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PubMed1993

Cloning, expression, and crystallization of jack bean (Canavalia ensiformis) canavalin.

Canavalin is the major storage protein of the jack bean (Canavalia ensiformis) and belongs to the classical vicilin fraction. A full-length cDNA for canavalin was generated by the polymerase chain reaction. The nucleotide sequence coding for canavalin and the corresponding amino acid sequence were determined and shown to be homologous with those of other seed storage proteins. The amino acid sequence contained an internal sequence duplication corresponding to the structural redundancy in the monomer demonstrated by crystallographic analysis. The coding region of the canavalin cDNA was inserted into a T7 RNA polymerase expression vector and used to transform Escherichia coli. A recombinant protein with a molecular mass of 47 kilodaltons was expressed and purified to 95% homogeneity. The protein exhibited the same physical, immunological, and biochemical properties as native jack bean canavalin. Recombinant canavalin, following treatment with trypsin, was crystallized in two forms. Crystals of a rhombohedral habit grew to 1 mm in the longest dimension and diffracted to beyond 3-A resolution. Three-dimensional diffraction data demonstrated crystals of the recombinant protein to be isomorphous with crystals of the natural plant protein, thereby confirming the identity of their structures.

Amino Acid Sequence↗
PubMed1993

Plasminogen-activator inhibitor type 2 (PAI-2) is a spontaneously polymerising SERPIN. Biochemical characterisation of the recombinant intracellular and extracellular forms.

Plasminogen-activator inhibitor type 2 (PAI-2) is a specific inhibitor of plasminogen activators (PA) that exists in an intracellular, low-molecular-mass form and a secreted, high-molecular-mass form that varies with respect to glycosylation. Here we have developed expression systems for both forms of PAI-2 and biochemically characterised the purified proteins. In order to obtain efficient secretion, we constructed an artificial signal sequence and fused it to the coding region of PAI-2. With this construct, more than 90% of PAI-2 was secreted as a glycosylated, 60-kDa molecular-mass form, but the level of expression was low and unstable. To obtain higher expression of secreted PAI-2, a novel expression vector based on the Semliki-forest-virus replicon was used. Secreted PAI-2 was purified to homogeneity and N-terminal sequence analysis showed that the artificial signal peptide was correctly removed. The intracellular, non-glycosylated form of PAI-2 was expressed in Escherichia coli and purified to homogeneity. Both the secreted and the intracellular forms of PAI-2 were found to inhibit plasminogen activators by forming SDS-resistant complexes and the second-order rate constants were similar for both forms, ranging over 2.4-2.7 x 10(6) M-1s-1 for urokinase-type PA, 2.5-2.7 x 10(5) M-1s-1 for two-chain tissue-type PA and 0.8-1.2 x 10(4) M-1s-1 for single-chain tissue-type PA. None of the purified PAI-2 forms bound to vitronectin. Circular-dichroism spectral analysis revealed that PAI-2 has a CD spectrum that resembles ovalbumin more than PA-inhibitor type 1, confirming the greater similarity between these two members of the serine-protease inhibitor family. Similar to what has been described for the Z-form of alpha 1-antitrypsin, purified PAI-2 was found to spontaneously form polymers during incubation at room temperature. Attempts to convert PAI-2 to a stable locked conformation resembling the conformation of latent PAI-1 by treatment with diluted guanidinium chloride were unsuccessful. Instead, this treatment enhanced the formation of PAI-2 polymers, possibly by the loop-sheet polymerisation mechanism described for alpha 1-antitrypsin.

Animals↗
PubMed1988

Nucleotide sequence of pig plasma gelsolin. Comparison of protein sequence with human gelsolin and other actin-severing proteins shows strong homologies and evidence for large internal repeats.

Pig plasma gelsolin (Mr = 81595; 739 residues) contains 704 identical residues out of a maximum 730 when compared to the cytoplasmic form of human gelsolin. The cDNA sequence also codes for a peptide of 33 residues N-terminal to the nine-residue plasma extension sequence previously reported: these 33 residues are highly homologous to the human signal peptide and plasma extension. Comparison of the gelsolin sequences with chicken brush border villin, severin from Dictyostelium discoideum and fragmin from Physarum polycephalum shows a strong evolutionary relationship between all these proteins. There are six large repeating segments in gelsolin and villin, and three similar segments in severin and fragmin. Although these multiple repeats cannot be related to any known function of these actin-severing proteins, this superfamily of proteins appears to have evolved from an ancestral sequence of 120 to 130 amino acid residues.

