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Characterization of the Drosophila melanogaster mitochondrial proteome.

We have combined high-resolution two-dimensional (2-D) gel electrophoresis with mass spectrometry with the aim of identifying proteins represented in the 2-D gel database of Drosophila melanogaster mitochondria. First, we purified mitochondria from third instar Drosophila larvae and constructed a high-resolution 2-D gel database containing 231 silver-stained polypeptides. Next, we carried out preparative 2-D PAGE to isolate some of the polypeptides and characterize them by MALDI-TOF analysis. Using this strategy, we identified 66 mitochondrial spots in the database, and in each case confirmed their identity by MALDI-TOF/TOF analysis. In addition, we generated antibodies against two of the mitochondrial proteins as tools for characterizing the organelle.

Amino Acid Sequence↗
PubMed1973

Formation of a hemolytically active cellular intermediate by the interaction between properdin factors B and D and the activated third component of complement.

The present studies demonstrate that the factor B-dependent C3 convertase can be affixed to an erythrocyte by use of an intermediate bearing C3b and that this convertase brings the hemolytic reaction to completion with an efficiency comparable to that of classical convertase. The evidence that the EAC43 intermediate was lysed by a new pathway includes requirements for factors B and D and cell-bound C3b for subsequent lysis by the terminal components, C3-C9. The linear stoichiometry of the effective molecule titrations of C3b and factor B, and the first-order kinetics displayed by the generation and decay of the factor B-dependent hemolytic site are characteristics consistent with the one-hit theory as initially developed for the classical complement system. The use of hemolytically active cellular intermediates to examine the reactions occurring with C3b and factors B and D has allowed extension of the one-hit theory to this molecular sequence, development of effective molecule titrations, recognition of the analogies to the functional characteristics of the classical C3 convertase, and discrimination of the probable mechanism of terminal complement activation from reactive lysis.

Complement System Proteins↗
PubMed1975

Purification and partial characterization of human and porcine C3a anaphylatoxin.

C3a anaphylatoxin is a protein fragment generated enzymatically in serum during activation of the third component of complement (C3). A four-step procedure is described for the purification of human and porcine C3a anaphylatoxins from their respective sera after activation with inulin. Because serum carboxypeptidase rapidly inactivates C3a, the inhibitor epsilon-aminocaproic acid (EACA) was added during C3 activation, thus permitting isolation of fully active C3a anaphylatoxins directly from serum. A 2000-fold purification of C3a was achieved with an average 30% recovery assuming total conversion of C3 during treatment of serum with inulin. Human C3a anaphylatoxin obtained through the action of the C3 activating enzyme of the "alternate" pathway appeared nearly identical with the C3a obtained from isolated C3 after treatment with the C4,2 enzyme of the "classical" pathway or with trypsin. Comparisons were made between various properties of human and porcine C3a anaphylatoxins. The molecular weights differed only slightly. Electrophoresis on a cellulose acetate strip at pH 8.6 indicated a difference of approximately one net charge between human and porcine C3a, with the human anaphylatoxin exhibiting the more basic behavior. Although the amino acid compositions are similar, significant differences exist. The most marked difference was the total absence of threonine residues in porcine C3a. The NH2-terminal sequences of 20 amino acid residues were examined; homology existed for 16 of the 20 positions. Although partial analysis of the primary structure of human and porcine C3a indicates approximately 80% homology, no immunological cross-reactivity between the anaphylatoxins could be detected with antisera produced to either human or porcine C3a. In spite of the structural differences, the biological activities of porcine and human C3a were essentially identical. Smooth muscle contraction, increase in vascular permeability, and release of histamine from mast cells were similarly induced by equal amounts of anaphylatoxin from either human or porcine origin. Porcine C3a, like human C3a, was shown to contain a COOH-terminal arginyl residue essential for smooth muscle contraction and for induction of histamine release from mast cells. The sequence adjacent to the COOH-terminal arginine was Leu-Ala-Arg-COOH for both humans and porcine C3a. Current evidence suggests common mechanisms exist for the generation of C3a in various animal species and that the two known C3 activating enzymes in serum exhibit trypsin-like specificity.

Amino Acid Sequence↗
PubMed1997

Separate magnocellular and parvocellular contributions from temporal analysis of the multifocal VEP.

