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PubMed2001

Site-directed mutational analysis of active site residues in the acetate kinase from Methanosarcina thermophila.

Acetate kinase catalyzes the magnesium-dependent transfer of the gamma-phosphate of ATP to acetate. The recently determined crystal structure of the Methanosarcina thermophila enzyme identifies it as a member of the sugar kinase/Hsc70/actin superfamily based on the fold and the presence of five putative nucleotide and metal binding motifs that characterize the superfamily. Residues from four of these motifs in M. thermophila acetate kinase were selected for site-directed replacement and analysis of the variants. Replacement of Asp(148) and Asn(7) resulted in variants with catalytic efficiencies less than 1% of that of the wild-type enzyme, indicating that these residues are essential for activity. Glu(384) was also found to be essential for catalysis. A 30-fold increase in the magnesium concentration required for half-maximal activity of the E384A variant relative to that of the wild type implicated Glu(384) in magnesium binding. The kinetic analysis of variants and structural data is consistent with nonessential roles for active site residues Ser(10), Ser(12), and Lys(14) in catalysis. The results are discussed with respect to the acetate kinase catalytic mechanism and the relationship to other sugar kinase/Hsc70/actin superfamily members.

Acetate Kinase↗
PubMed1985

Origin of human triosephosphate isomerase isozymes: further evidence for the single structural locus hypothesis with Japanese variants.

Four electrophoretic variants of human erythrocyte triosephosphate isomerase (TPI) have been studied to investigate the origin of the multiple forms of human TPI, in particular the constitutive TPI-B isozyme and the cell division-associated TPI-A isozyme. The variant phenotype expressed by the constitutive TPI-B isozyme in both erythrocytes and peripheral lymphocytes was also expressed by the cell division-associated isozymes in mitogen-stimulated lymphocytes and hair root cells. These results strongly support the hypothesis of Decker and Mohrenweiser (1981) that TPI-B and TPI-A originated from the same structural gene. We also found that the isozyme e is different from TPI-A with respect to both its electrophoretic mobility and heat stability. This finding is in contrast to the recent conclusion of Yuan et al. (1981) that both the isozyme e and TPI-A are deamidation products of TPI-B.

Carbohydrate Epimerases↗
PubMed1980

Structure and function of normal and variant human phosphoglycerate kinase.

Complete amino acid sequence of normal human phosphoglycerate kinase (PGK) was determined. The enzyme consists of 417 amino acid residues with acetylserine at the NH2-terminal and isoleucine at the COOH-terminal. The structural abnormality of PGK-II, which is fairly common in Southern Pacific populations, is a single amino acid substitution from threonine in the normal enzyme to asparagine in the variant enzyme at the 352nd position. The substitution induced no change in the enzyme activity, but induced strong binding of the variant enzyme with citrate. The structural abnormality of PGK-München is a single amino acid substitution from aspartic acid in the normal enzyme to asparagine in the variant enzyme at the 267th position. PGK-München is associated with red cell enzyme deficiency (about 20% of normal), and substantial heat instability. Therefore, the negative charge of an aspartyl residue at the 267th position must play a role in maintaining the stability of the enzyme molecule. Possible mechanisms of hemolysis due to hereditary deficiency of PGK are discussed.

Amino Acid Sequence↗
PubMed1995

Hydroxyarginine-containing polyphenolic proteins in the adhesive plaques of the marine mussel Mytilus edulis.