Animals↗
PubMed2026

theBIGbam: compression and interactive exploration of large-scale sequencing alignments with circular mapping support.

SUMMARY: theBIGbam (github.com/bhagavadgitadu22/theBIGbam) is a genome browser and alignment viewer designed for massive metagenomic and metatranscriptomic datasets. The tool takes BAM files containing read alignments, together with genome assemblies in FASTA format or annotated genome sequences in GenBank format. Alternatively, it can start from raw FASTQ reads and generate alignments using a modified mapper that supports circular genomes, enabling seamless read mapping across genome ends. theBIGbam can compress hundreds of gigabytes of input files 10- to 100-fold into dedicated databases while retaining key per-position information, including coverage depth and recurrent mismatches, insertions, and deletions between reads and the reference. These databases can be served to a local web browser, enabling interactive exploration of any contig in any sample using DNAFeaturesViewer for genome maps and Bokeh for mapping-derived features. Contig-sample pairs available for visualization can be filtered using a range of summary metrics calculated per contig, per sample, and per contig-sample pair to guide users toward the most relevant signals. Through its interactive visualization, theBIGbam facilitates the exploration of complex datasets, while its integrated database-combining assembly features, annotated features, and mapping-derived features-provides the information needed to investigate biological hypotheses systematically. Designed to complement existing browsing tools like IGV and Anvi'o, theBIGbam is particularly suited for examining misassemblies, subpopulations, microdiversity, and contig topology in large-scale datasets. AVAILABILITY AND IMPLEMENTATION: theBIGbam is an open-source Rust/Python package that can be installed from Bioconda or PyPI. The source code and documentation are available on GitHub (github.com/bhagavadgitadu22/theBIGbam).

Software↗
PubMed1978

Color vision mechanisms in monkey striate cortex: dual-opponent cells with concentric receptive fields.

1. I have recorded with tungsten microelectrodes from single cells in the monkey's visual cortex and have specifically studied those neurons which were sensitive to the color of the stimulus. In the primate striate cortex there are four classes of color-coded cells. The cells described in this paper have concentric receptive fields with one red-green opponent-color system in the field center and the opposite organization in the surround. These dual-opponent cells were nost sensitive to the simultaneous presentation of two different colors, one covering the field center and the other illuminating the surround. They are probable involved in the perception of simultaneous color-contrast phenomena. 2. Spectral sensitivity curves revealed that both the field centers and the surrounds received opposite types of inputs from red-sensitive and green-sensitive cones. None of the cells tested had inputs from rods. 3. Area-sensitivity curves showed that peripheral suppression was present for both phases of the center opponent-color system. The boundary between the center and the surround was the same for both sets of opponent systems. Some cells had "silent" surrounds, which did not respond to annular stimuli. 4. Multiple-unit recordings from a concentric cell and one of its presumed afferents yielded information regarding its possible synaptic inputs. In some cases the cells appeared to receive contacts from red/green opponent-color geniculated fibers with circular receptive fields that lacked an antagonistic surround (similar to Wiesel and Hubel's (37) type II class). In other instances the afferents had on-center, off surround receptive fields or the reverse, but received inputs from only one cone type, either red or green (similar to Wiesel and Hubel's type III class). 5. Concentric cells were always driven by only one eye. 6. The laminar distribution of these cells was limited almost entirely to layer IV and its subdivisions. 7. The cumulative evidence presented in this paper indicates that the concentric cells probably received direct geniculate inputs and, therefore, they are the first cortical stage in the integration of color-contrast information.

Animals↗
PubMed1995

Complete sequence of the maize chloroplast genome: gene content, hotspots of divergence and fine tuning of genetic information by transcript editing.

