John M. Walker

Antibacterial activity from marine microalgae in laboratory culture

Culture supernatants and methanolic and hexane extracts of 132 marine microalgae have been screened against six strains of bacteria. In vitro antibacterial activity was detected in 28 of the organic solvent extracts, primarily those of the hexane. The extracts were most effective against Staphylococcus aureus and to a lesser extent Bacillus subtilis. No antimicrobial activity was detected in the culture supernatants and no extract inhibited the growth of Escherichia coli, Streptococcus faecalis, Klebsiella pneumoniae or Pseudomonas aeruginosa.

Inhibitors of the Na+ K+-atpase

Abstract 1. The major ionmotive ATPase, in animal cells, is the Na+, K+-ATPase or sodium pump. 2. This membrane bound enzyme is responsible for the translocation of Na+ ions and K+ ions across the plasma membrane, an active transport mechanism that requires the expenditure of the metabolic energy stored within the ATP molecule. 3. This ubiquitous enzyme controls directly or indirectly many essential cellular functions, such as, cell volume, free calcium concentration and membrane potential. 4. It is, therefore, apparent that alterations in its regulation may play key roles in pathological processes.

Proteolytic enzymes for peptide production.

1. I n t r o d u c t i o n There are three main reasons why a protein chemist might wish to cleave a protein of interest into peptide fragments. The first reason is to generate, by extensive proteolysis, a large number of relatively small (5-20 residues) peptides either for peptide mapping (see vol. 1, Chapter 5) or for purification and subsequent manual sequence determination by the dansyl-Edman method (see vol. 1, Chapter 24). The second reason is to generate relatively large peptides (50-150 residues) by limited proteolysis for automated sequence analysis, such as with the gas-phase sequencer. The third reason is to prepare, again by limited proteolysis, specific fragments for studies relating structure to function. In each case, the specificity of the enzyme used to generate the peptides is a prime consideration, since the aim is to provide high yields of discrete fragments. It can be appreciated that significantly <100% cleavage at some or all of the cleavage sites on the protein being digested will generate a far more complex mixture of a larger number of polypeptides, each in relatively low yield. It is for this reason that enzymes of high specificity, such as trypsin, which cleaves at the C-terminal side of arginine and lysine residues, are mainly used for peptide production. However, other proteases with considerably less specificity have also found use in peptide production, particularly when limited proteolysis is being used, or where native protein is used as the substrate when only a limited number of susceptible peptide

Proteinase K (EC 3.4.21.14)

The original publication is available at www.springerlink.com Copyright Humana Press [Full text of this article is not available in the UHRA]

The Dansyl-Edman Method for Peptide Sequencing

: The dansyl-Edman method for peptide sequencing uses the Edman degradation (see Chapter 26 ) to sequentially remove amino acids from the N-terminus of a peptide. Following the cleavage step of the Edman degradation, the thiazolinone derivative is extracted with an organic solvent and discarded. This contrasts with the direct Edman degradation method ( Chapter 26 ), where the thiazolinone is collected, converted to the more stable PTH derivative, and identified. Instead, a small fraction ( approximately 5%) of the remaining peptide is taken and the newly liberated N-terminal amino acid determined in this sample by the dansyl method (see Chapter 23 ). Although the dansyl-Edman method results in successively less peptide being present at each cycle of the Edman degradation, this loss of material is more than compensated for by the fact that the dansyl method for identifying N-terminal amino acids is about one hundred times more sensitive than methods for identifying PTH amino acids. The dansyl-Edman method described here was originally introduced by Hartley (1).

High-throughput microtiter plate-based chromogenic assays for glycosidase inhibitors

Rapid microtiter plate-based colorimetric assays have been developed that allow the ccreening of large numbers of samples for the presence of inhibitors of α-glucosidase, α-amylase, and β-galactosidase. The assays are particularly useful for screening large numbers of microbial culture filtrates.

The Dansyl Method for Identifying N -Terminal Amino Acids

The reagent l-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride, DNS-C1) reacts with the free amino groups of peptides and proteins as shown in Fig. 1. Total acid hydrolysis of the substituted peptide or protein yields a mixture of free amino acids plus the dansyl derivative of the N-terminal amino acid, the bond between the dansyl group and the N-terminal amino acid being resistant to acid hydrolysis. The dansyl amino acid is fluorescent under UV light and is identified by thin-layer chromatography on polyamide sheets. This is an extremely sensitive method for identifying amino acids and in particular has found considerable use in peptide sequence determination when used in conjunction with the Edman degradation (see Chapter 24 ). The dansyl technique was originally introduced by Gray and Hartley (1), and was developed essentially for use with peptides. However, the method can also be applied to proteins (see Note No. 12). Fig. 1. Reaction sequence for the labeling of N-terminal amino acids with dansyl chloride.

Production of Protein Hydrolysates Using Enzymes

Traditionally, protein hydrolysates for amino acid analysis are produced by hydrolysis in 6 N HCl. However, this method has the disadvantage that tryptophan is totally destroyed, serine and threonine partially (5–10%) destroyed, and most importantly, asparagine and glutamine are hydrolyzed to the corresponding acids. Digestion of the protein/peptide with enzymes to produce protein hydrolysate overcomes these problems, and is particularly useful when the concentration of asparagine and glutamine is required. For peptides less than about 35 residues in size, complete digestion can be achieved by digestion with aminopeptidase M and prolidase. For larger polypeptides and proteins, an initial digestion with the nonspecific protease Pronase is required, followed by treatment with aminopeptidase M and prolidase. Since it is important that all enzymes have maximum activity, the following sections will discuss the general characteristics of these enzymes.

Purification of cloned trypanosomal calmodulin and preliminary NMR studies

Cloned trypanosomal calmodulin was expressed in Escherichia coli and purified to homogeneity using hydrophobic interaction chromatography on phenyl-Sepharose. The purified protein was subjected to NMR analysis which allows detailed changes to be observed when, firstly, calcium, and secondly, the drug calmidazolium bind. These spectral changes are the result of conformational changes in the protein and proximity effects due to the drug.

Synthesis and analysis of the enantiomers of calmidazolium, and a 1H NMR demonstration of a chiral interaction with calmodulin

Calmidazolium [R24571, 1-[bis(4-chlorophenyl)methyl]-3-[2-(2,4-dichlorophenyl)-2-[(2,4- dichlorophenyl)methoxy]ethyl]-1H-imidazolium chloride] is a potent calmodulin inhibitor. This paper describes the synthesis and properties of the enantiomers of calmidazolium from the enantiomers of miconazole [1(N)-(2-(2,4-dichlorobenzyloxy)-2-(2,4 dichlorophenyl))-ethyl imidazole], prepared from the racemate by chiral preparative scale high performance liquid chromatography. Overlap between ligand and protein resonances in the aromatic region of the 1H NMR spectrum of the calmidazolium-calmodulin complexes has been obviated by preparation of the protein with all of its nine phenylalanine rings deuterated (Phe-d5 calmodulin). This has been accomplished by the overexpression of calmodulin derived from Trypanosoma brucei rhodiesiense in E. coli in a medium supplemented with ring-deuterated phenylalanine. The kinetics of binding of each enantiomer are slow on the 1H NMR time scale as judged by the behaviour of the H2 resonance of Histidine-107, which is clearly visible under the sample conditions used. The aromatic spectral regions of the protein-bound (+) and (-) enantiomers contrast strikingly, reflecting differences in bound environment and/or conformation.