An unusual polymorphic protein family of nine or more variants has been isolated from the byssal adhesive plaques and foot of the marine mussel Mytilus edulis. In accordance with established terminology, the family is referred to as M. edulis foot protein 3 or simply Mefp-3. Variants of Mefp-3 have molecular masses of about 6 kDa, isoelectric points greater than 10.5, and an amino acid composition dominated by six amino acids: glycine, asparagine, 3,4-dihydroxyphenylalanine (Dopa), tryptophan, arginine, and an unknown basic amino acid. The latter has been isolated and identified as 4-hydroxyarginine using fast atom bombardment mass spectrometry and appropriate standards. The primary structure of variant Mefp-3F has been determined by peptide mapping using automated Edman sequencing in combination with fast atom bombardment and matrix-assisted laser desorption ionization mass spectrometry: ADYYGPNYGPPRRYGGGNYNRYNRYGRRYGGYKGWNNGWNRGRRGKYW where Y represents Dopa, and R represents hydroxyarginine. Notably, the 4 occurrences of RY are marked by a resistance to trypsin digestion. Although the conversion of tyrosines to Dopa is essentially complete, hydroxylation of arginines varies between 40 and 80%. In contrast to other mussel adhesive proteins such as Mefp-1 and -2 which have large numbers of highly conserved, tandemly repeated peptide motifs, Mefp-3 has only short sporadic repeats. The specific function of Mefp-3 in byssal adhesion is unknown.

Amino Acid Sequence↗
PubMed2002

Infections of feline leukemia virus and feline immunodeficiency virus.

Feline retrovirus infections have been extensively studied for more than 30 years as an animal model for the persistent infections and pathogenesis caused by retroviruses in general. Two retroviruses, feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV), have been recognized as causative agents of a variety of diseases including proliferative and degenerative diseases. Recent studies revealed the receptors of FeLV, its variants and FIV. FeLVs utilize at least three distinct receptors, two of which have been successfully cloned and characterized. Furthermore, an FeLV variant which induces severe immunodeficiency, utilizes a truncated envelope of the endogenous FeLV as coreceptor or cofactor for viral entry. FIV utilizes as receptor one of the chemokine receptors, CXCR4 which also is a coreceptor for the T-lymphotropic human immunodeficiency virus. This review provides an overview to the infections of FeLV and FIV, specifically focuses on the viral genomic structures, FeLV variants, the immune responses and recent findings on the receptors for FeLV and FIV. Better understanding of retroviral persistence and pathogenesis will aid the development of prophylactic vaccines and therapeutic medicine to interfere with retrovirus infections.

Animals↗
PubMed1986

Structure and activities of a variant ubiquitin sequence from bakers' yeast.

Ubiquitin is an extremely conserved protein, with an identical sequence throughout the animal kingdom. However, the gene sequence of the yeast protein [Ozkaynak, E., Finley, D., & Varshavsky, A. (1984) Nature (London) 312, 663-666] predicts three amino acid differences. This implies that some functions or binding interactions of ubiquitin are different in yeast and animal cells. In an effort to define these differences, ubiquitin has been purified to homogeneity from bakers' yeast and characterized. Amino acid analysis of the protein and the isolated tryptic peptides confirms the primary structure of this protein as predicted from the gene sequence. This result indicates that the gene sequenced is the transcriptionally active gene from yeast. The conformation of yeast ubiquitin is similar to human ubiquitin as judged by circular dichroism, sensitivity to trypsin, and Stokes radius. Yeast and animal ubiquitins show identical activities in supporting ubiquitin-dependent protein degradation and in the ATP-pyrophosphate exchange reaction catalyzed by the purified ubiquitin-adenylating enzyme. Thus, the three conservative amino acid differences between yeast and animal ubiquitins have very little effect on the structure of ubiquitin or its activity in the ubiquitin-dependent proteolytic system. These results suggest that at least some of the evolutionary pressure preventing sequence variation among animal ubiquitins stems from one or more of its nonproteolytic functions.

Amino Acid Sequence↗
PubMed1997

The primary structure of Trypanosoma (Nannomonas) congolese variant surface glycoproteins.

