The gamma-tubulin complex is a large multiprotein complex that is required for microtubule nucleation at the centrosome. Here we report the purification and characterization of the human gamma-tubulin complex and the identification of its subunits. The human gamma-tubulin complex is a ring of ~25 nm, has a subunit structure similar to that reported for gamma-tubulin complexes from other species, and is able to nucleate microtubule polymerization in vitro. Mass spectrometry analysis of the human gamma-tubulin complex components confirmed the presence of four previously identified components (gamma-tubulin and gamma-tubulin complex proteins [GCPs] 2, 3, and 4) and led to the identification of two new components, GCP5 and GCP6. Sequence analysis revealed that the GCPs share five regions of sequence similarity and define a novel protein superfamily that is conserved in metazoans. GCP5 and GCP6, like other components of the gamma-tubulin complex, localize to the centrosome and associate with microtubules, suggesting that the entire gamma-tubulin complex takes part in both of these interactions. Stoichiometry experiments revealed that there is a single copy of GCP5 and multiple copies of gamma-tubulin, GCP2, GCP3, and GCP4 within the gamma-tubulin complex. Thus, the gamma-tubulin complex is conserved in structure and function, suggesting that the mechanism of microtubule nucleation is conserved.

A number of proteins from a silver-stained two-dimensional (2-D) electrophoresis gel of mouse liver whole-cell lysate were identified by peptide mass mapping and sequence database searching. The excised protein spots were processed by in situ reduction and alkylation, followed by Lys-C digestion. The masses of the resulting peptide mixtures were measured with a matrix-assisted laser desorption/ionization (MALDI) reflection-time-of-flight mass spectrometer. These masses were used successfully to search a protein sequence database. Optimized silver staining and digestion protocols allowed proteins to be identified routinely at the low picomole level. The high mass accuracy and resolution provided by delayed extraction were important for high specificity in the database search. Fragment ion data obtained by MALDI post-source decay (PSD) measurements not only provided confirmation of peptide identification, but could be used to identify the protein from a single peptide without spectral interpretation.

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We report here for the first time that Zn2+ is an effective inhibitor of renin and the protease from HIV-1, two aspartyl proteinases of considerable physiological importance. Inhibition of renin is noncompetitive and is accompanied by binding of 1 mol of Zn2+/mol of enzyme. Depending on the substrate, inhibition of the HIV protease by Zn2+ can be either competitive or noncompetitive, but in neither case is loss of activity due to disruption of the protease dimer. Inhibition of both enzymes is first order with respect to Zn2+ and is rapidly reversed by addition of EDTA. Ki values are strongly pH dependent and optimal in the range of 20 microM at or above pH 7. All of the data in hand suggest that the inhibitory effect of Zn2+ is a consequence of its binding at, or near, the active-site carboxyl groups of these aspartyl proteinases. This inhibition of the viral enzyme may help to explain some of the beneficial effects seen in AIDS patients who have received Zn2+ therapy.

The natriuretic peptide receptor-C (NPR-C) constitutes greater than 95% of the natriuretic peptide binding sites in vivo. This cell surface glycoprotein is a disulfide-linked homodimer with a subunit molecular weight of 68,000. Two sources and types of ANP affinity-purified human NPR-C were used to map disulfide linkages and glycosylation sites of this receptor by mass spectrometry: the extracellular domain obtained by papain cleavage of a receptor-IgG fusion protein expressed in Chinese hamster ovary cells, and a baculovirus/Sf9-expressed cytoplasmic truncation mutant in which 34 of 37 cytoplasmic domain amino acids were deleted. Two intramolecular disulfide bonded loops were found in the 435 amino acid extracellular domain (C63-C91, C168-C216). The juxtamembrane residues C428 and C431 are involved in homodimer formation, confirmed by site-directed mutagenesis of full-length NPR. Three of the four potential Asn-linked glycosylation sites are occupied: N41 (complex), N248 (high mannose), and N349 (complex; partial occupancy). These data describe the intra- and intermolecular linkages in NPR-C, providing a model for the homologous guanylyl cyclase receptors, NPR-A and NPR-B; both of the cyclase receptors likely contain the first amino-terminal 29 amino acid loop, but only NPR-A possesses the second 49 amino acid loop in common with NPR-C.