On the size of the active site in proteases. I. Papain.

This contribution is a completely updated and expanded version of the four prior analogous reviews that were published in this journal in 1997, 2003, 2007, and 2012. In the case of all approved therapeutic agents, the time frame has been extended to cover the 34 years from January 1, 1981, to December 31, 2014, for all diseases worldwide, and from 1950 (earliest so far identified) to December 2014 for all approved antitumor drugs worldwide. As mentioned in the 2012 review, we have continued to utilize our secondary subdivision of a “natural product mimic”, or “NM”, to join the original primary divisions and the designation “natural product botanical”, or “NB”, to cover those botanical “defined mixtures” now recognized as drug entities by the U.S. FDA (and similar organizations). From the data presented in this review, the utilization of natural products and/or their novel structures, in order to discover and develop the final drug entity, is still alive and well. For example, in the area of cancer, over t...

Natural Products as Sources of New Drugs from 1981 to 2014

Molecular chaperones, including the heat-shock proteins (Hsps), are a ubiquitous feature of cells in which these proteins cope with stress-induced denaturation of other proteins. Hsps have received the most attention in model organisms undergoing experimental stress in the laboratory, and the function of Hsps at the molecular and cellular level is becoming well understood in this context. A complementary focus is now emerging on the Hsps of both model and nonmodel organisms undergoing stress in nature, on the roles of Hsps in the stress physiology of whole multicellular eukaryotes and the tissues and organs they comprise, and on the ecological and evolutionary correlates of variation in Hsps and the genes that encode them. This focus discloses that (a) expression of Hsps can occur in nature, (b) all species have hsp genes but they vary in the patterns of their expression, (c) Hsp expression can be correlated with resistance to stress, and (d) species' thresholds for Hsp expression are correlated with levels of stress that they naturally undergo. These conclusions are now well established and may require little additional confirmation; many significant questions remain unanswered concerning both the mechanisms of Hsp-mediated stress tolerance at the organismal level and the evolutionary mechanisms that have diversified the hsp genes.

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Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology

In order to obtain a novel binding protein against a chosen target, DNA molecules, each encoding a protein comprising one of a family of similar potential binding domains and a structural signal calling for the display of the protein on the outer surface of a chosen bacterial cell, bacterial spore or phage (genetic package) are introduced into a genetic package. The protein is expressed and the potential binding domain is displayed on the outer surface of the package. The cells or viruses bearing the binding domains which recognize the target molecule are isolated and amplified. The successful binding domains are then characterized. One or more of these successful binding domains is used as a model for the design of a new family of potential binding domains, and the process is repeated until a novel binding domain having a desired affinity for the target molecule is obtained. In one embodiment, the first family of potential binding domains is related to bovine pancreatic trypsin inhibitor, the genetic package is M13 phage, and the protein includes the outer surface transport signal of the M13 gene III protein.

Directed evolution of novel binding proteins

Fractionation of MOPC 41 DL-1 tumors revealed that the mRNA for the light chain of immunoglobulin is localized exclusively in membrane-bound ribosomes. It was shown that the translation product of isolated light chain mRNA in a heterologous protein-synthesizing system in vitro is larger than the authentic secreted light chain; this confirms similar results from several laboratories. The synthesis in vitro of a precursor protein of the light chain is not an artifact of translation in a heterologous system, because it was shown that detached polysomes, isolated from detergent-treated rough microsomes, not only contain nascent light chains which have already been proteolytically processed in vivo but also contain unprocessed nascent light chains. In vitro completion of these nascent light chains thus resulted in the synthesis of some chains having the same mol wt as the authentic secreted light chains, because of completion of in vivo proteolytically processed chains and of other chains which, due to the completion of unprocessed chains, have the same mol wt as the precursor of the light chain. In contrast, completion of the nascent light chains contained in rough microsomes resulted in the synthesis of only processed light chains. Taken together, these results indicate that the processing activity is present in isolated rough microsomes, that it is localized in the membrane moiety of rough microsomes, and, therefore, that it was most likely solubilized during detergent treatment used for the isolation of detached polysomes. Furthermore, these results established that processing in vivo takes place before completion of the nascent chain. The data also indicate that in vitro processing of nascent chains by rough microsomes is dependent on ribosome binding to the membrane. If the latter process is interfered with by aurintricarboxylic acid, rough microsomes also synthesize some unprocessed chains. The data presented in this paper have been interpreted in the light of a recently proposed hypothesis. This hypothesis, referred to as the signal hypothesis, is described in greater detail in the Discussion section.