The nucleotide sequence of the chloroplast (cp) DNA from maize (Zea mays) has been completed. The circular double-stranded DNA, which consists of 140,387 base-pairs, contains a pair of inverted repeat regions (IRA and IRB) with 22,748 base-pairs each, which are separated by a small and a large single copy region (SSC and LSC) of 12,536 and 82,355 base-pairs, respectively. The gene content and the relative positions of a total of 104 genes (70 peptide-encoding genes, 30 tRNA genes and four rRNA genes) are identical with the chloroplast DNA of the closely related species rice (Oryza sativa). A detailed analysis of the two graminean plastomes allows the identification of hotspots of divergence which predominate in one region containing a cluster of tRNA genes and in two regions containing degenerated reading frames. One of these length differences is thought to reflect a gene transfer event from the plastome to the nucleus, which is followed by progressive degradation of the respective chloroplast gene resulting in gene fragments. The other divergent plastome region seems to be due to the complete loss of a plastid gene and its functional substitution by a nuclear encoded eukaryotic homologue. The rate of neutral nucleotide substitutions is significantly reduced for protein coding genes located in the inverted repeat regions. This indicates that the existence of inverted repeat regions confers increased genetic stability of the genes positioned in these regions as compared to genes located in the two single copy regions. Editing events cause the primary structures of several transcripts to deviate from the corresponding genomic sequences by C to U transitions. The unambiguous deduction of amino acid sequences from the nucleotide sequences of the corresponding genes is, therefore, not possible. A survey of the 25 editing positions identified in 13 different transcripts of the maize plastome shows that representatives of all protein coding gene classes are subject to editing. A strong bias exists for the second codon position and for certain codon transitions. Based on the number and the codon transition types, and taking into account the frequency of putative editing sites in all peptide encoding genes and unidentified reading frames, a total number of only few more than the experimentally verified 25 editing sites encoded in the maize plastome is estimated. This corresponds to 0.13% of amino acid positions which cannot be derived from the corresponding codons present in the corresponding genes.

Base Sequence↗
PubMed2002

[B-flow and contrast medium-enhanced power Doppler (Optison(R))--preoperative diagnosis of high-grade stenosis of the internal carotid artery].

PURPOSE: To improve sonographic diagnosis of high-grade stenosis of the internal carotid artery (ICA) by vascular sonography in "B-flow"-mode and in contrast medium (Optison(R))-enhanced power Doppler (PD). MATERIAL AND METHODS: 88 patients suspected of a high-grade stenosis of the extracranial ICA were examined by independent examiners with color-coded Doppler (CCD), B-flow and PD to compare the methods for morphology and degree of stenosis. In 21 cases contrast medium was used in PD. Sonographic results of all 88 patients were compared with selective angiography (DSA), in 53 cases with operative findings. RESULTS: A complete occlusion was found in 9 patients and a 50 - 70 % degree non-exulcerated stenosis in 26 cases. 53 of 88 patients presented findings in both sonography and DSA leading to operation of the ICA. Among these 47 had a stenosis > 70 % and 6 a stenosis of 60 - 70 % with exulcerated plaques. Even a small dose of contrast medium (0.3 ml) gave a significant signal increase in PD improving flow detection even in extensive vascular calcification. In both the early phase (up to the first minute p. i.) flow contrast was optimal and without significant artifacts. Thus, the extent and the morphology of the stenosis was better appreciated. Hypoechogenic plaques, ulcerations and thrombi are as easily recognized by B-flow as flow in preocclusive stenosis, free of vibration and blooming artifacts. This gave a better correlation with DSA and a higher diagnostic accuracy in grading a stenosis than CCD and PD. Circular calcifications or vessel kinking made diagnosis of flow more difficult. CONCLUSION: B-flow allows a reliable diagnosis of the degree of stenosis of ICA and a better appreciation of plaque morphology than CCD. In some cases with extensive calcifications contrast medium-enhanced PD is able to detect better intrastenotic flow. Only small quantities of contrast medium (Optison(R)) are required.

Aged↗
PubMed1989

Parallel DNA: generation of a duplex between two Drosophila sequences in vitro.

We have observed the existence of a parallel complementary region between two Drosophila DNA sequences, fragments of the suffix [(1986) EMBO J, 5, 2341-2347] and a 5'-non-coding sequence of the alcohol dehydrogenase gene [(1983) Cell 33, 125-133]. The region includes approximately 40 bp, 76% of which are complementary in the same polarity. Synthetic complementary 16 bp oligonucleotides corresponding to this region which were bound by the 5'-ends through a 1.6-hexanediol bridge form a duplex which displays both melting and annealing as judged by UV absorbance. Anti parallel complementary 16 bp long oligonucleotides bound by the 5'-3' ends through the same bridge and a single-strand sequence were used as controls. The Hoechst 33,258 drug binds to this parallel duplex of DNA; however, the properties of such a complex testify against the B-form of the duplex.

