Zinc inhibition of renin and the protease from human immunodeficiency virus type 1

We report here for the first time that Zn2+ is an effective inhibitor of renin and the protease from HIV-1, two aspartyl proteinases of considerable physiological importance. Inhibition of renin is noncompetitive and is accompanied by binding of 1 mol of Zn2+/mol of enzyme. Depending on the substrate, inhibition of the HIV protease by Zn2+ can be either competitive or noncompetitive, but in neither case is loss of activity due to disruption of the protease dimer. Inhibition of both enzymes is first order with respect to Zn2+ and is rapidly reversed by addition of EDTA. Ki values are strongly pH dependent and optimal in the range of 20 microM at or above pH 7. All of the data in hand suggest that the inhibitory effect of Zn2+ is a consequence of its binding at, or near, the active-site carboxyl groups of these aspartyl proteinases. This inhibition of the viral enzyme may help to explain some of the beneficial effects seen in AIDS patients who have received Zn2+ therapy.     

Calcium-free calmodulin is a substrate of proteases from human immunodeficiency viruses 1 and 2

Calcium-free calmodulin-(CaM) is rapidly hydrolyzed by proteases from both human immunodeficiency viruses (HIV) 1 and 2. Kinetic analysis reveals a sequential order of cleavage by both proteases which initiates in regions of the molecule known from X-ray crystallographic analysis of Ca2+/CaM to be associated with calcium binding. Although HIV-1 and HIV-2 proteases hydrolyze two bonds in common, the initial site of cleavage required for subsequent events differs in each case. The first bond hydrolyzed by the HIV-1 protease is the Asn-Tyr linkage in the sequence, -N-I-D-G-D-G-Q-V-N-Y-E-E-, found in the fourth calcium binding loop. In contrast, it is an Ala-Ala bond in the third calcium loop, -D-K-D-G-N-G-Y-I-S-A-A-E-, that is first hydrolyzed by the HIV-2 enzyme, followed in short order by cleavage of the same Asn-Tyr linkage described above. Thereafter, both enzymes proceed to hydrolyze additional peptide bonds, some in common, some not. Considerable evidence exists that inhibitors are bound to the protease in an extended conformation and yet all of the cleavages we observed occur within, or at the beginning of helices in Ca2+/CaM, regions that also appear to be insufficiently exposed for protease binding. Molecular modeling studies indicate that CaM in solution must adopt a conformation in which the first cleavage site observed for each enzyme is unshielded and extended, and that subsequent cleavages involve further unwinding of helices.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specificity and inhibition of proteases from human immunodeficiency viruses 1 and 2

Highly purified, recombinant preparations of the virally encoded proteases from human immunodeficiency viruses (HIV) 1 and 2 have been compared relative to 1) their specificities toward non-viral protein and synthetic peptide substrates, and 2) their inhibition by several P1-P1' pseudodipeptidyl-modified substrate analogs. Hydrolysis of the Leu-Leu and Leu-Ala bonds in the Pseudomonas exotoxin derivative, Lys-PE40, is qualitatively the same for HIV-2 protease as published earlier for the HIV-1 enzyme (Tomasselli, A. G., Hui, J. O., Sawyer, T. K., Staples, D. J., FitzGerald, D. J., Chaudhary, V. K., Pastan, I., and Heinrikson, R. L. (1990) J. Biol. Chem. 265, 408-413). However, the rates of cleavage at these two sites are reversed for the HIV-2 protease which prefers the Leu-Ala bond. The kinetics of hydrolysis of this protein substrate by both enzymes are mirrored by those obtained from cleavage of model peptides. Hydrolysis by the two proteases of other synthetic peptides modeled after processing sites in HIV-1 and HIV-2 gag polyproteins and selected analogs thereof demonstrated differences, as well as similarities, in selectivity. For example, while the two proteases were nearly identical in their rates of cleavage of the Tyr-Pro bond in the HIV-1 gag fragment, Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val, the HIV-1 protease showed a 64-fold enhancement over the HIV-2 enzyme in hydrolysis of a Tyr-Val bond in the same template. Accordingly, the HIV-2 protease appears to have a different specificity than the HIV-1 enzyme; it is better able to hydrolyze substrates with small amino acids in P1 and P1', but is variable in its rate of hydrolysis of peptides with bulky substituents in these positions. In addition to these comparisons of the two proteases with respect to substrate specificity, we present inhibitor structure-activity data for the HIV-2 protease. Relative to P1-P1' statine or Phe psi [CH2N]Pro-modified pseudopeptidyl inhibitors, compounds having Xaa psi[CH(OH)CH2]Yaa inserts were found to show significantly higher affinities to both enzymes, generally binding from 10 to 100 times stronger to HIV-1 protease than to the HIV-2 enzyme. Molecular modeling comparisons based upon the sequence homology of the two enzymes and x-ray crystal structures of HIV-1 protease suggest that most of the nonconservative amino acid replacements occur in regions well outside the catalytic cleft, while only subtle structural differences exist within the active site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Brain Ligatin: A Membrane Lectin That Binds Acetylcholinesterase

