Robert L. Heinrikson

Amino acid analysis by reverse-phase high-performance liquid chromatography: precolumn derivatization with phenylisothiocyanate

Methods for the quantitative derivatization of amino acids with phenylisothiocyanate and for the separation and quantitation of the resulting phenylthiocarbamyl derivatives by reverse-phase high-performance liquid chromatography are described. Phenylthiocarbamylation of amino acids proceeds smoothly in 5 to 10 min at room temperature. Coupling solvents, reagent, and some byproducts are removed by rotary evaporation under high vacuum, and the phenylthiocarbamyl derivatives are dissolved in 0.05 M ammonium acetate, pH 6.8, for injection onto the octyl or octadecylsilyl reverse-phase column. Columns are equilibrated with the same solvent and the effluent stream is monitored continuously at 254 nm for detection of the amino acid derivatives. Elution of all of the phenylthiocarbamyl amino acids is achieved in about 30 min utilizing gradients of increasing concentrations of ammonium acetate and acetonitrile or methanol. This approach to amino acid analysis offers select advantages, both with respect to methods which employ reverse-phase separation of prederivatized samples and to the classical ion-exchange procedure. All amino acids, including proline, are converted quantitatively to phenylthiocarbamyl compounds and these are stable enough to eliminate any need for in-line derivatization. Furthermore, results comparable in sensitivity and precision to those obtained by state-of-the-art ion-exchange analyzers may be generated with equipment that need not be dedicated to a single application.

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.

Heterodimer formation is essential for heparanase enzymatic activity

Heparanase is an endo-beta-D-glucuronidase involved in cleavage of heparan sulfate residues and hence participates in extracellular matrix degradation and remodeling. The heparanase cDNA encodes for a polypeptide of 543 amino acids that appears as a approximately 65 kDa band in SDS-PAGE analysis. The protein undergoes a proteolytic cleavage that is likely to occur at two potential cleavage sites, Glu(109)-Ser(110) and Gln(157)-Lys(158), yielding an 8 kDa polypeptide at the N-terminus, a 50 kDa polypeptide at the C-terminus, and a 6 kDa linker polypeptide that resides in-between. The active form of heparanase has long been thought to be a 50 kDa polypeptide isolated from cells and tissues. However, attempts to obtain heparanase activity after expression of the 50 kDa polypeptide failed, suggesting that the N-terminal region is important for heparanase enzymatic activity. It has been hypothesized that heterodimer formation between the 8 and 50 kDa heparanase subunits is important for heparanase enzymatic activity. By individually or co-expressing the 8 and 50 kDa heparanase subunits in mammalian cells, we demonstrate specific association between the heparanase subunits by means of co-immunoprecipitation and pull-down experiments. Moreover, a region in the 50 kDa heparanase subunit that mediates interaction with the 8 kDa subunit was identified. Altogether, our results clearly indicate that heterodimer formation is necessary and sufficient for heparanase enzymatic activity in mammalian cells.

Prediction of the tertiary structure and substrate binding site of caspase-8

The caspases represent a family of sulfhydryl proteases that play important regulatory roles in the cell. The tertiary structure of the protease domain of caspase‐8, also called FLICE, has been predicted by a segment match modeling procedure. First, the atomic coordinates of the catalytic domain of caspase‐3, also called CPP32, a member of the family that is closely related to caspase‐8, were determined based upon the crystal structure of human caspase‐1 (interleukin converting enzyme). Then, the caspase‐3 structure was used as a template for modeling the protease domain of caspase‐8. The resulting structure shows the expected level of similarity with the conformations of caspases‐1 and ‐3 for which crystal structures have been determined. Moreover, the subsite contacts between caspase‐8 and the covalently linked inhibitor, Ac‐DEVD‐aldehyde, are only slightly different from those seen in the caspase‐3 enzyme/inhibitor complex. The model of caspase‐8 can serve as a reference for subsite analysis relative to design of enzyme inhibitors that may find therapeutic application.

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.

