ABSTRACT
A protein reacting with a monoclonal antibody against human placental diamine oxidase was purified from the specific granules of human neutrofil granulocytes using affinity chromatography on aminohexyl-divinylsulfonyl-agarose. The protein had an M(r) determined by SDS/PAGE, corresponding to diamine oxidase, but had other properties which indicated that it might be a different protein. A combination of protein chemical techniques, including N-terminal sequencing, identified the protein as lactoferrin, an iron-containing protein with an M(r) of approximately 800000, a high isoelectric point and ferroxidase activity. Purified commercial lactoferrin was shown to bind to aminohexyl-divinylsulfonyl-agarose, and to be eluted in a heterogenous way from the matrix by amines and salt. Alignment of the sequences of diamine oxidase and lactoferrin showed that they are similar, indicating a common ancestry for these two different classes of metallo-oxidases.
Subject(s)
Amine Oxidase (Copper-Containing)/chemistry , Lactoferrin/isolation & purification , Amine Oxidase (Copper-Containing)/immunology , Amines/metabolism , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Blotting, Western , Chromatography, Affinity , Chromatography, Ion Exchange , Diamines/metabolism , Electrophoresis, Agar Gel , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Humans , Lactoferrin/chemistry , Molecular Sequence Data , Neutrophils/chemistry , Placenta/enzymology , Sepharose/analogs & derivatives , Sepharose/metabolism , Sequence Alignment , Sequence AnalysisABSTRACT
We have developed a program, HookSpace, which provides a simplistic approach to assessing the diversity of molecular databases. The spatial relationship between pairs of intramolecular functional groups can be analysed in a variety of ways to provide both qualitative and quantitative measures of diversity. Results are described and contrasted for two commercially available databases and a combinatorial library of benzodiazepam derivatives. HookSpace highlights the main differences in molecular content of these data sets.
Subject(s)
Databases, Factual , Molecular Structure , Software , Crystallography, X-Ray , Diazepam/analogs & derivatives , Diazepam/chemistry , Drug Design , Ligands , Models, MolecularABSTRACT
Denatured and reduced N-terminal extended insulin-like growth factor-1 (AE-IGF-1) was purified from Escherichia coli extracts and subjected to in vitro folding. The renaturation process was shown to be a function of the redox potential of the solution. Folding by different methods had no significant effect on the renaturation. A maximal yield of 60% (w/w) was obtained. The folded AE-IGF-1 was enzymatically converted to IGF-1. The major by-product (20% w/w) was identified as scrambled IGF-1. Enzymatic digestion at alkaline and acidic pH suggested two possible disulphide bond arrangements; (i) Cys6-Cys47, Cys18-Cys61, Cys48-Cys52; or (ii) Cys6-Cys52, Cys18-Cys61, Cys47 and Cys48 being in their reduced forms. Energy minimization and molecular modelling suggested that the scrambled IGF-1, having reduced cysteines at positions 47 and 48, was the energetically most stable conformation of the two.
Subject(s)
Insulin-Like Growth Factor I/metabolism , Protein Folding , Amino Acid Sequence , Aminopeptidases/pharmacology , Binding, Competitive , Circular Dichroism , Cysteine , Endopeptidases/genetics , Humans , Insulin-Like Growth Factor I/analogs & derivatives , Insulin-Like Growth Factor I/drug effects , Insulin-Like Growth Factor I/genetics , Models, Molecular , Molecular Sequence Data , Molecular Weight , Peptide Mapping , Protein Denaturation , Protein Engineering , Receptor, IGF Type 1/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
True lipases attach triacylglycerols and act at an oil-water interface; they constitute a ubiquitous group of enzymes catalysing a wide variety of reactions, many with industrial potential. But so far the three-dimensional structure has not been reported for any lipase. Here we report the X-ray structure of the Mucor miehei triglyceride lipase and describe the atomic model obtained at 3.1 A resolution and refined to 1.9 A resolution. It reveals a Ser..His..Asp trypsin-like catalytic triad with an active serine buried under a short helical fragment of a long surface loop.
Subject(s)
Lipase , Mucor/enzymology , Serine Endopeptidases , Amino Acid Sequence , Binding Sites , Disulfides , Molecular Sequence Data , Molecular Structure , Peptide Fragments , Protein Conformation , X-Ray DiffractionABSTRACT
Neural networks provide a basis for semiempirical studies of pattern matching between the primary and secondary structures of proteins. Networks of the perceptron class have been trained to classify the amino-acid residues into two categories for each of three types of secondary feature: alpha-helix or not, beta-sheet or not, and random coil or not. The explicit prediction for the helices in rhodopsin is compared with both electron microscopy results and those of the Chou-Fasman method. A new measure of homology between proteins is provided by the network approach, which thereby leads to quantification of the differences between the primary structures of proteins.