Ligatin, a lectin that recognizes phosphorylated sugars, has been demonstrated in mammalian tissues to bind specific hydrolases to cell surfaces. Ligatin exists as a filament that can be released from membranes still complexed with its bound hydrolases by treatment of membrane preparations with CaCl₂ and/or pH 8.0. The ligatin-hydrolase complexes subsequently can be dissociated with ethyleneglycol-bis(β-amino-ethyl ether) N, N′-tetraacetic acid, resulting in a concurrent depolymerization of the ligatin filament. From membrane preparations of cerebrum, this procedure solubilized ligatin and a membrane-bound acetylcholinesterase (EC 3.1.1.7). Binding of the cosolubilized acetylcholinesterase to ligatin could be demonstrated in vitro by affinity chromatography using the immobilized lectin. Ligatin-hydrolase complexes have been shown to be dissociated by specific phosphorylated sugars (mannose 6-phosphate and glucose 1-phosphate). These sugars were also effective in eluting bound brain acetylcholinesterase from ligatin affinity columns. Analysis of labeled glycitols produced by tritiated borohydride reduction confirmed the presence of phosphorylated sugars on the ligatin-cosolubilized material from brain.     

Lipid-polyethylene glycol interactions: I. Induction of fusion between liposomes

Summary Fusion between unilamellar vesicles of both egg phosphatidylcholine and bovine phosphatidylserine was induced by polyethylene glycol. Aggregation and fusion events were monitored by electron microscopy and turbidity measurements. The threshold concentration of polyethylene glycol for aggregation and fusion is found to be independent of lipid concentration. Typically, aggregation of phosphatidylcholine vesicles starts at 2.5% (wt/wt) polyethylene glycol, but fusion is not significant until the polyethylene glycol concentration reaches 35%. Multilamellar vesicles were formed as a result of fusion.Abbreviations PEG Polyethylene glycol - IMP Intramembranous particle - PC Phosphatidylcholine - PS Phosphatidylserine - SUV Small unilamellar vesicles - MLV Multilamellar vesicles - DPPC Dipalmitoyl phosphatidylcholine - DSC Differential scanning calorimetry     

Lipid-polyethylene glycol interactions: II. Formation of defects in bilayers

Summary Polyethylene glycol, a known cell fusogen, is found to induce the formation of structural defects in egg phosphatidylcholine multilamellar vesicles, as shown by freeze-fracture microscopy.³¹P NMR spectra of these vesicles reveal the existence of a nonbilayer (isotropic) phase. The observed disruption in the bilayers is believed to be associated with an intermediate stage of membrane fusion.Abbreviations PEG Polyethylene glycol - IMP Intramembranous particle - PC Phosphatidylcholine - PS Phosphatidylserine - SUV Small unilamellar vesicles - MLV Multilamellar vesicles - DPPC Dipalmitoyl phosphatidylcholine - DSC Differential scanning calorimetry - DMPC Dimyristoylphosphatidylcholine - T _c Phase transition temperature     