Alcohol Dehydrogenase↗
PubMed1991

[1.10/1.15 promille--consequences for the current and future promille regulation].

In the Federal Republic so far it is a matter of jurisdiction and not of legislation, to fix the blood alcohol limit for punishable absolute inability to drive in the sense of section 315 c par. 1 no. 1 a StGB (German Criminal Code). This fact has considerable disadvantages for traffic-safety and legal security. The level of the critical value inevitably varies due to new results of alcohol- and traffic-research. It is pushed up by the fact, that the critical value of jurisdiction has to be a generalized one, which has taken even special alcohol-digestibility into consideration. The critical value of jurisdiction therefore is too high and too instable. The fixation-process of the critical value by the law courts could lead to a very long and rattling period of different judicial opinions, just like it was after the 1.1 promille-suggestion by Salger. But the legislator has to assure clear and safe conditions, even when alcohol is in the game. The principal elements of the 1.1 promille-decision by the BGH (German Federal Law Court) on the 28th of June 1990 correspond mainly with the arguments, which Salger used for this 1.1 promille-suggestion in January 1990. Going beyond of that, some medical and natural-scientific arguments concerning the lowering of the security-addition are included. Under the condition that the institute, which analyses the blood-alcohol forensically, participates successfully in circular tests, the BGH has fixed the punishable critical value to 1.0 promille (fundamental value 1.0 promille plus a safety-addition of 1.0 promille). To prove their successfully participation in circular tests, the institutes, which are carrying through blood-alcohol-analyses, are obliged to assure the judicial authorities of their participation in circular tests and furthermore they have to demonstrate with 4 resp. 5 single measured values of each blood-alcohol-determination, that their deviation is below the maximal value (= 0,048 promille), which has been given by the expert opinion of the Federal Health Board 1989. For blood analyses of institutes, which are not yet successfully participating in circular tests, the punishable critical value has been fixed to 1.15 promille (fundamental value 1.0 promille plus a safety-addition of 0.15 promille). This is valid for a transitional period, in which these institutes should get the opportunity, to participate in a circular test successfully. The critical values mentioned above are also valid for those proceedings, which had not yet been finished in the moment of the decision of the BGH.(ABSTRACT TRUNCATED AT 400 WORDS)

Accidents, Traffic↗
PubMed1994

Identification and classification of interstitial cells in the canine proximal colon by ultrastructure and immunocytochemistry.

The ultrastructure and immunocytochemistry of interstitial cells (ICs) in the canine proximal colon were investigated. Three types of ICs were found within the tunica muscularis. (1) ICs were located along the submucosal surface of the circular muscle (IC-SM). These cells shared many features of smooth muscle cells, including myosin thick filaments and immunoreactivity to smooth muscle gamma actin, myosin light chain, and calponin antibodies. IC-SM were clearly different from smooth muscle cells in that contractile filaments were less abundant and intermediate filaments consisted of vimentin instead of desmin. (2) ICs in the region of the myenteric plexus (IC-MY) were similar to IC-SM, but these cells had no thick filaments or immunoreactivity to smooth muscle gamma actin or calponin antibodies. (3) The fine structures and immunoreactivity of ICs within the muscle layers (IC-BU) were similar to IC-MY, but IC-BU lacked a definite basal lamina and membrane caveolae. IC-BU and IC-MY were both immunopositive for vimentin. Since all ICs were immunopositive for vimentin, vimentin antibodies may be a useful tool for distinguishing between ICs and smooth muscle cells. Each class of ICs was closely associated with nerve fibers, made specialized contacts with smooth muscle cells, and formed multicellular networks. A combination of ultrastructural and immunocytochemical techniques helps the identification and classification of ICs by revealing the fine structures and determining the "chemical coding" of each class of ICs.

Actins↗
PubMed2004

Studies on activities, cell uptake and DNA binding of four trans-planaramineplatinum(II) complexes of the form: trans-PtL(NH3)Cl2, where L=2-hydroxypyridine, imidazole, 3-hydroxypyridine and imidazo(1,2-alpha)pyridine.