Temporal analysis of the multifocal cortical visual evoked potential (VEP) was studied using pseudo-random (m-sequence) achromatic stimulation. The effects of variation of luminance contrast on the first-order response were complex. At low to mid contrasts (< 60%), a wave doublet (P100-N115) predominated. A second wave complex (N100-P120-N160) dominated at high contrasts. The second-order responses, however, showed an extremely simple variation with luminance contrast. Intrinsic differences in the adaptation time of the generators of these two components caused a distinct separation in the slices of the second-order response. A rapidly adapting nonlinearity saturating at low contrasts was only observable when measuring the responses from two consecutive flashes. Its latency coincided with the contrast saturating first-order response component. By comparison, the nonlinearity derived from the responses to the stimuli with longer interstimulus intervals (second and third slices) yielded a much more linear contrast response function with lower contrast gain and latencies, which clearly corresponded to the longer latency component of the first-order response. Thus, the second-order responses show a first slice which is predominantly driven by neural elements that have a latency and contrast function that mimic those of the magnocellular neurons of the primate LGN and a second slice which is dominated by a generator whose properties resemble primate parvocellular function. This division into magno and parvocellular contribution to the VEP is based on function (interaction time) as distinct from other currently available analyses, with potential for neural analysis of visual disease.

Adult↗
PubMed1979

Axonal transport of actin in rabbit retinal ganglion cells.

We labeled proteins in the cell bodies of rabbit retinal ganglion cells with [35S]methionine and subsequently observed the appearance of radioactive actin in tissues containing the axons and synaptic terminals of these neurons, i.e., the optic nerve (ON), optic tract (OT), lateral geniculate nucleus (LGN) and the superior colliculus (SC). The temporal sequence of appearance of labeled actin (which was identified by its specific binding to DNase I, its electrophoretic mobility, and its peptide map) in these tissues indicated that actin is an axonally transported protein with a maximum transport velocity of 3.4--4.3 mm/d. The kinetics of labeling actin were similar to the kinetics of labeling two proteins (M1 and M2) which resemble myosin; these myosin-like proteins were previously found to be included in the groups of proteins (groups III and IV) transported with the third and fourth most rapid maximum velocities. The similarity in transport between actin and myosin-like proteins supports the idea that a number of proteins in the third and fourth transport groups may be functionally related by virtue of their involvement in a force-generating mechanism and suggests the possibility that these proteins may be axonally transported as a preformed force-generating unit.

Actins↗
PubMed1988

The use of various properties of amino acids in color and monochrome dot-matrix analyses for protein homologies.

Software has been developed to allow the use of a number of parameters in the comparative representation of proteins in color and monochrome dot matrices. They include the parameters of partial specific volume, residue bulkiness, the mean area buried of side chains, seven additional hydropathy scales, mutability, polarity, secondary structure propensities, energy/residue, energy/atom, Rf values, the pKs at the N and C terminals, user-defined parameters and, if desired, randomly generated values. Many of these parameters can be combined in n space using an algorithm based on the Euclidian distance relationship in order to derive consensus values. The problem of scoring matched identities is addressed and the user may stipulate that they score 100 on a 0-100 scale or be determined from the Dayhoff MDM78 values with the rest of the matrix scaled appropriately. The PAMs matrix has been incorporated in such a way to allow the user to stipulate various PAM's values or estimated percentage difference between two peptide sequences, and converting to log odds values. In addition, the similarity ring developed by Swanson and the matrix proposed by Bacon and Anderson have been adapted for use in the program. Color indices have been utilized to give a 'third dimension' to the projections, allowing the user to judge the degree of similarity of different regions which are represented. The software also provides for the plotting of nucleotides in which case color is used to code individual nucleotides, purines versus pyrimidines, or similar colors are used to differentiate between A and T bases on the one hand, and G and C on the other.

Algorithms↗
PubMed1996

Orientation dependence in homologous recombination.