The complete nucleotide sequences were determined for three transcripts each encoding a different variant surface glycoprotein (VSG) of Trypanosoma (Nannomonas) congolense. The nucleotide sequence was determined also for a transcript encoding a fourth VSG, but this was truncated. The data obtained confirm absence of the canonical polyadenylation signal, lack of conserved sequence elements in the 3' untranslated region, and heterogeneity in the spliced-leader acceptor site in the T. congolense VSG transcripts examined. A comparison of the amino acids deduced from the nucleotide sequences of the four VSGs and those of other VSGs published previously reveals a strong conservation of several structural domains, particularly cysteine residues located throughout most of the molecules. The majority of T. congolense VSGs analyzed in this study resemble most the N-terminal cysteine residue domain type B of T. brucei, characterized by a cysteine residue located toward the N-terminal end, a cluster of cysteine residues in the central region, and at least three cysteine residues between positions 250 and 300 of the molecules. One of the VSGs analyzed, ILNat3.3, did not fit into any of the classification schemes proposed for the VSGs so far studied, and thus may represent a different class of these surface molecules. Unlike VSGs of T. brucei, the T. congolense VSGs have no cysteine residues at the carboxy-terminal end. These data now make it possible to predict general primary structural features of T. congolense VSGs.

Amino Acid Sequence↗
PubMed1987

Structures of human alcohol and aldehyde dehydrogenases.

Human alcohol dehydrogenase is a dimeric zinc metalloenzyme for which forms of three classes, I, II and III, have been distinguished. Subunits hybridize within but not between classes. There are three types of subunit, alpha, beta, and gamma, in class I. The primary structures of all three forms have been established, as well as the overall properties and the effects of the amino acid substitutions between the various forms. Each subunit has 374 residues, of which 35 exhibit differences among the alpha, beta and gamma chains. Corresponding cDNA structures are also known, as are the genetic organization and details of the gene structures. Allelic variants occur at the beta and gamma loci. Corresponding amino acid substitutions have been characterized, and enzymatic differences between the allelic forms are explained by defined residue exchanges. The results also illustrate recent and repeated isozyme evolution, a subject where alcohol dehydrogenases exceptionally well offer detailed examples. Human aldehyde dehydrogenase occurs of two types, a mitochondrial and a cytosolic form. The enzymes are tetramers, do not contain functional metals, and have subunits which do not form inter-type hybrids. The primary structures have been determined, revealing a positional identity of 68% (in 500 residues) between the mitochondrial and cytosolic forms. The N-terminus is heterogeneous and is not blocked in the subunit of the mitochondrial enzyme, in contrast to that of the cytosolic enzyme or those of all the alcohol dehydrogenases (also cytosolic). A reactive cysteine residue at position 302 has been ascribed functional importance at or close to the active site, is conserved in the two aldehyde dehydrogenases, and is associated with the action of disulfiram on the enzyme. In Oriental populations, a mutant allelic variant of the mitochondrial protein with impaired enzyme function has also been characterized.

Alcohol Dehydrogenase↗
PubMed1999

Design and NMR analysis of HDV ribozymes for structural investigation.

Three variants of minimized hepatitis delta virus (HDV) RNA ribozyme systems (Rz-1 to approximately Rz-3) (Fig. 1) were designed on the basis of the "pseudoknot" structure model and synthesized. Rz-1 is a cis-acting ribozyme system (a cleaved form, 56-mer) in which stem IV is deleted from the active domain of genomic HDV RNA. Rz-1 was uniformly labeled with stable isotopes, 13C and 15N. The 2D-NOESY and 2D-HSQC data for Rz-1 suggest that Rz-1 forms the pseudoknot structure and G38 which is opposite to the cleavage site makes a base-pair. Rz-2 is a trans-acting ribozyme system which consists of three RNA oligomer strands (substrate: 8-mer, the cytidine residue at the cleavage site is replaced by 2'-O-methylcytidine; enzyme: 16-mer plus 35-mer). Rz-3 is a ribozyme in which the three RNA strands of Rz-2 are connected. It turns out that Rz-3 forms an inactive structure with low cleavage activity (k(obs) = 0.009) and final cleavage yield (6%). Rz-3 has the highest cleavage activity at pH 5.5. The optimal activity at acidic pH is similar to that of the wild type ribozyme. We also synthesized and examined the activity and structure of Rz-4 (designed by Perrotta and Been) which consists of two RNA strands (1).

Base Pairing↗
PubMed1995

A lipooligosaccharide-binding site on HepG2 cells similar to the gonococcal opacity-associated surface protein Opa.

