Localization of a 64-kDa phosphoprotein in the lumen between the outer and inner envelopes of pea chloroplasts

The identification and localization of a marker protein for the intermembrane space between the outer and inner chloroplast envelopes is described. This 64-kDa protein is very rapidly labeled by [γ-32P]ATP at very low (30 nM) ATP concentrations and the phosphoryl group exhibits a high turnover rate. It was possible to establish the presence of the 64-kDa protein in this plastid compartment by using different chloroplast envelope separation and isolation techniques. In addition comparison of labeling kinetics by intact and hypotonically lysed pea chloroplasts support the localization of the 64-kDa protein in the intermembrane space. The 64-kDa protein was present and could be labeled in mixed envelope membranes isolated from hypotonically lysed plastids. Mixed envelope membranes incorporated high amounts of 32P from [γ-32P]ATP into the 64-kDa protein, whereas separated outer and inner envelope membranes did not show significant phosphorylation of this protein. Water/Triton X-114 phase partitioning demonstrated that the 64-kDa protein is a hydrophilic polypeptide. These findings suggest that the 64-kDa protein is a soluble protein trapped in the space between the inner and outer envelope membranes. After sonication of mixed envelope membranes, the 64-kDa protein was no longer present in the membrane fraction, but could be found in the supernatant after a 110000 × g centrifugation

Involvement of Mhc Loci in immune responses that are not Ir-gene-controlled

Twenty-nine randomly chosen, soluble antigens, many of them highly complex, were used to immunize mice of two strains, C3H and B10.RIII. Lymphnode cells from the immunized mice were restimulated in vitro with the priming antigens and the proliferative response of the cells was determined. Both strains were responders to 28 of 29 antigens. Eight antigens were then used to immunize 11 congenic strains carrying different H-2 haplotypes, and the T-cell proliferative responses of these strains were determined. Again, all the strains responded to seven of the eight antigens. These experiments were then repeated, but this time -antibodies specific for the A (AA) or E (EE) molecules were added to the culture to block the in vitro responsiveness. In all but one of the responses, inhibition with both A-specific and E-specific antibodies was observed. The response to one antigen (Blastoinyces) was exceptional in that some strains were nonresponders to this antigen. Furthermore, the response in the responder strains was blocked with A-specific, but not with E-specific, antibodies. The study demonstrates that responses to antigens not controlled by Irr genes nevertheless require participation of class II Mhc molecules. In contrast to Ir gene-controlled responses involving either the A- or the E-molecule controlling loci (but never both), the responses not Ir-controlled involve participation of both A- and E-controlling loci. The lack of Ir-gene control is probably the result of complexity of the responses to multiple determinants. There is thus no principal difference between responses controlled and those not controlled by Ir genes: both types involve the recognition of the antigen, in the context of Mhc molecules

Proteome Profiling of Breast Tumors by Gel Electrophoresis and Nanoscale Electrospray Ionization Mass Spectrometry

We have conducted proteome-wide analysis of fresh surgery specimens derived from breast cancer patients, using an approach that integrates size-based intact protein fractionation, nanoscale liquid separation of peptides, electrospray ion trap mass spectrometry, and bioinformatics. Through this approach, we have acquired a large amount of peptide fragmentation spectra from size-resolved fractions of the proteomes of several breast tumors, tissue peripheral to the tumor, and samples from patients undergoing noncancer surgery. Label-free quantitation was used to generate protein abundance maps for each proteome and perform comparative analyses. The mass spectrometry data revealed distinct qualitative and quantitative patterns distinguishing the tumors from healthy tissue as well as differences between metastatic and non-metastatic human breast cancers including many established and potential novel candidate protein biomarkers. Selected proteins were evaluated by Western blotting using tumors grouped according to histological grade, size, and receptor expression but differing in nodal status. Immunohistochemical analysis of a wide panel of breast tumors was conducted to assess expression in different types of breast cancers and the cellular distribution of the candidate proteins. These experiments provided further insights and an independent validation of the data obtained by mass spectrometry and revealed the potential of this approach for establishing multimodal markers for early metastasis, therapy outcomes, prognosis, and diagnosis in the future. © 2008 American Chemical Society