Homologous recombination was investigated in Escherichia coli with two plasmids, each carrying the homologous region (two defective neo genes, one with an amino-end deletion and the other with a carboxyl-end deletion) in either direct or inverted orientation. Recombination efficiency was measured in recBC sbcBC and recBC sbcA strains in three ways. First, we measured the frequency of cells carrying neo+ recombinant plasmids in stationary phase. Recombination between direct repeats was much more frequent than between inverted repeats in the recBC sbcBC strain but was equally frequent in the two substrates in the recBC sbcA strain. Second, the fluctuation test was used to exclude bias by a rate difference between the recombinant and parental plasmids and led to the same conclusion. Third, direct selection for recombinants just after transformation with or without substrate double-strand breaks yielded essentially the same results. Double-strand breaks elevated recombination in both the strains and in both substrates. These results are consistent with our previous findings that the major route of recombination in recBC sbcBC strains generates only one recombinant DNA from two DNAs and in recBC sbcA strains generates two recombinant DNAs from two DNAs.

Crossing Over, Genetic↗
PubMed1987

Structure and evolutionary origin of the gene encoding mouse NF-M, the middle-molecular-mass neurofilament protein.

We describe the complete sequence of the gene encoding mouse NF-M, the middle-molecular-mass neurofilament protein. The coding sequence is interrupted by two intervening sequences which align perfectly with the first two intervening sequences in the gene encoding NF-L (the low-molecular-mass neurofilament protein); there is no intron in the gene encoding NF-M corresponding to the third intron in NF-L. Therefore, both the number of introns and their arrangement in the genes coding NF-L and NF-M contrast sharply with the number and arrangement of introns in the genes of known sequence, encoding other members of the intermediate filament multigene family (desmin, vimentin, glial fibrillary acidic protein and the acidic and basic keratins); with the exception of a single truncated keratin gene that lacks an encoded tailpiece, these genes all contain eight introns, of which at least six are placed at homologous locations. Assuming the existence of a primordial intermediate filament gene containing most (if not all) the introns found in contemporary non-neurofilament intermediate filament genes, it seems likely that an RNA-mediated transposition event was involved in the generation of an ancestral gene encoding the NF polypeptides. A combination of insertional transposition and gene-duplication events could then explain the anomalous number and placement of introns within these genes. Consistent with this notion, we show that the genes encoding NF-M and NF-L are linked.

Amino Acid Sequence↗
PubMed1992

Analysis of mutations in the V3 domain of gp160 that affect fusion and infectivity.

The third hypervariable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein has been proposed to play an important role in mediating viral entry. Antibodies to the V3 domain block HIV-1 infection but not virus binding to CD4. At the center of the V3 domain is a relatively conserved sequence of amino acids, GPGRA. It has previously been shown that mutation of some of these amino acids reduced the ability of gp160 expressed on the surface of cells to induce fusion with CD4-bearing cells. In order to analyze the role of V3 domain sequences in mediating HIV entry, we introduced several amino acid substitution mutations in the GPGRA sequence of gp160 derived from HIV-1 strain HXB2 and in the analogous sequence of strain SF33, GPGKV. Virus was generated by cotransfecting the env constructs and a selectable env-negative HIV vector, HIV-gpt. When complemented with a retrovirus env gene, infectious virus capable of a single round of replication was produced. The viral particles produced were analyzed biochemically for core and envelope proteins and for infectious titer. The transfected envs were also analyzed for ability to bind to CD4 and mediate cell fusion. Several of the amino acid substitutions resulted in moderate to severe decreases in virus infectivity and fusion activity. Envelope glycoprotein assembly onto particles and CD4 binding were not affected. These results provide evidence that V3 sequences are involved in mediating the fusion step of HIV-1 entry.

Amino Acid Sequence↗
PubMed2005

Resistance gene analogues in sugarcane and sorghum and their association with quantitative trait loci for rust resistance.

