa hemopexin-binding protein of 706705 recognized a 57-kDa protein on Western blots of the cell envelope proteins of 706705, DL42, and Eagan; no reaction was observed with the 100-kDa hemopexin-binding protein of DL42. These data suggest that some H. influenzae strains possess at least two hemopexin receptors, the expression of which is determined by the prevailing growth environment.
Haemophilus influenzae can acquire heme from hemopexin for use as a source of both essential porphyrin and iron. In classical ligand-binding studies, we observed time-dependent, saturable, and displaceable binding of human 125 I-labelled hemopexin to intact cells of H. influenzae type b (Hib) strain 760705 grown in an iron-restricted medium. From these experiments, which demonstrate that hemopexin associates with a single class of binding site, the affinities (K d s) and receptor numbers were calculated for heme-hemopexin (K d , 205 nM; 3,200 receptors per cell) and apohemopexin (K d , 392 nM; 4,400 receptors per cell). Thus, Hib expresses a specific hemopexin receptor which shows some preference for the heme-protein complex. Affinity chromatography on hemopexin-Sepharose 4B of detergent-solubilized membranes from Hib strain 760705 results in the copurification of three proteins with molecular masses of 57, 38, and 29 kDa. Trypsinization of whole cells of Hib 760705 abolishes hemopexin binding and correlates with the disappearance of the 57-kDa hemopexinbinding protein and appearance of a 52-kDa species which does not bind either hemopexin in ligand blot assays or a monoclonal antibody (MAbT11-30) raised against the 57-kDa protein. From immunoblotting assays and NH 2-terminal amino acid sequence analysis, the 38-kDa protein isolated following hemopexin affinity chromatography was identified as the porin protein P2. These data, taken together with the receptor-binding studies which support a single class of hemopexin-binding site, suggest that P2 and the 29-kDa protein function as accessory proteins to the 57-kDa hemopexin-binding protein to facilitate the uptake of heme from receptorbound hemopexin. To determine whether hemopexin binding and the 57-kDa protein are conserved in Haemophilus strains, whole-cell dot blots and immunoblots of the outer membrane proteins prepared from strains belonging to each of 21 different Hib outer membrane protein subtypes, six nontypeable strains, and five Haemophilus parainfluenzae strains were probed with either hemopexin or MAbT11-30. Only the H. parainfluenzae strains which lack the 57-kDa protein do not bind hemopexin. Since H. influenzae has also been shown to produce a soluble 100-kDa hemopexin-binding protein, cell-free culture supernatants were also examined for the presence of this protein. Apart from Hib 760705 and H. parainfluenzae, the 100-kDa hemopexin-binding protein was detected in all the other Haemophilus strains. The abilities of Hib 760705 to both bind and acquire heme from hemopexin without expressing a 100-kDa soluble hemopexin-binding protein show that in strain 760705, this 100-kDa protein is not essential for the utilization of heme from hemopexin.
Intact neurofilaments (NF) purified from mammalian brain and spinal cord promote the assembly of microtubules in solutions of pure phosphocellulose (PC)-purified tubulin. This assembly is temperature-dependent and is inhibited by mitotic spindle inhibitors. The ability of NF to induce microtubule formation is 20% of that of purified microtubule-associated proteins (MAPs), whereas MAPs comprise less than 5% of the protein in the NF preparations. The inducing activity of NF is rapidly lost on boiling. When intact NF are incubated with PC-tubulin and then centrifuged, tubulin is sedimented together with the filaments. This association is inhibited by colchicine and podophyllotoxin and is cold-sensitive. NF purified to homogeneity under denaturing conditions and then reassembled completely lack the ability to promote the assembly of PC-tubulin or to bind tubulin on a centrifugation assay. No MAPs are present in these preparations, though these filaments have the ability to bind exogenous MAPs. While these experiments do not rule out an intrinsic microtubule-assembly-promoting activity, they suggest that this activity is due to nontriplet proteins in the preparation, most likely filament-associated MAPs.
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