Four trans-planaramineplatinum(II) complexes code named YH9, YH10, YH11 and YH12 each of the form trans-PtL(NH(3))Cl(2), where L=2-hydroxypyridine and 3-hydroxypyridine, imidazole, and imidazo(1,2-alpha)pyridine for YH9, YH10, YH11 and YH12, respectively, have been synthesized and the activity of the compounds against human cancer cell lines, cell uptake, DNA-binding and nature of interaction with pBR322 plasmid DNA have been studied. The compound having imidazo(1,2-alpha)pyridine ligand as one the carrier ligands in the trans-configuration is found to be significantly more active than cis-platin against ovarian A2780(cisR) cancer cell line corresponding with higher Pt-DNA binding. All other compounds have resistance factors less than that for cis-platin in the A2780 and A2780(cisR) cell lines. A greater prevention of BamH1 digestion with increasing concentration of the compounds indicates that as the compounds bind with nucleobases in DNA, the DNA conformation is changed sufficiently so as to prevent BamH1 digestion at the specific GG site. Gel electrophoresis results also indicate that as the compounds bind to DNA, unwinding of supercoiled form I DNA takes place to change it from the negatively supercoiled form I through relaxed circular form I to the positively supercoiled form I.

Antineoplastic Agents↗
PubMed2002

A hierarchic approach to the design of hexameric helical barrels.

The design of large macromolecular assemblies is an endeavor with implications for protein engineering as well as nanotechnology. A hierarchic approach was used to design an antiparallel hexameric, tubular assembly of helices. In previous studies, a domain-swapped, dimeric three-helix bundle was designed from first principles. In the crystal lattice, three dimers associate around a 3-fold rotational axis to form a hexameric assembly. Although this hexameric assembly was not observed in solution, it was possible to stabilize its formation by changing three polar residues per monomer to hydrophobic (two Phe and one Trp) residues. Molecular models based on the crystallographic coordinates of DSD (PDB accession code 1G6U) show that these side-chains pack in the central cavity (the "supercore") of the hexameric bundle. Analytical ultracentrifugation, fluorescence spectroscopy, CD spectroscopy, and guanidine-HCl denaturation were used to determine the assembly of the hexamer. To probe the requirements for stabilizing the hexamer, we systematically varied the polarity and steric bulk of one of the Phe residues in the supercore of the hexamer. Depending on the nature of this side-chain, it is possible to modulate the stability of the hexamer in a predictable manner. This family of hexameric proteins may provide a useful framework for the construction of proteins that change their oligomeric states in response to binding of small molecules.

Amino Acid Sequence↗
PubMed2004

DEC-205 expression on migrating dendritic cells in afferent lymph.

Previous studies have identified a 210 000-molecular weight molecule expressed at a high level on the surface of dendritic cells (DCs) in afferent lymph of cattle and evident on cells with the morphology of DCs in lymphoid tissues. Expression is either absent from other immune cells or is present at a lower level. The molecular weight and cellular distribution suggested that the molecule, called bovine WC6 antigen (workshop cluster), might be an orthologue of human DEC-205 (CD205). To establish whether this was the case, the open reading frame of bovine DEC-205 was amplified, by polymerase chain reaction, from thymic cDNA (accession no. AY264845). The cDNA sequence of bovine DEC-205 had 86% and 78% nucleic acid identity with human and mouse molecules, respectively. COS-7 cells transfected with a plasmid containing the cattle DEC-205 coding region expressed a molecule that stained with WC6-specific monoclonal antibody, showing that ruminant WC6 is an orthologue of DEC-205. Two-colour flow cytometry of mononuclear cells from afferent lymph draining cattle skin, and from blood, confirmed the high level of expression on large cells in lymph that were uniformly DC-LAMP positive and major histocompatibility complex class II positive. Within this DEC-205+ DC-LAMP+ population were subpopulations of cells that expressed the mannose receptor or SIRPalpha. The observations imply that DCs in afferent lymph are all DEC-205high, but not a uniform population of homogeneous mature DCs.

Amino Acid Sequence↗
PubMed2005

Transcription and RNA-processing in fission yeast mitochondria.