The lacto-N-neotetraose-containing lipooligosaccharide (LOS) present on the surface of most Neisseria gonorrhoeae organisms may serve many important functions in gonococcal pathogenesis. This surface glycolipid contains the cross-reactive epitope to human paragloboside and can be sialylated by gonococci grown in the presence of CMP-N-acetylneuraminic acid. Another possible role for this glycolipid could be to mimic human asialocarbohydrates and act as a ligand for asialoglycoprotein receptors contained on numerous human cells. The most noted of this large family of receptors is that expressed on the surface of hepatic cells. In a model cell system, using the hepatoma tissue culture cell line HepG2, we wanted to investigate if the presence of this asialoglycoprotein receptor influenced the adherence and/or invasion of gonococci expressing the lacto-N-neotetraose structure. Piliated variants of the gonococcal wild-type strain 1291 and its isogeneic LOS mutant 1291E were used in adherence-invasion assays. This gonococcal strain is somewhat unusual in that it expresses large amounts of predominantly one species of LOS, thus reducing the complexity of interpreting the data. The data from these assays suggested that the Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc carbohydrate structure on the wild-type LOS affected the adherence-invasion of gonococci into the HepG2 cells. In studies to determine whether the major hepatic asialoglycoprotein receptor was involved in these interactions, we found that the HepG2 cells contained two receptors which bound gonococcal LOS. One of these was the asialoglycoprotein receptor, and the data concerning this receptor will be reported elsewhere. The data on the second receptor are reported here. Purified, 125I-labeled gonococcal LOS was used to identify specific high-affinity LOS-binding sites. These binding experiments revealed one major binding site corresponding to a protein with a molecular mass of 70 kDa (p70). Several lines of evidence in this study suggested that the oligosaccharide region of LOS played an important role in LOS binding to the p70 of HepG2 cells. In addition, we show that this human LOS receptor has some similarities to the gonococcal Opa proteins.

Antigens, Bacterial↗
PubMed2006

Expression and processing of hepatitis C virus structural proteins in Pichia pastoris yeast.

Development of heterologous systems to produce useful HCV vaccine candidates is an important part of HCV research. In this study different HCV structural region variants were designed to express the first 120 aa, 176 aa, 339 aa, and 650 aa of HCV polyprotein, and aa 384 to 521, or aa 384-605 or aa 384-746 of HCV E2 protein fused to the leader sequence of sucrose invertase 2 allowing the secretion of recombinant E2 proteins. Low expression levels were observed for HCV core protein (HCcAg) variants expressing the first 120 aa and 176 aa (HCcAg.120 and HCcAg.176, respectively). Higher expression levels were observed when HCcAg was expressed as a polypeptide with either E1 or E1 and E2 proteins. In addition, HCcAg was processed to produce two antigenic bands with 21 and 23kDa (P21 and P23, respectively) when expressed as a polypeptide with HCV E1 and E2 proteins. Results also suggest E1 processing in the context of HCcAg.E1.E2 polyprotein. On the other hand, E2.521, E2.605, and E2.680 were efficiently excreted to the culture medium. However, the entire E2.746 variant predominantly localized in the insoluble fraction of ruptured cells. Results suggest that the hydrophobic C-terminal E2 region from aa 681 to 746 is critical for intracellular retention of recombinant E2.746 protein in Pichia pastoris cells. Endo H or PNGase F treatment suggests that E2.746 was modified with high-mannose type oligosaccharides in P. pastoris. These data justify the usefulness of P. pastoris expression system to express HCV structural viral proteins which may be useful targets for HCV vaccine candidates.

Antigens, Viral↗
PubMedDate not supplied

Somatotype of children with gonadal dysgenesis and structural anomalies of genitalia.