Fifty-four different sugarcane resistance gene analogue (RGA) sequences were isolated, characterized, and used to identify molecular markers linked to major disease-resistance loci in sugarcane. Ten RGAs were identified from a sugarcane stem expressed sequence tag (EST) library; the remaining 44 were isolated from sugarcane stem, leaf, and root tissue using primers designed to conserved RGA motifs. The map location of 31 of the RGAs was determined in sugarcane and compared with the location of quantitative trait loci (QTL) for brown rust resistance. After 2 years of phenotyping, 3 RGAs were shown to generate markers that were significantly associated with resistance to this disease. To assist in the understanding of the complex genetic structure of sugarcane, 17 of the 31 RGAs were also mapped in sorghum. Comparative mapping between sugarcane and sorghum revealed syntenic localization of several RGA clusters. The 3 brown rust associated RGAs were shown to map to the same linkage group (LG) in sorghum with 2 mapping to one region and the third to a region previously shown to contain a major rust-resistance QTL in sorghum. These results illustrate the value of using RGAs for the identification of markers linked to disease resistance loci and the value of simultaneous mapping in sugarcane and sorghum.

Chromosome Mapping↗
PubMed1994

Identification of the alpha 1c-adrenoceptor in rabbit arteries and the human saphenous vein using the polymerase chain reaction.

The expression of the alpha 1C-adrenoceptor subtype in human and rabbit blood vessels has been analyzed using the reverse transcriptase/polymerase chain reaction technique (RT/PCR). The 20 bp primers employed were designed from the bovine alpha 1C-adrenoceptor and flank a least conserved region--the putative third cytoplasmic loop. RT/PCR products generated from rabbit and human brain mRNA both had 93% homology to the bovine alpha 1C-adrenoceptor and were used as species and subtype specific probes in Southern blot analysis of vascular RT/PCR products. Poly A+ RNA was purified from the human saphenous vein and rabbit aorta, renal, pulmonary and central ear arteries and amplified by RT/PCR. Size analysis by agarose gel electrophoresis, together with Southern hybridization of the resulting cDNA products confirm the expression of the alpha 1C-adrenoceptor in these vessels.

Animals↗
PubMed1999

Identification of SLC7A7, encoding y+LAT-1, as the lysinuric protein intolerance gene.

Lysinuric protein intolerance (LPI; OMIM 222700) is a rare, recessive disorder with a worldwide distribution, but with a high prevalence in the Finnish population; symptoms include failure to thrive, growth retardation, muscle hypotonia and hepatosplenomegaly. A defect in the plasma membrane transport of dibasic amino acids has been demonstrated at the baso-lateral membrane of epithelial cells in small intestine and in renal tubules and in plasma membrane of cultured skin fibroblasts from LPI patients. The gene causing LPI has been assigned by linkage analysis to 14q11-13. Here we report mutations in SLC7A7 cDNA (encoding y+L amino acid transporter-1, y+LAT-1), which expresses dibasic amino-acid transport activity and is located in the LPI region, in 31 Finnish LPI patients and 1 Spanish patient. The Finnish patients are homozygous for a founder missense mutation leading to a premature stop codon. The Spanish patient is a compound heterozygote with a missense mutation in one allele and a frameshift mutation in the other. The frameshift mutation generates a premature stop codon, eliminating the last one-third of the protein. The missense mutation abolishes y+LAT-1 amino-acid transport activity when co-expressed with the heavy chain of the cell-surface antigen 4F2 (4F2hc, also known as CD98) in Xenopus laevis oocytes. Our data establish that mutations in SLC7A7 cause LPI.

Adolescent↗
PubMed1996

Identification of cholecystokinin-B/gastrin receptor domains that confer high gastrin affinity: utilization of a novel Xenopus laevis cholecystokinin receptor.

A hallmark of the mammalian brain cholecystokinin (CCK) receptor, CCK-B/gastrin (CCK-BR), is its high affinity for two structurally related peptides, CCK and gastrin. Previous radioligand binding experiments suggested that the predominant CCK receptor from Xenopus laevis brain shares high affinity for sulfated cholecystokinin octapeptide but has > or = 1000-fold lower affinity for gastrin. To determine the molecular basis for this pharmacological divergence between mammalian and lower vertebrate receptors, we isolated a cDNA encoding the X. laevis brain CCK receptor (CCK-XLR). CCK-XLR shares approximately 50% homology at the amino acid level with both the human CCK-BR and the peripheral CCK-A receptor subtypes. The recombinant X. laevis receptor has a distinct pharmacological profile of agonist and antagonist affinities and as such offers a useful tool for structure-function studies. We used CCK-XLR to map the human CCK-BR domains that confer high affinity for gastrin. A series of chimeric CCK-BR/CCK-XLR constructs was generated and pharmacologically characterized. While maintaining wild-type affinity for sulfated cholecystokinin octapeptide, receptors with increasing amino-terminal contributions from CCK-BR demonstrated a stepwise increase in gastrin affinity. Further dissection of the amino-terminal third of the human receptor, a domain that confers a > 250-fold increase in gastrin affinity, revealed the importance of interactions among at least three subdomains. Additional structural requirements for gastrin affinity mapped to a segment spanning transmembrane domains IV and V.

