Proteomic analysis of mitochondria from the Bos taurus heart. II. Identification of mitochondrial soluble proteins

A fraction of the so-called mitochondrial soluble proteins was obtained after the destruction of purified mitochondria by sonication according to the previously found approach to the identification of protein subsets of the Bos taurus heart proteome. A tryptic destruction of these proteins was achieved. Approximately half of the tryptic hydrolysate was separated into two fractions of cysteine-containing and cysteine-free peptides by covalent chromatography on Thiopropyl Sepharose 4B. The cysteine-containing peptides were modified by iodoacetamide. The peptides were mass-spectrometrically identified in all the three fractions of tryptic hydrolysate, and the proteins were searched for in the amino acid sequence databases. There were 213 unique proteins reliably identified.     

Mitochondrial H+-ATPase: Identification of Subunits of the F0 Subcomplex that Contact Membrane Lipids

The subunits of the F₀ membrane sector of bovine heart mitochondrial H⁺-ATPase that contact the lipids of the mitochondrial inner membrane were identified with the use of specially synthesized proteoliposomes that contained active mitochondrial H⁺-ATPase and a photoreactive lipid, which was 1-acyl-2-[12-[di-azocyclopentadiene-2-carbonylamino)-[12-¹⁴C]dodecanoyl]-sn-glycero-3-phosphocholine, 1-acyl-2-[11-([¹²⁵I]diazoiodocyclopentadiene-2-carbonyloxy)undecanoyl]-sn-glycero-3-phosphocholine, or 1-acyl-2-[12-(diazocyclopentadiene-2-carbonylamino)dodecanoyl]-sn-glycero-3-phosphocholine, where acyl is a mixture of the residues of palmitic (70%) and stearic (30%) acids. An analysis of the cross-linked products obtained upon the UV-irradiation of these proteoliposomes indicated that subunits c and a of the F₀ membrane sector contact the lipids. The crosslinked products were identified by SDS-PAGE and MALDI mass spectrometry.     

Preparation of soybean seed samples for FT-IR microspectroscopy

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.     

The structure of p53 tumour suppressor protein reveals the basis for its functional plasticity

p53 major tumour suppressor protein has presented a challenge for structural biology for two decades. The intact and complete p53 molecule has eluded previous attempts to obtain its structure, largely due to the intrinsic flexibility of the protein. Using ATP-stabilised p53, we have employed cryoelectron microscopy and single particle analysis to solve the first three-dimensional structure of the full-length p53 tetramer (resolution 13.7 A). The p53 molecule is a D2 tetramer, resembling a hollow skewed cube with node-like vertices of two sizes. Four larger nodes accommodate central core domains, as was demonstrated by fitting of its X-ray structure. The p53 monomers are connected via their juxtaposed N- and C-termini within smaller N/C nodes to form dimers. The dimers form tetramers through the contacts between core nodes and N/C nodes. This structure revolutionises existing concepts of p53's molecular organisation and resolves conflicting data relating to its biochemical properties. This architecture of p53 in toto suggests novel mechanisms for structural plasticity, which enables the protein to bind variably spaced DNA target sequences, essential for p53 transactivation and tumour suppressor functions.     

Mitochondrial H+-ATPase: identification of subunits of the F0 subcomplex contacting with membrane lipids

The subunits of the F0 membrane sector of bovine heart mitochondrial H(+)-ATPase that contact the lipids of the mitochondrial inner membrane were identified with the use of specially synthesized proteoliposomes that contained active mitochondrial H(+)-ATPase and a photoreactive lipid, which was 1-acyl-2-[12-(diazocyclopentadiene-2-carbonylamino)-[12-14C]dodecanoyl]-sn- glycero-3-phosphocholine, 1-acyl-2-[11-([125I]diazoiodocyclopentadiene-2-carbonyloxy)undecanoyl]-sn- glycero-3-phosphocholine, or 1-acyl-2-[12-(diazocyclopentadiene-2-carbonylamino)dodecanoyl]-sn-glycero- 3-phosphocholine, where acyl is a mixture of the residues of palmitic (70%) and stearic (30%) acids. An analysis of the cross-linked products obtained upon the UV-irradiation of these proteoliposomes indicated that subunits c and a of the F0 membrane sector contact the lipids. The cross-linked products were identified by SDS-PAGE and MALDI mass spectrometry.     

The precursor of the F1β subunit of the ATP synthase is covalently modified upon binding to plant mitochondria

We present evidence for a unique covalent modification of a nuclear-encoded precursor protein targeted to plant mitochondria. We investigated the early events of in vitro import for the mitochondrial precursor of the ATP synthase F₁ subunit from Nicotiana plumbaginifolia (pF₁) into plant mitochondria. When pF₁ of 59 kDa was incubated with mitochondria isolated from different higher-plant species, a band of 61 kDa was generated. The 61 kDa protein was a covalently modified form of the 59 kDa pF₁. The modification was dependent on the 25 amino acid long N-terminal region of the presequence of pF₁. The modification was catalysed by an enzyme located in the outer mitochondrial membrane which was specific for higher plants and could not be washed off from the membrane by urea, KCl or EDTA. The modification was ATP- and Ca²⁺-dependent, but it was not affected by inhibitors of protein kinases. No inhibition of the modification was observed with phosphatase, methylation or acylation inhibitors. The modification occurs prior to translocation through the mitochondrial outer membrane. Inhibition of the modification process does not affect the import of the precursor protein, hence precursor modification was not a prerequisite for import. Both the modified and the unmodified pF₁ proteins were strongly associated with the mitochondrial outer membrane.     

Anti-tumor therapy with macroencapsulated endostatin producer cells

Background  

Theracyte is a polytetrafluoroethylene membrane macroencapsulation system designed to induce neovascularization at the tissue interface, protecting the cells from host's immune rejection, thereby circumventing the problem of limited half-life and variation in circulating levels. Endostatin is a potent inhibitor of angiogenesis and tumor growth. Continuous delivery of endostatin improves the efficacy and potency of the antitumoral therapy. The purpose of this study was to determine whether recombinant fibroblasts expressing endostatin encapsulated in Theracyte immunoisolation devices can be used for delivery of this therapeutic protein for treatment of mice bearing B16F10 melanoma and Ehrlich tumors.