We systematically examined transcription and RNA-processing in mitochondria of the petite-negative fission yeast Schizosaccharomyces pombe. Two presumptive transcription initiation sites at opposite positions on the circular-mapping mtDNA were confirmed by in vitro capping of primary transcripts with guanylyl-transferase. The major promoter (Pma) is located adjacent to the 5'-end of the rnl gene, and a second, minor promoter (Pmi) upstream from cox3. The primary 5'-termini of the mature rnl and cox3 transcripts remain unmodified. A third predicted accessory transcription initiation site is within the group IIA1 intron of the cob gene (cobI1). The consensus promoter motif of S. pombe closely resembles the nonanucleotide promoter motifs of various yeast mtDNAs. We further characterized all mRNAs and the two ribosomal RNAs by Northern hybridization, and precisely mapped their 5'- and 3'-ends. The mRNAs have leader sequences with a length of 38 up to 220 nt and, in most instances, are created by removal of tRNAs from large precursor RNAs. Like cox2 and rnl, cox1 and cox3 are not separated by tRNA genes; instead, transcription initiation from the promoters upstream from rnl and cox3 compensates for the lack of tRNA-mediated 5'-processing. The 3'-termini of mRNAs and of SSU rRNA are processed at distinct, C-rich motifs that are located at a variable distance (1-15 nt) downstream from mRNA and SSU-rRNA coding regions. The accuracy of RNA-processing at these sites is sequence-dependent. Similar 3'-RNA-processing motifs are present in species of the genus Schizosaccharomyces, but not in budding yeasts that have functionally analogous A+T-rich dodecamer processing signals.

Base Sequence↗
PubMed1995

Expression and DNA-binding properties of the 14K carboxyl terminal fragment of Escherichia coli DNA topoisomerase I.

DNA sequence coding for the last 121 amino acids of Escherichia coli topoisomerase I was synthesized by PCR and cloned into a plasmid under the control of the T7 promoter. Induction of T7 RNA polymerase in E. coli carrying the plasmid clone resulted in over-expression of this C-terminal domain fragment previously shown to confer higher DNA binding affinity to the enzyme. Purification to homogeneity was achieved by phosphocellulose and single-stranded DNA agaraose chromatography. Direct interaction between this 14K domain and poly(dA) was demonstrated by UV spectroscopy. Noncovalent complexes formed between this fragment and oligo(dT) 8 and oligo(dT) 16 can also be trapped by photo-crosslinking.

Bacteriophage T7↗
PubMed2004

The genome sequence of Mycoplasma mycoides subsp. mycoides SC type strain PG1T, the causative agent of contagious bovine pleuropneumonia (CBPP).

Mycoplasma mycoides subsp. mycoidesSC (MmymySC)is the etiological agent of contagious bovine pleuropneumonia (CBPP), a highly contagious respiratory disease in cattle. The genome of Mmymy SC type strain PG1(T) has been sequenced to map all the genes and to facilitate further studies regarding the cell function of the organism and CBPP. The genome is characterized by a single circular chromosome of 1211703 bp with the lowest G+C content (24 mole%)and the highest density of insertion sequences (13% of the genome size)of all sequenced bacterial genomes. The genome contains 985 putative genes, of which 72 are part of insertion sequences and encode transposases. Anomalies in the GC-skew pattern and the presence of large repetitive sequences indicate a high genomic plasticity. A variety of potential virulence factors was identified, including genes encoding putative variable surface proteins and enzymes and transport proteins responsible for the production of hydrogen peroxide and the capsule, which is believed to have toxic effects on the animal.

Animals↗
PubMed1998

Overexpression, Purification and Characterization of Thermostable Catechol 2,3-dioxygenase.