Amino Acid Sequence↗
PubMed1992

Epitope mapping of monoclonal antibodies to the Escherichia coli F1 ATPase alpha subunit in relation to activity effects and location in the enzyme complex based on cryoelectron microscopy.

The interaction of Escherichia coli F1 ATPase (ECF1) with several different monoclonal antibodies (mAbs) specific for the alpha subunit has been examined. The epitopes for each of the mAbs have been localized by using molecular biological approaches to generate fragments of the alpha subunit. The binding of several of the mAbs has also been examined by cryoelectron microscopy of ECF1 Fab complexes. One of the mAbs, alpha II, bound in the region Asn 109-Val 153 without affecting ATPase activity. Most of the mAbs bound in the C-terminal third of the alpha subunit. MAb alpha 1 bound between residues Gln 443 and Trp 513. This mAb activated ATPase activity and was visualized in cryoelectron microscopy, superimposed on the alpha subunit, indicating that the epitope was on the top or bottom of ECF1 in the hexagonal projection. Other mAbs to the C-terminus, including alpha D which also activated the enzyme, reacted between Gly 371 and Trp 513 but failed to bind to small overlapping fragments within this sequence. The epitopes for these mAbs are probably formed by the folded polypeptide which occurs only in Western analysis when long stretches of the alpha subunit are present, suggesting that the C-terminus of alpha is a self-folding domain. In cryoelectron microscopy, Fab fragments for alpha D were seen extending from the sides of the ECF1 complex in hexagonal projection.

Antibodies, Monoclonal↗
PubMed1996

Design of peptides: synthesis, crystal structure and molecular conformation of N-Boc-L-Val-delta Phe-L-Val-OC H3.

The dehydropeptide Boc-L-Val-delta Phe-L-Val-OC H3 was synthesized by azlactone method in solution phase. The peptide crystallized from its solution in a methanol/water mixture (70:30) in space group P2(1)2(1)2(1) with a = 13.638(1)A, b = 22.864(3)A, c = 27.600(2)A, V = 8606(1)A3. The structure was determined by direct methods and refined to an R value of 0.089 for 3326 observed [1 > or = 2 sigma(I)] reflections. The structure contains three crystallographically independent molecules. Two molecules (A and B) adopt identical conformations with phi 1(A) = -130(1), phi 1(B) = -139(1), psi 1(A) = 153(1), psi 1(B) = 145(1), phi 2(A) = 62(1), phi 2(B) = 56(1), psi 2(A) = 33(1), psi 2(B) = 33(1), phi 3(A) = -75(1), phi 3(B) = -77(1) psi 3T(A) = 152(1) and psi 3T(B) = 163(1) degrees. The conformation of the third molecule (C) is different as in that torsion angle psi 1 is rotated by 180 degrees. The backbone torsion angles are phi 1 = -128(2), psi 1 = -37(2), phi 2 = 65(1), psi 2 = 35(1), phi 3 = -84(1) and psi 3T = 169(1). It is significant that a characteristic beta-turn II conformation as observed in peptides with a delta Phe residue at (i+2) position has not been observed in this case. Thus the present structure demonstrates that an (i+2) substituted delta Phe with branched beta-carbon residue Val on both sides of it in a three peptide unit sequence does not adopt a folded conformation. The three independent molecules in the asymmetric unit form three hydrogen bonds between each pair of molecules and generates a tightly interacting unit of three molecules. These units pack into the crystalline state with hydrogen bonds involving N1, N2 and N3 of molecule C and O1' and O2' of symmetry related molecule A. The structure confirms that a peptide containing a delta Phe residue at (i+2) position with branched beta-carbon residues as its immediate neighbours on both sides does not adopt a folded conformation. This structure further demonstrates that the unfolded structures without any intramolecular hydrogen bond can be influenced by intermolecular forces, thus causing conformational variations in saturated residues. It is noteworthy that the conformation of the delta Phe residue remains unchanged in all the three molecules.

