ABSTRACT
Giant taro (Alocasia macrorrhiza) contains a protein which inhibits both trypsin and chymotrypsin. This trypsin/chymotrypsin inhibitor exists as a dimer of two identical monomers each with slight polymorphism and is an attractive candidate for conferring insect resistance in transgenic plants. The 184 amino-acid sequence (molecular mass of 19774 Da for the Met-24, Glu-50 form) has been determined and is compared with those of other Kunitz-type trypsin, chymotrypsin and subtilisin inhibitors. There appears to be greater 'homology' between the giant taro inhibitor and those inhibitors from other monocotyledons than inhibitors from dicotyledons. The P1 loop region is different from that of other Kunitz-type inhibitors and contains a sequence Leu-Ala-Phe-Phe-Pro at residues 56-60. This section of sequence differs only by a Leu/Ile replacement to a tight binding inhibitor of neutrophil elastase, recently produced by genetic engineering. The most likely candidate for the P1 residue in the giant taro trypsin/chymotrypsin inhibitor is Leu-56.
Subject(s)
Chymotrypsin/antagonists & inhibitors , Plant Proteins/chemistry , Trypsin Inhibitors/chemistry , Amino Acid Sequence , Molecular Sequence Data , Sequence Alignment , Trypsin Inhibitors/pharmacologyABSTRACT
The enzyme methanol dehydrogenase (EC 1.1.99.8) from Hyphomicrobium X was used in an attempt to develop a rapid colorimetric test for methanol. The enzyme was stabilized for storage by lyophilization in the presence of the disaccharide trehalose. It was found that the enzyme retained significantly greater activity in the dried state with trehalose than without. The enzyme was partially purified by ammonium sulphate fractionation, after which it was found to be more stable in solution at pH 9 than at pH 7. A procedure is given which involves mixing a defined amount of enzyme with the methanol-containing water together with phenazine methosulphate (PMS), 2-6-dichlorophenol-indophenol (DCPIP) and cyanide, and observing the resultant colour change from blue to yellow if methanol is present. The sensitivity of the procedure is such that 9 mg L-1 of methanol can be readily detected.
Subject(s)
Alcohol Oxidoreductases/metabolism , Bacteria/enzymology , Bacterial Proteins/metabolism , Methanol/analysis , Trehalose/pharmacology , 2,6-Dichloroindophenol , Alcohol Oxidoreductases/drug effects , Alcohol Oxidoreductases/isolation & purification , Bacterial Proteins/drug effects , Bacterial Proteins/isolation & purification , Buffers , Colorimetry , Freeze Drying , Methanol/metabolism , Nitroblue TetrazoliumABSTRACT
A purification procedure has been developed by which urease activity in extracts from the cyanobacterium Anabaena cylindrica was enriched 500-fold. The procedure involves MgSO4 precipitation at 55 degrees C and chromatography on hydroxylapatite and diethylaminoethyl sephadex. Its molecular weight was measured by sedimentation equilibrium in an airfuge to be 197,000 +/- 2000 with an estimated subunit molecular weight of 32,000 as determined by polyacrylamide gel electrophoresis. The pH- and temperature-dependence of the enzyme were determined and the activity found to be optimal at pH 8 and 30 degrees C, respectively. The concentration-dependence of the activation of the enzyme by Mg++ was measured, as were the effects on activity of a range of other metal ions.
Subject(s)
Anabaena/enzymology , Urease/isolation & purification , Chromatography, Liquid , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Hydrogen-Ion Concentration , Magnesium/metabolism , Molecular Weight , Temperature , Urease/chemistry , Urease/metabolismABSTRACT
A new series of expression vectors that direct high-level overproduction of gene products in Escherichia coli is described. All contain strong bacteriophage lambda promoters, PR and PL, arranged in tandem so that both promote transcription into genes inserted into or between unique restriction sites. The vectors also direct expression of the lambda cI857 gene (from its natural promoter, PM), which enables their use in any E. coli host strain to effect controlled expression by shifting the temperature of cultures from 30 to 42 degrees C. The vectors pCE30, pND201, pPT150 and pMA200U are derivatives of the high-copy-number plasmid pUC9. Vector pCE33 is an analogous derivative of the heat-inducible runaway-replication plasmid, pMOB45, and directs overproduction of proteins by virtue of increase in both gene dosage and transcription following treatment at 42 degrees C. The vectors pND201 and pPT150 bear a ribosome-binding site (RBS) perfectly complementary to the 3' end of E. coli 16-S rRNA a few bp upstream from a unique HpaI site. Ways in which they may be used to improve the efficiency of translation of mRNA by substitution of a natural RBS with selection for optimal spacing from an ATG (or GTG) start codon are described. The phagemid vector pMA200U is a direct analog of pCE30 designed to facilitate preparation of single-stranded DNA templates for use in oligodeoxyribonucleotide-directed mutagenesis of overexpressed genes.
