Ilene M. Reardon

Structural and functional properties of a phospholipase A2 purified from an inflammatory exudate

The cell-free supernatant of sterile inflammatory peritoneal exudates contains a phospholipase A2 that participates in the digestion of Escherichia coli killed by polymorphonuclear leukocytes or by the purified bactericidal/permeability increasing protein (BPI) of these cells. This phospholipase A2 has been purified, and the sequence of the NH2-terminal 39 amino acids has been determined and compared with sequences of both BPI-responsive and BPI-nonresponsive phospholipases A2 from snake venoms and mammalian pancreas. The high concentration and location of basic residues in the NH2-terminal region is a common feature of BPI-responsive phospholipases A2 and may characterize those phospholipases A2 participating in inflammatory events.

Predicting human immunodeficiency virus protease cleavage sites in proteins by a discriminant function method.

Based on the sequence‐coupled (Markov chain) model and vector‐projection principle, a discriminant function method is proposed to predict sites in protein substrates that should be susceptible to cleavage by the HIV‐1 protease. The discriminant function is defined by Δ = ϕ+ – ϕ−, where ϕ+ and ϕ− are the cleavable and noncleavable attributes for a given peptide, and they can be derived from two complementary sets of peptides, S+ and S−, known to be cleavable and noncleavable, respectively, by the enzyme. The rate of correct prediction by the method for the 62 cleavable peptides and 239 noncleavable peptides in the training set are 100 and 96.7%, respectively. Application of the method to the 55 sequences which are outside the training set and known to be cleaved by the HIV‐1 protease accurately predicted 100% of the peptides as substrates of the enzyme. The method also predicted all but one of the sites hydrolyzed by the protease in native HIV‐1 and HIV‐2 reverse transcriptases, where the HIV‐1 protease discriminates between nearly identical sequences in a very subtle fashion. Finally, the algorithm predicts correctly all of the HIV‐1 protease processing sites in the native gag and gag/pol HIV‐1 polyproteins, and all of the cleavage sites identified in denatured protease and reverse transcriptase. The new predictive algorithm provides a novel route toward understanding the specificity of this important therapeutic target.

Large scale purification and refolding of HIV-1 protease from Escherichia coli inclusion bodies

The protease encoded by the human immunodeficiency virus type 1 (HIV-1) was engineered inEscherichia coli as a construct in which the natural 99-residue polypeptide was preceded by an NH2-terminal methionine initiator. Inclusion bodies harboring the recombinant HIV-I protease were dissolved in 50% acetic acid and the solution was subjected to gel filtration on a column of Sephadex G-75. The protein, eluted in the second of two peaks, migrated in SDS-PAGE as a single sharp band ofMr ≈ 10,000. The purified HIV-1 protease was refolded into an active enzyme by diluting a solution of the protein in 50% acetic acid with 25 volumes of buffer atpH 5.5. This method of purification, which has also been applied to the purification of HIV-2 protease, provides a single-step procedure to produce 100 mg quantities of fully active enzyme.

Characterization of the structure and function of three phospholipases A2 from the venom of Agkistrodon halys pallas

Three monomeric phospholipases A2 with isoelectric points 4.5, 6.9 and 9.3 were purified from the venom of Agkistrodon halys pallas. The complete amino acid sequence of the acidic enzyme and partial amino acid sequences of the neutral and basic phospholipases were determined in order to relate differences in enzymatic reactivities, pharmacologic activities and cytotoxicities to aspects of structure. Studies reported here and elsewhere demonstrate that the three phospholipases A2 exhibit pronounced differences relative to function. The acidic enzyme maintains the highest reactivity toward hydrolysis of monolayers at the air-water interface and may share a feature in common with the acidic enzyme from A. h. blomhoffii, namely the inhibition of platelet aggregation. The neutral phospholipase A2 designated agkistrotoxin, is characterized by potent activity as a pre-synaptic neurotoxin. Agkistrotoxin is the first single polypeptide chain, neurotoxic phospholipase A2 to be documented with a Group II disulfide pattern and, in several respects, may be considered functionally and structurally analogous to notexin from the Australian tiger snake venom. Finally, the basic membranes in the presence of a bactericidal-permeability-increasing protein from neutrophil sources.

Purification and Characterization of Invertase from a Novel Industrial Yeast, Schwanniomyces Occidentalis

The use of yeast as an expression system for heterologous proteins offers several potential advantages with respect to industrial scale-up and genetics over other expression systems, but suffers from several drawbacks. For example, the secreted proteins of S. cerevisiae, found in the periplasm, are hyperglycosylated and the organism has a limited range of usable substrates. Other yeasts have similar disadvantages in addition to producing a variety of proteases. We have investigated the use of Schwanniomyces occidentalis as a host for developing a gene expression system in which these and several disadvantages are minimized. The present paper describes the isolation and characterization of an invertase from cell free supernatants of the yeast Schwanniomyces occidentalis grown on lactose. The enzyme is a beta-D-fructofuranoside-fructohydrolyase, composed of two identical subunits of 76,000 to 78,000 da. with a native molecular mass of 125,000 +/- 25,000 da. of which approximately 17% can be attributed to N-linked carbohydrate. The enzyme has a Vmax of 0.49 +/- 0.025 units, a Km of 21 +/- 1.5 mM, and temperature and pH optima of 55 degrees C and 3.9-4.5, respectively. The amino acid sequences of the amino terminal region and an internal tryptic peptide support an 81% identity with the invertase from Saccharomyces cerevisiae. The enzyme is induced by low glucose and is catabolite repressed.

An examination of structural interactions presumed to be of importance in the stabilization of phospholipase A2 dimers based upon comparative protein sequence analysis of a monomeric and dimeric enzyme from the venom ofAgkistrodon p. piscivorus

Phospholipases A2 may exist in solution both as monomers and dimers, but enzymes that form strong dimers (KD approximately 10−9 M) have been found, thus far, only in venoms of the snake family Crotilidae. The complete amino acid sequences of a basic monomeric and an acidic dimeric phospholipase A2 fromAgkistrodon piscivorus piscivorus (American cotton-mouth water moccasin) venom have been determined by protein sequencing methods as part of a search for aspects of structure contributing to formation of stable dimers. Both the monomeric and dimeric phospholipases A2 are highly homologous to the dimeric phospholipases A2 fromCrotalus atrox andCrotalus adamanteus venoms, and both have the seven residue carboxy-terminal extension characteristic of the crotalid and viperid enzymes. Thus, it is clear that the extension is not a prerequisite for dimerization. Studies to date have revealed two characteristic features of phosphilipases A2 that exist in solution as strong dimers. One is the presence in the dimers of a Pro-Pro sequence at position 112 and 113 which just precedes the seven residue carboxy-terminal extension (residues 116–122). The other is a low isoelectric point; only the acidic phospholipases A2 have been observed, thus far, to form stable dimers. These, alone or together, may be necessary, though not sufficient conditions for phospholipase A2 dimer formation. Ideas regarding subunit interactions based upon crystallographic data are evaluated relative to the new sequence information on the monomeric and dimeric phospholipases A2 fromA. p. piscivorus venom.