Crystallography, X-Ray↗
PubMed1991

A phosphoglycerate kinase-like molecule localized to glycosomal microbodies: evidence that the topogenic signal is not at the C-terminus.

The phosphoglycerate kinase (PGK) gene complex of Trypanosoma brucei contains three tandemly linked related genes. One gene encodes a cytoplasmic PGK, while another encodes a PGK isozyme localized to glycosomal microbodies. In this communication, we report that the third gene in this complex encodes a 56-kDa molecule which is also localized to the glycosomal core. DNA sequence analysis indicates that this gene contains multiple substitutions and a large insertion in the amino domain, but that it is very similar to the other PGK isozymes in the carboxy domain. The C-terminal tripeptide is identical to that of the cytoplasmic isozyme, and only one conservative change occurs in the last 25 amino acids. The encoded protein, p56, thus contrasts with the many peroxisomal microbody proteins in which the C-terminal tripeptide contains sufficient information for targeting to peroxisomes. Multiple mechanisms may exist for targeting proteins to the protein cores of microbody organelles. Comparisons of the DNA sequences of several alleles suggest that homologous recombination plays a role in the generation of allelic diversity.

Alleles↗
PubMed1998

Alpha 1-proteinase inhibitor is the serum regulator of the activity of p57, a C3-cleaving proteinase present in human erythrocyte membranes.

Human erythrocytes express at the membrane level a p57 serine proteinase which cleaves C3, the third component of complement. We demonstrated herein that human serum carries an inhibitory activity against this p57 membrane proteinase. Purification allowed to identify this inhibitor as the alpha 1-proteinase inhibitor (alpha 1-PI) on the basis of its molecular weight, antigenicity and amino acid sequence identity. Data demonstrated that alpha 1-PI is the unique and strong serum inhibitor of the p57 proteinase activity: inhibition studies showed that alpha 1-PI inhibited p57 proteinase activity with a kass value of 10(5) M-1 s-1. Inhibition of p57 proteinase by alpha 1-PI was due to formation of a SDS-stable complex between both components. We suggest that inhibition of the membrane p57 proteinase activity by serum alpha 1-PI may be involved in the regulation of C3 fragment generation and/or in clearance in liver of C3b bearing immune complexes by erythrocyte-CR1.

Complement C3↗
PubMed1987

Morphf mutants of Rous sarcoma virus: nucleotide sequencing analysis suggests that a class of morphf mutants was generated through splicing of a cryptic intron.

The nature of the lesions involved in producing the fusiform phenotype of three mutants (WO101, WO201, and tsST529) of the Schmidt-Ruppin A strain of Rous sarcoma virus (RSV) was determined by molecular cloning and DNA sequencing. WO101 and WO201 contained an in-frame deletion of the v-src region coding for amino acids 116 to 140 of p60v-src. The deleted segment was flanked by consensus splice donor and acceptor sequences and contained an appropriately positioned branchpoint acceptor consensus sequence, suggesting that the deletion occurred through an aberrant RNA splicing event. S1 mapping experiments performed on RNA isolated from chicken cells infected with molecularly cloned wild-type RSV DNA suggested that the splice acceptor involved in the generation of this deletion was utilized at a low frequency (less than 1.0%) in wild-type RSV-infected cells. These results suggested that stable mutations may have arisen in the coding sequence of a eucaryotic viral transforming gene as a result of a probable aberrant RNA splicing event followed by reverse transcription into DNA. ST529 was found to harbor the same deletion present in WO101 and WO201 but also contained a point mutation which resulted in the substitution of lysine for glutamic acid at position 93. This change and the resulting large change in local charge were presumably required for the temperature-sensitive transformation phenotype of ST529. These results, together with other known deletions that produce fusiform mutants, suggested that a region within the amino-terminal one-third coding region of the src gene contributed to a structural domain of p60v-src that was important for controlling some morphological parameters of transformation in cells infected with RSV.

Avian Sarcoma Viruses↗