ABSTRACT
One novel Kunitz BPTI-like peptide designated as BBPTI-1, with chymotrypsin inhibitory activity was identified from the venom of Burmese Daboia russelii siamensis. It was purified by three steps of chromatography including gel filtration, cation exchange and reversed phase. A partial N-terminal sequence of BBPTI-1, HDRPKFCYLPADPGECLAHMRSF was obtained by automated Edman degradation and a Ki value of 4.77nM determined. Cloning of BBPTI-1 including the open reading frame and 3' untranslated region was achieved from cDNA libraries derived from lyophilized venom using a 3' RACE strategy. In addition a cDNA sequence, designated as BBPTI-5, was also obtained. Alignment of cDNA sequences showed that BBPTI-5 exhibited an identical sequence to BBPTI-1 cDNA except for an eight nucleotide deletion in the open reading frame. Gene variations that represented deletions in the BBPTI-5 cDNA resulted in a novel protease inhibitor analog. Amino acid sequence alignment revealed that deduced peptides derived from cloning of their respective precursor cDNAs from libraries showed high similarity and homology with other Kunitz BPTI proteinase inhibitors. BBPTI-1 and BBPTI-5 consist of 60 and 66 amino acid residues respectively, including six conserved cysteine residues. As these peptides have been reported to have influence on the processes of coagulation, fibrinolysis and inflammation, their potential application in biomedical contexts warrants further investigation.
Subject(s)
Chymotrypsin/antagonists & inhibitors , Peptides/pharmacology , Serine Proteinase Inhibitors/genetics , Serine Proteinase Inhibitors/isolation & purification , Viper Venoms/chemistry , Amino Acid Sequence , Animals , Chymotrypsin/metabolism , Cloning, Molecular , Intercellular Signaling Peptides and Proteins , Molecular Sequence Data , Peptides/chemistry , Peptides/genetics , Peptides/isolation & purification , Daboia , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacology , Viper Venoms/genetics , Viper Venoms/isolation & purification , Viper Venoms/pharmacologyABSTRACT
Two trypsin inhibitors and one chymotrypsin inhibitor from Chinese Daboia russellii siamensis venom, denoted as CBPTI-1, CBPTI-2 and CBPTI-3 were purified, characterized and cloned from lyophilized venom-derived cDNA libraries. The N-terminus of CBPTI-1 was modified and not amenable to Edman degradation sequencing, however an internal partial sequence was found to be SGRCRGHLRRIYYNPDSNKCE. The N-termini of CBPTI-2 and CBPTI-3 were unmodified and their partial sequences were established as HDRPTFCNLAPESGRCRAH and HDRPKFCYLPADPGECMAYIRSFYYDS respectively. From cloning studies CBPTI-1 was found to consist of 66 amino acid residues, while CBPTI-2 and CBPTI-3 precursors consist of 60 amino acid residues, including 6 cysteine residues. Another cDNA sequence (CBPTI-4) was also obtained. Alignment of cDNA sequences showed that CBPTI-3 exhibited similar sequence homology to CBPTI-4 cDNA except for an 8 nucleotide deletion in the open-reading frame. CBPTI-1 and CBPTI-2 were demonstrated to be potent trypsin inhibitors, but were also shown to be effectively potent in chymotrypsin inhibition. The K(i) values of CBPTI-1 and CBPTI-2 for trypsin inhibition were 4.07 × 10(-7) M and 6.66 × 10(-7) M, respectively, and they were non-competitive in their activity. CBPTI-3 showed chymotrypsin inhibition activity with a K(i) value of 2.55 × 10(-9) M, but did not show trypsin inhibitor activity.
Subject(s)
Chymotrypsin/antagonists & inhibitors , Daboia/metabolism , Elapid Venoms/chemistry , Peptide Fragments/metabolism , Trypsin Inhibitors , Trypsin/metabolism , Amino Acid Sequence , Animals , Cattle , Chromatography, High Pressure Liquid , Chymotrypsin/analysis , Chymotrypsin/metabolism , Cloning, Molecular , Elapid Venoms/genetics , Elapid Venoms/metabolism , Molecular Sequence Data , Peptide Fragments/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Trypsin/analysis , Trypsin Inhibitors/genetics , Trypsin Inhibitors/isolation & purification , Trypsin Inhibitors/metabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the incidence of hot spot mutation of PDS gene by genetic screening testing method in Chifeng City, Inner Mongolia. The feasibility and effectiveness of genetic screening method in finding enlarged vestibular aqueduct syndrome were confirmed by temporal bone CT scan.</p><p><b>METHODS</b>DNA were extracted from peripheral blood of 141 students of Chifeng Deaf and Dumb school. PDS IVS7-2 A-G mutation, the most common PDS mutation in Chinese population, was analyzed by direct sequencing for PDS exon 7, exon 8 with intron 7. The individuals found with homozygous or heterozygous PDS IVS7-2 A-G mutation were given further temporal CT scan, ultrasound scan of thyroid and thyroid hormone assays. The results of PDS genetic screening and temporal bone CT scan were compared with each other.</p><p><b>RESULTS</b>The sequencing results revealed twenty cases carrying PDS IVS7-2 A-G mutation, of whom nine cases were homozygous mutation and eleven cases were heterozygous mutation. Eighteen cases underwent temporal bone CT scan except two cases that left the school due to other health problem. Sixteen cases were confirmed to be enlarged vestibular aqueduct syndrome (EVAS) by CT scan and the shape and function of thyroid were clinically normal by ultrasound scan of thyroid and thyroid hormone assays, respectively.</p><p><b>CONCLUSIONS</b>The patients suffered from EVAS can be diagnosed by the screening for the PDS hot spot mutation which has unique advantage in epidemiologic study in large scale deaf population. The preliminary data of this study suggested relatively high incidence of EVAS in Chifeng area.</p>
Subject(s)
Adolescent , Child , Child, Preschool , Female , Humans , Young Adult , China , Genetic Testing , Hearing Loss , Genetics , Membrane Transport Proteins , Genetics , Point Mutation , Syndrome , Vestibular Aqueduct , Pathology , Vestibular Diseases , GeneticsABSTRACT
The importance of cellulase as a means for the efficient utilization of abundant cellulose resources in the world has been well recognized. Many researchers devote themselves to studying the mechanism of the action of cellulase to cellulose so that such expensive enzyme can be used much more widely. The first step is to obtain cellulase of high purity. So purification of cellulase is the key point in this field. However, the major problem in isolation is that cellulase is a complicated enzyme system and needs too many steps for separation, and that every cellulase needs special purification processing which cannot be used for the others. A novel method for the separation of the cellulase from crude extraction of Aspergillus niger with normal qualitative filter paper processed by 5 mol/L sodium hydroxide without precipitation and desalting steps was developed. Further purification of the cellulase was achieved by using an anion-exchange column of POROS 20HQ. The cellulase purified was identified as a new endoglucanase that had relatively high endurance to pH and temperature. Its relative molecular mass was estimated to be 60,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme exhibited very high activity towards carboxymethyl cellulose (CMC) with specific activity of 350 U.mg-1 and the recovery of activity of 9.7%. Its optimum pH and temperature were 4.0 and 70 degrees C, respectively. This is a simple, rapid and efficient method for purifying cellulase with high activity.