The somatotype of 92 children with sexual structural anomalies of whom 28 with female Turner's syndrome, 22 with Klinefelter's syndrome, 20 with adreno-genital syndrome, 14 with hypospadias, 2 with Reinfenstein's syndrome and 6 with feminizing testis was established on the basis of biometric and genetically determined morphologic characteristics (dermatoglyphics, pigment complex). A morphogram utilizing 12 anthropometric and 5 dermatoglyphic parameters was constructed; it can be used in phenotypical differentiation of the anatomo-clinical variants of structural anomalies of the genitalia and gonads. The 12 anthropometric parameters were: weight, stature, height of the head + neck segment, the sternum-symphysis distance, length of arms and legs, the biacromial and bitrochanterian diameters, the circumference of the thorax, abdomen, hips and head. The dermatoglyphic parameters were: the digital delta index, TFRC, a-b ridge count, the ATD angle and the A line obliquity. For longitudinal assessment of the phenotype of children with structural anomalies of genitalia and gonads, the authors propose utilization of the percentile diagrams regarding weight, stature, pubes to floor distance, and the biacromial and bitrochanterian diameters.

Adolescent↗
PubMed1988

Structural and immunologic evidence for a small molecular weight ("21K") variant of prolactin.

The mol wt (Mr) of intact murine PRL is approximately 23,000. Immunoblotting analysis shows a 21,000 Mr band in fresh rat and mouse pituitary extracts that cross-reacts strongly with PRL antibodies. The band becomes markedly altered by stimuli known to influence PRL secretion, such as nursing, estradiol benzoate, and perphenazine. By two-dimensional electrophoresis, it splits into at least three components, two of which have more acidic pI than PRL. The tyrosine peptide maps of the three proteins resemble that of PRL. These results indicate that the 21,000 Mr band consists of a cluster of proteins that are structural variants of PRL, raising the possibility that these variants result from alternative splicing of the PRL gene transcript.

Animals↗
PubMed1996

Structure-activity relationships for inhibition of inosine monophosphate dehydrogenase by nuclear variants of mycophenolic acid.

Structure-activity relationships in the region of the phthalide ring of the inosine monophosphate dehydrogenase inhibitor mycophenolic acid have been explored. Replacement of the lactone ring with other cyclic moieties resulted in loss of potency, especially for larger groups. Replacement of the ring by acyclic substituents also indicated a strong sensitivity to steric bulk. A phenolic hydroxyl group, with an adjacent hydrogen bond acceptor, was found to be essential for high potency. The aromatic methyl group was essential for activity; the methoxyl group could be replaced by ethyl to give a compound with 2-4 times the potency of mycophenolic acid in vitro and in vivo.

Animals↗
PubMed1991

Expression and function of a variant T cell receptor complex lacking CD3-gamma.

A T cell line termed DIL2 has been derived from an infant with a polyclonal T cell receptor (TCR)/CD3 cell surface expression defect. Indirect immunofluorescence showed that the expression of certain TCR/CD3 epitopes (like those detected by WT31 and BMA031 monoclonals) was strongly reduced (around five-fold) on DIL2, whereas other epitopes (like those detected by SP34 and Leu4) were only around two-fold lower than in normal T cell lines. Specific immunoprecipitates of surface-radioiodinated DIL2 cells contained TCR-alpha, TCR-beta, CD3-delta, CD3-epsilon and TCR-zeta chains, but lacked CD3-gamma. This structural TCR/CD3 variant was, however, capable of transducing certain activation signals, since normal proliferation and a low but significant calcium flux was observed in DIL2 cells after engagement with specific antibodies. Our data suggest that a functional TCR/CD3 complex can be expressed on the surface of T cells in the absence of CD3-gamma.

Antibodies, Monoclonal↗
PubMed1994

[Variability of rDNA genes, detected as a result of analyzing a pseudogene nucleotide sequence in Drosophila melanogaster].