Gene duplication in the evolution of the two complementing domains of Gram-negative bacterial tetracycline efflux proteins

The resistance of Gram- bacteria to the broad-spectrum antibiotic tetracycline (Tc) results from energy-dependent drug efflux mediated by the tet gene product, the cytoplasmic membrane Tet protein. Amino acid (aa) sequences deduced from total tet nucleotide sequences of three different resistance determinants (classes A, B and C) indicate that the protein products [Tet(A), Tet(B), and Tet(C)] share a common ancestor. Hydropathic analysis of Tet sequences predicts twelve transmembrane segments in each protein, with six occurring in each half of the molecule. More importantly, the linear distributions of these segments in the N- and C-terminal halves are nearly identical, suggesting that the two halves of each Tet protein are related by a process of tandem gene duplication and divergence. Indeed, a variable but significant conservation of sequence was detected among the N- and C-terminal halves for all possible comparisons of the three proteins. Such conservation was not observed within other prokaryotic integral membrane proteins or when other prokaryotic proteins were compared to Tet halves. Similarity, both in sequence and in predicted transmembrane structural organization, strongly suggests that a common ancestor of Tet(A), Tet(B), and Tet(C) arose by duplication of a gene reading frame specifying a transmembrane protein of approximately 200 aa residues. The two halves of Tet proteins correspond to the two domains, alpha and beta, which have distinct, complementary roles in Tc efflux. Nevertheless, selective constraints to function in the cytoplasmic membrane have apparently led to maintenance of similar patterns of secondary structural organization in these complementary domains.

An examination of the expected degree of sequence similarity that might arise in proteins that have converged to similar conformational states: The impact of such expectations on the search for homology between the structurally similar domains of rhodanese

Abstract Recognition of structural similarity in proteins invites the inference of homology even when the amino acid sequences are not highly similar. The influence of structural similarity on both the genetic tests for amino acid sequence similarity and the inference of homology was examined by statistical methods. Structure-dependent compositions of amino acid sequences representing segments of secondary and supersecondary structure were examined for the preferential occurrence of structurally similar amino acids that are also similar according to the genetic criteria of Fitch (1970), Dayhoff (1979) and McLachlan (1971). These analyses revealed that: (1) the preferential occurrence of structurally similar amino acids in analogous secondary structures should not give rise to a statistically significant sequence similarity; (2) some positional amino acid preferences in secondary and supersecondary structures score highly in sequence similarity tests; (3) the preferential occurrence of non-polar, β-branched amino acids in the parallel β-pleated sheets of α β proteins constitutes an important structural bias in genetic tests for sequence similarity. These findings may be applied to sequence comparisons whenever the conformational states are either known experimentally or may be inferred from predictive analysis of the amino acid sequence. The corrections for structure-dependent compositions were made in a search for homology between the two structurally similar, globular domains of bovine liver rhodanese. Despite these corrections and earlier failures to observe significant sequence similarity, a statistically significant sequence similarity was detected, supporting the inference that the domains are internally paralogous, i.e. intraspecies products of a partial internal duplication of an ancestral gene.

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.

SPECIFICITY OF RETROVIRAL PROTEASES : AN ANALYSIS OF VIRAL AND NONVIRAL PROTEIN SUBSTRATES

Publisher Summary This chapter focuses on current ideas regarding the specificity of the HIV protease and retroviral enzymes. It tries to identify a substrate of the HIV protease that has a well-defined tertiary structure where one can have confidence that the structure clearly relates to the conformation recognized by the protease. The retroviral enzymes require a long stretch of structure in a substrate that is accessible and of the “right” sequence and that is either extended or extendable as a consequence of binding to the enzyme. Such regions will most likely be found in interdomain segments akin to those linking the individual protein components of viral polyproteins. HIV protease will degrade some proteins extensively only when those proteins begin to lose their structural organization, thus the retroviral enzymes are sensors of structural integrity in their protein substrates. Structural proteins, proteins with acid-rich domains, proteins that begin to unfold by pH denaturation, and proteins from which tight-binding and structure-stabilizing ligands have been removed are ready targets for these enzymes.

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.