The Quaternary Structure of Chicken Acetylcholinesterase and Butyrylcholinesterase; Effect of Collagenase and Trypsin

Acetylcholinesterase (EC 3.1.1.7.; AChE) and butyrylcholinesterase (EC 3.1.1.8.; BuChE) from chicken muscle exist as sets of structurally homologous forms with very similar properties. The collagenase sensitivity and aggregation properties of the 'heavy' forms of both enzymes indicate that they possess a collagen-like tail, and their stepwise dissociation by trypsin confirms that they correspond to triple (A12) and double (A8) collagen-tailed tetramers. In addition to this dissociating effect, trypsin digests an important fraction of the catalytic units of AChE, in a progressive manner, removing as much as 30% of the enzyme's mass, without inactivation of the tetramers and of the tailed molecules. The trypsin-modified AChE forms closely resemble the corresponding mammalian AChE forms in their hydrodynamic properties. It is not known whether the trypsin-digestible peptides, which do not appear to be involved in the ionic or hydrophobic interactions of the enzymes, are a fragment of the catalytic subunit or whether they constitute distinct polypeptides.     

Postsynaptic effects of crotoxin and of its isolated subunits.

Crotoxin is a potent neurotoxin from the venom of Crotalus durissus terrificus. It is composed of two subunits: a basic phospholipase A2 with low toxicity (component B) and an acidic protein seemingly devoid of intrinsic biological activity (component A). Crotoxin and its isolated phospholipase subunit block the depolarisation caused by cholinergic agonists on the isolated electroplaque from Electrophorus electricus. The other component, which is inactive when applied alone, enhances the pharmacological activity of the phospholipase when the two components are used together. Crotoxin also blocks the increase of 22Na+ efflux caused by carbamylcholine from excitable microsacs prepared from Torpedo marmorata electric organ. Crotoxin therefore acts postsynaptically, but does not interfere with the binding of alpha-toxin from Naja nigricollis to the nicotinic cholinergic receptor site. Instead, like local anesthetics, it stabilizes a desensitized form of the acetylcholine receptor characterized by its high affinity for agonists. The phospholipase component B binds in a non-saturable manner to receptor-rich membranes. In contrast, component A does not bind to acetylcholine receptor-rich membranes, but completely prevents the non-saturable binding of component B. When the two components are applied together, a saturable binding of the latter is observed with the acetylcholine receptor-rich membranes.     

Modifiable chromatophore proteins in photosynthetic bacteria.

The chromatophores of Chromatium vinosum, as well as six other photosynthetic bacteria, contained two or more proteins which were insoluble when heated in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (beta-ME). When the chromatophores were dissolved at room temperature in SDS-beta-ME, these proteins were present in the SDS-polyacrylamide gel electrophoresis profiles, but when the samples were dissolved at 100 degrees C, they were absent or considerably diminished. When one-dimensional gels of chromatophores solubilized at room temperature were soaked in the SDS-beta-ME solution and heated to 100 degrees C and the gels were run in a second dimension, the proteins became immobilized in the original first-dimension gel, where they could be detected by staining. The two major proteins so affected in C. vinosum had apparent molecular weights of 28,000 and 21,000. The chromatophores of several other photosynthetic bacteria also contained predominant proteins between 30,000 and 19,000 molecular weight, which became insoluble when heated in the presence of SDS and beta-ME. In at least two of the species examined, these appeared to be reaction center proteins. The conditions causing the proteins to become insoluble were complex and involved temperature, SDS concentration, and the presence of sulfhydryl reagents. The chromatophores of four of the Chromatiaceae species and two strains of one of the Rhodospirillaceae species examined had a protein-pigment complex that was visible in SDS-polyacrylamide gel profiles of samples dissolved at room temperature but was absent in samples dissolved at 100 degrees C.