Entanglement, elasticity and viscous relaxation of actin solutions

We have investigated the viscosity and the plateau modulus of actin solutions with a magnetically driven rotating disc rheometer. For entangled solutions we observed a scaling of the plateau modulus versus concentration with a power of 7/5. The measured terminal relaxation time increases with a power 3/2 as a function of polymer length. We interpret the entanglement transition and the scaling of the plateau modulus in terms of the tube model for semiflexible polymers.Comment: 5 pages, 4 figures, published versio

Different Transport Pathways of Individual Precursor Proteins in Mitochondria

Transport of mitochondrial precursor proteins into mitochondria of Neurospora crassa was studied in a cellfree reconstituted system. Precursors were synthesized in a reticulocyte lysate programmed with Neurospora mRNA and transported into isolated mitochondria in the absence of protein synthesis. Uptake of the following precursors was investigated: apocytochrome c, ADP/ATP carrier and subunit 9 of the oligomycin-sensitive ATPase. Addition of high concentrations of unlabelled chemically prepared apocytochrome c (1–10 μM) inhibited the appearance in the mitochondrial of labelled cytochrome c synthesized in vitro because the unlabelled protein dilutes the labelled one and because the translocation system has a limited capacity [apparent V is 1–3 pmol × min−1× (mg mitochondrial protein)−1]. Concentrations of added apocytochrome c exceeding the concentrations of precursor proteins synthesized in vitro by a factor of about 104 did not inhibit the transfer of ADP/ATP carrier or ATPase subunit 9 into mitochondria. Carbonylcyanide m-chlorophenylhydrazone, an uncoupler of oxidative phosphorylation, inhibited transfer in vitro of ADP/ATP carrier and of ATPase subunit 9, but not of cytochrome c. These findings suggest that cytochrome c and the other two proteins have different import pathways into mitochondria. It can be inferred from the data presented that different 'receptors' on the mitochondrial surface mediate the specific recognition of precursor proteins by mitochondria as a first step in the transport process

The nucleotide and partial amino acid sequences of rat fetuin

Fetuins are among the major plasma proteins, yet their biological role has remained elusive. Here we report the molecular cloning of rat fetuin and the sequence analysis of a full-length clone, RF619 of 1456 bp with an open reading frame of 1056 bp encoding 352 amino acid residues. The coding part of RF619 was identical with the cDNA sequence of the natural inhibitor of the insulin receptor tyrosine kinase from rat (pp63) except for four substitutions and a single base insertion causing divergence of the predicted protein sequences. Partial amino acid sequences of rat plasma fetuin were in agreement with the predictions based on the RF619 cDNA. Purified rat fetuin inhibited the insulin receptor tyrosine kinase in vitro. Therefore, we conclude that RF619 and pp63 cDNA encode the same protein, i.e. authentic rat fetuin which is a functional tyrosine kinase inhibitor

Cell-Free Synthesis of the Mitochondrial ADP/ATP Carrier Protein of Neurospora crassa

ADP/ATP carrier protein was synthesized in heterologous cell-free systems programmed with Neurospora poly(A)-containing RNA and homologous cell-free systems from Neurospora. The apparent molecular weight of the product obtained in vitro was the same as that of the authentic mitochondrial protein. The primary translation product obtained in reticulocyte lysates starts with formylmethionine when formylated initiator methionyl-tRNA (fMet-tRNAfMet) was present. The product synthesized in vitro was released from the ribosomes into the postribosomal supernatant. The evidence presented indicates that the ADP/ATP carrier is synthesized as a polypeptide with the same molecular weight as the mature monomeric protein and does not carry an additional sequence