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Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma.

On the active site of proteases. III. Mapping the active site of papain; specific peptide inhibitors of papain

cDNA clones encoding the variable and constant regions of chicken immunoglobulin (Ig) gamma-chains were obtained from spleen cDNA libraries. Southern blots of kidney DNA show that the variable region sequences of eight cDNA clones reveal the same set of bands corresponding to approximately 30 cross-hybridizing VH genes of one subgroup. Since the VH clones were randomly selected, it is likely that the bulk of chicken H-chains are encoded by a single VH subgroup. Nucleotide sequence determinations of two cDNA clones reveal VH, D, JH and the constant region. The VH segments are closely related to each other (83% homology) as expected for VH or the same subgroup. The JHs are 15 residues long and differ by one amino acid. The Ds differ markedly in sequence (20% homology) and size (10 and 20 residues). These findings strongly indicate multiple (at least two) D genes which by a combinatorial joining mechanism diversify the H-chains, a mechanism which is not operative in the chicken L-chain locus. The most notable among the chicken Igs is the so-called 7S IgG because its H-chain differs in many important aspects from any mammalian IgG. The sequence of the C gamma cDNA reported here resolves this issue. The chicken C gamma is 426 residues long with four CH domains (unlike mammalian C gamma which has three CH domains) and it shows 25% homology to the chicken C mu. The chicken C gamma is most related to the mammalian C epsilon in length, the presence of four CH domains and the distribution of cysteines in the CH1 and CH2 domains. We propose that the unique chicken C gamma is the ancestor of the mammalian C epsilon and C gamma subclasses, and discuss the evolution of the H-chain locus from that of chicken with presumably three genes (mu, gamma, alpha) to the mammalian loci with 8-10 H-chain genes.

Chicken immunoglobulin gamma-heavy chains: limited VH gene repertoire, combinatorial diversification by D gene segments and evolution of the heavy chain locus.

Antibodies to Sequential and Conformational Determinants

A water insoluble derivative of a papain inhibitor was prepared by covalently linking Gly-Gly-Tyr(Bzl)-Arg to an agarose resin. The immobilized inhibitor binds active papain specifically. When papain prepared by the method of Kimmel and Smith was activated and applied to a column of the immobilized inhibitor at moderate ionic strength (20 mM EDTA, pH 4.3), about 50% of the total protein was not bound and was found to be catalytically inactive. The bound enzyme was released with distilled water. It contained one mole of SH per mole of protein. When assayed with α-N-benzoyl-l-arginine ethyl ester (25°, pH 6.0) the purified enzyme had a kcat of 28.5 sec-1 and a Km of 18 mM. The specific activity of the purified papain was about twice that of the original preparation and the Km was unchanged. The enzyme, inactivated by an equimolar amount of mercuric chloride could be fully activated even after prolonged storage.

Israel Schechter

Papers

On the size of the active site in proteases. I. Papain.

On the active site of proteases. III. Mapping the active site of papain; specific peptide inhibitors of papain

Chicken immunoglobulin gamma-heavy chains: limited VH gene repertoire, combinatorial diversification by D gene segments and evolution of the heavy chain locus.

Antibodies to Sequential and Conformational Determinants

The purification of papain by affinity chromatography.