Catechol 2,3-dioxygenase(EC 1.13.11.2) from Bacillus stearothermophilus has been shown to be highly thermostable. In order to obtain sufficient enzyme for its characterization, and to analyze the molecular basis of its intrinsic stability, the gene coding for this enzyme was sub-cloned and overexpressed in E. coli. After heat denaturation, ammonium sulfate fractionation, DEAE-52 ion-exchange chromatography and phenyl-Sepharose CL-4B hydrophobic chromatography, the enzyme was purified to homogeneity and the yield was 16%. The enzyme is a homotetramer. The molecular weight of each subunit is 36.4 kD as determined by SDS-PAGE. Using the tyrosine difference spectrum method, the extinction coefficient of the enzyme at 279.2 nm is estimated as 89(mmol/L)(-1).cm(-1), the optimum temperature and pH of the enzyme is 60 degrees and 8.0, respectively. The K(m) for catechol of the enzyme is 1.24x10(-5) mol/L, K(cat) is 24 000 min(-1). Acetone competitively inhibited the enzyme activity to catechol, with an inhibition constant K(i) of about 165 mmol/L. The thermostability of the enzyme is reflected by its unaltered catalytic activity over 1 h at 65 degrees. Irreversible thermal denaturation becomes significant between 70-80 degrees. THE pH stability of the enzyme and its resistance toward chemical denaturation such as urea and SDS confirm the intrinsic stability of the enzyme. It was also studied that the effect of different pHs on the molecular structure of the enzyme, using circular dichroism spectra and intrinsic fluorescence analysis.

Journal Article↗
PubMed2002

Sterol carrier protein-2: structure reveals function.

The multiple actions of sterol carrier protein-2 (SCP-2) in intracellular lipid circulation and metabolism originate from its gene and protein structure. The SCP-x/pro-SCP-2 gene is a fusion gene with separate initiation sites coding for 15-kDa pro-SCP-2 (no enzyme activity) and 58-kDa SCP-x (a 3-ketoacyl CoA thiolase). Both proteins share identical cDNA and amino acid sequences for 13-kDa SCP-2 at their C-termini. Cellular 13-kDa SCP-2 derives from complete, posttranslational cleavage of the 15-kDa pro-SCP-2 and from partial posttranslational cleavage of 58-kDa SCP-x. Putative physiological functions of SCP-2 have been proposed on the basis of enhancement of intermembrane lipid transfer (e.g., cholesterol, phospholipid) and activation of enzymes involved in fatty acyl CoA transacylation (cholesterol esters, phosphatidic acid) in vitro, in transfected cells, and in genetically manipulated animals. At least four important SCP-2 structural domains have been identified and related to specific functions. First, the 46-kDa N-terminal presequence present in 58-kDa SCP-x is a 3-ketoacyl-CoA thiolase specific for branched-chain acyl CoAs. Second, the N-terminal 20 amino acid presequence in 15-kDa pro-SCP-2 dramatically modulates the secondary and tertiary structure of SCP-2 as well as potentiating its intracellular targeting coded by the C-terminal peroxisomal targeting sequence. Third, the N-terminal 32 amino acids form an amphipathic a-helical region, one face of which represents a membrane-binding domain. Positively charged amino acid residues in one face of the amphipathic helices allow SCP-2 to bind to membrane surfaces containing anionic phospholipids. Fourth, the hydrophobic faces of the N-terminal amphipathic a helices along with beta strands 4, 5, and helix D form a ligand-binding cavity able to accommodate multiple types of lipids (e. g., fatty acids, fatty acyl CoAs, cholesterol, phospholipids, isoprenoids). Two-dimensional 1H-15N heteronuclear single quantum coherence spectra of both apo-SCP-2 and of the 1:1 oleate-SCP-2 complex, obtained at pH 6.7, demonstrated the homogenous formation of holo-SCP-2. While comparison of the apo- and holoprotein amide fingerprints revealed about 60% of the resonances remaining essentially unchanged, 12 assigned amide residues underwent significant chemical-shift changes upon oleic acid binding. These residues were localized in three regions: the juncture of helices A and B, the mid-section of the beta sheet, and the interface formed by the region of beta strands 4, 5, and helix D. Circular dichroism also showed that these chemical-shift changes, upon oleic acid binding, did not alter the secondary structure of SCP-2. The nuclear magnetic resonance chemical shift difference data, along with mapping of the nearby hydrophobic residues, showed the oleic acid-binding site to be comprised of a pocket created by the face of the beta sheet, helices A and B on one end, and residues associated with beta strands 4, 5, and helix D at the other end of the binding cavity. Furthermore, the hydrophobic nature of the previously ill-defined C-terminus suggested that these 20 amino acids may form a 'hydrophobic cap' which closes around the oleic acid upon binding. Thus, understanding the structural domains of the SCP-x/pro-SCP-2 gene and its respective posttranslationally processed proteins has provided new insights into their functions in intracellular targeting and metabolism of lipids.

Acetyl-CoA C-Acetyltransferase↗