ABSTRACT
Hydroxylapatite (also called hydroxyapatite), a form of calcium phosphate, can be used as a matrix for the chromatography of both proteins and nucleic acids. Protocols are provided for both standard low-pressure chromatography of a protein mixture using a hydroxylapatite column prepared in the laboratory, and an HPLC method, applicable to proteins and nucleic acids, that uses a commercially available column. Alternate protocols describe column chromatography using a step gradient or batch binding and step-gradient elution.
Subject(s)
Chromatography, High Pressure Liquid/methods , Durapatite/chemistry , Proteins/isolation & purification , Nucleic Acids/chemistry , Nucleic Acids/isolation & purification , Proteins/chemistryABSTRACT
A colorimetric assay for HIV proteinase using small protected peptide substrates is described. Substrates are cleaved to release N-terminal prolyl peptides which react with isatin to form a blue product which is measured spectrophotometrically. The assay is suitable for use with pure enzyme or crude extracts derived from genetically engineered Escherichia coli.
Subject(s)
HIV Protease/analysis , Oligopeptides/analysis , Amino Acid Sequence , Chromatography, High Pressure Liquid , Colorimetry/methods , Escherichia coli/enzymology , Isatin/chemistry , Kinetics , Molecular Sequence Data , Oligopeptides/chemical synthesis , Proline/analysis , Substrate SpecificityABSTRACT
Kinetic constants (Km,Kcat) are derived for the hydrolysis of a number of chromogenic peptide substrates by the aspartic proteinase from HIV-2. The effect of systematic replacement of the P2 residue on substrate hydrolysis by HIV-1 and HIV-2 proteinases is examined.
Subject(s)
Endopeptidases/metabolism , Gene Products, pol/metabolism , HIV-1/enzymology , HIV-2/enzymology , Amino Acid Sequence , HIV Protease , In Vitro Techniques , Kinetics , Molecular Sequence Data , Oligopeptides/metabolism , Protein Conformation , Structure-Activity Relationship , Substrate SpecificityABSTRACT
A series of peptide derivatives based on the transition-state mimetic concept has been designed that inhibit the proteinase from the human immunodeficiency virus (HIV). The more active compounds inhibit both HIV-1 and HIV-2 proteinases in the nanomolar range with little effect at 10 micromolar against the structurally related human aspartic proteinases. Proteolytic cleavage of the HIV-1 gag polyprotein (p55) to the viral structural protein p24 was inhibited in chronically infected CEM cells. Antiviral activity was observed in the nanomolar range (with one compound active below 10 nanomolar) in three different cell systems, as assessed by p24 antigen and syncytium formation. Cytotoxicity was not detected at 10 and 5 micromolar in C8166 and JM cells, respectively, indicating a high therapeutic index for this new class of HIV proteinase inhibitors.
Subject(s)
Antiviral Agents , Endopeptidases/metabolism , Gene Products, pol/metabolism , HIV-1/enzymology , HIV-2/enzymology , Peptides/pharmacology , Protease Inhibitors/pharmacology , Amino Acid Sequence , Cell Line , Drug Design , Gene Products, gag/metabolism , HIV Protease , HIV-1/drug effects , Molecular Sequence Data , Molecular Structure , Structure-Activity RelationshipABSTRACT
Kinetic constants were determined for the interaction of the HIV-2 aspartic proteinase with a synthetic substrate and a number of inhibitors at several pH values. Acetyl-pepstatin was more effective towards HIV-2 proteinase than the renin inhibitor, H-261; this effect is exactly the opposite from that observed previously for the proteinase from the HIV-1 AIDS virus.
Subject(s)
HIV-2/enzymology , Protease Inhibitors , Aspartic Acid Endopeptidases , Endopeptidases , Hydrogen-Ion Concentration , Kinetics , Molecular Weight , Protease Inhibitors/pharmacology , Substrate SpecificityABSTRACT
The cyclohexane triones are a novel group of synthetic antibacterial agents that are active against gram-positive bacteria, Haemophilus influenzae, and Mycobacterium smegmatis. In general, these compounds behaved in a manner similar to that of hexachlorophene, inhibiting the transport of low-molecular-weight hydrophilic substances into bacteria. Unlike cationic detergents, such as chlorhexidine, they did not cause disruption of the bacterial cytoplasmic membrane over a short time period. The most potent antibacterial cyclohexane trione studied had a reduced ability to inhibit solute transport in comparison with certain less active analogs. Cyclohexane triones may express more than a single type of antibacterial effect.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Cell Membrane/drug effects , Cyclohexanes/pharmacology , Cyclohexanones/pharmacology , Culture Media , Cytoplasm/drug effects , Membranes/drug effects , Microbial Sensitivity Tests , Pseudomonas aeruginosa/drug effects , Staphylococcus/drug effects , Staphylococcus aureus/drug effectsABSTRACT
The 5'-PAA and 5'-PFA phosphate esters of 5-bromo-2'-deoxyuridine (BUdR) were synthesized and their antiherpes virus activity was evaluated in vitro. Both compounds showed activity of the same order as the parent nucleoside, BUdR, against HSV-2 but were 4 to 12 times less potent against HSV-1. The 5'-PAA phosphate ester of BUdR (Ro 21-9875) was also active against varicella-zoster virus (VZV) and human cytomegalovirus (HCMV). The 5'-PAA phosphate ester of 5-bromovinyl-2'-deoxyuridine (BVdU) was also synthesized and showed good antiviral activity against HSV-1 only (ID50 = 1.3 mg/l). Further evaluation against selected mutants (TK- or PAAr) indicated a requirement for the expression of the virus-coded thymidine kinase (TK) for the antiviral activity of Ro 21-9875. Kinetic studies revealed non-competitive mixed inhibition of the viral enzyme by this compound. This suggests that it may have some intrinsic TK mediated activity though breakdown to its component parts is undoubtedly a significant contributing factor.