A pseudogene bearing the bulk of the 18S RNA gene was detected outside the rDNA cluster. It comprised irregularly distributed nucleotide substitutions as well as short insertions and deletions. No sequence alterations were observed in the 5' region of the pseudogene, whereas the frequency of substitutions and alterations per nucleotide number in the 3' region and in the middle of the sequence was 7.6% and 1.8%, respectively. The observed sharp irregularity in distribution of substitutions and alterations was considered the result of successive recombinations between the functional 18S rRNA gene and its diverged or damaged variants. This phenomenon provides experimental evidence that recombinations between the pseudogene and functioning repeats of rDNA are implicated in the mechanism of rDNA sequence correction. A segment of the pseudogene sequence was shown to contain substitutions primarily in regions coding for single-strand parts of the RNA molecule. The same segment contained a deletion and an insertion of a nucleotide, approximating it to the most of the studied eukaryotic 18S rRNA sequences. These observations allowed us to supposed that a structural rDNA variant, a fragment of which appears in the pseudogene sequence, is present in the genome. The data obtained suggest both the presence of 18S rDNA variants, and recombination between them, determining the concerted evolution of rRNA genes.

Animals↗
PubMed2000

A five-dimensional structural investigation of the misfit layer compound

The structure of the misfit layer compound [Bi0.87SrO2]2[CoO2]1.82, bismuth strontium cobaltite, was determined by single-crystal X-ray diffraction using the five-dimensional superspace-group formalism. This composite crystal, of monoclinic symmetry, is composed of two subsystems exhibiting incommensurate periodicities along b, the binary axis direction. The first composite part [Bi0.87SrO2] displays an intrinsic modulation of planar monoclinic type characterized by the wavevector q* = 0.293a* + 0.915c*. The second composite part [CoO2] shows two different centered lattice variants. The structure of the misfit layer crystal can be described as an alternation along c of distorted rock-salt-type slabs, formed from [BiO] and [SrO] layers (first subsystem), and of [CoO2] layers (second subsystem) displaying a distorted CdI2-type structure. Two main structural results are obtained. First, as a consequence of the intrinsic modulation, disordered zones, characterized by Bi vacancies, are regularly distributed in the [BiO] layers. Second, strong chemical bonds are implied between the strontium atoms of the first subsystem and the oxygen atoms of the second one.

Journal Article↗
PubMed1988

Histone H1 heterogeneity in the midge, Chironomus thummi. Structural comparison of the H1 variants in an organism where their intrachromosomal localization is possible.

1. Seven subfractions of histone H1 have been isolated and purified from larvae of Chironomus thummi (Diptera). They have been denominated I-1, II-1, II-2, II-3, III-1, III-2, and III-3, according to the order of migration in two steps of preparative electrophoresis. 2. The amino acid compositions are similar to those of other H1 histones. Subfractions I-1 and II-1 were found to contain one methionine and two tyrosine residues, II-2 contained two methionine and three tyrosine residues, and III-1 one methionine and three tyrosine residues. The other subfractions contained one or two methionine and two or three tyrosine residues. For subfractions I-1 and II-1 a chain length of about 252 amino acids was estimated. 3. Peptide pattern analyses after chemical cleavage at the methionine and tyrosine residues, and enzymatic cleavage with thrombin and chymotrypsin, respectively, showed that all subfractions have different individual primary structures. A comparison of peptide sizes and of the positions in the peptide patterns of epitopes recognized by monoclonal antibodies was made to check whether some of the subfractions could arise by proteolytic degradation of others. This possibility can be excluded for five of the subfractions and is very improbable for the two others. Treatment of C. thummi H1 with alkaline phosphatase did not change the pattern of subfractions, while the phosphorylated subfraction of histone H2A disappeared after this treatment. Most and very probably all subfractions are thus H1 sequence variants. 4. Inbred strains and individual larvae of C. thummi were found to comprise all seven variants. The H1 heterogeneity can therefore not be due to allelic polymorphism. Salivary gland nuclei were found to contain variant I-1 and at least some of the other variants. 5. H1 from Drosophila melanogaster and from calf thymus were used as reference molecules in all cleavage experiments and yielded the peptide patterns expected from the sequence. The comparison discriminates the group of C. thummi H1 histones clearly from Drosophila and calf thymus H1. Limited trypsin digestion yielded a protected peptide of uniform size in six of the seven variants which was considerably smaller than the protected central domain of calf thymus H1. 6. Two other species of Chironomidae, C. pallidivittatus and Glyptotendipes barbipes were found to contain five and three H1 subfractions, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