Michaelis complexes of porcine pancreatic elastase with 7-[(alkylcarbamoyl)amino]-4-chloro-3-ethoxyisocoumarins: translational sampling of inhibitor position and kinetic measurements.

A step leading to the formation of the covalent complexes between porcine pancreatic elastase (PPE) and 7-[(alkylcarbamoyl)amino]-4-chloro-3-ethoxyisocoumarins (alkylHNCO-EICs) is the formation of the noncovalent Michaelis complex. No average structures are available for the Michaelis complexes of PPE with alkylHNCO-EICs. We present the results of an initial step in obtaining these structures and have determined kinetic constants as well. The kinetic results indicate that formation of the Michaelis complex is what differentiates the effectiveness of these inhibitors in inactivating PPE. The structural and kinetic results together suggest that the structure of the Michaelis complex is necessary for the design of potent alkylHNCO-EIC inhibitors of PPE. Two novel alkylHNCO-EICs are predicted to be the best inhibitors of this series. An alternate mechanism for serine protease inhibition is also proposed. Evidence for, and studies that may add support to, the hypothesized mechanism are discussed.     

Cloning and sequencing of the cDNA encoding the major albumin of Theobroma cacao

The major albumin, a polypeptide of 21 kilodaltons (kDa), from the seeds of cocoa (Theobroma cacao L.), has been identified and partially purified by preparative gel electrophoresis. Some N-terminal sequence was obtained, permitting the construction of an oligonucleotide probe. This probe was used to isolate the corresponding copy DNA (cDNA) clone from a library made from poly(A)⁺ RNA from immature cocoa beans. The cDNA sequence has a single major open reading frame, that translates to give a 221-amino-acid polypeptide of M_r 24003. The existence of a precursor to the 21-kDa polypeptide of this size was confirmed by immunoprecipitation from total poly(A)⁺ RNA translation products. The polypeptide has a hydrophobic signal sequence of 26 amino acids before the mature start, and the mature polypeptide would have an M_r of 21223. The protein sequence is homologous with sequences of the Kunitz protease and -amylase inhibitor family, and the protein probably functions to defend the seed's protein reserves from the digestive enzymes of invading pests. However because the protein comprises 25–30% of the total seed protein it may itself also function as a storage protein. Electron micrographs of immunogold-labelled embryo sections show that the protein is located in membrane-enclosed organelles.Abbreviations cDNA copy DNA - IgG immunoglobulin G - kb kilobase pairs - kDa kilodaltons - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacylamide gel electrophoresis The authors are very grateful to Dr R. Jennings of the Virology Department, Sheffield University Medical School, for help in raising antibodies, and to Dr G. Cope, of the Biological Sciences Electron Microscopy Unit, Sheffield University, for taking the electron micrographs.To whom correspondence should be addressed.     

Cloning and characterization of an extracellular temperature-labile serine protease gene from Aeromonas hydrophila

Aeromonas virulence is thought to depend on multigenic functions. The gene for an extracellular protease from Aeromonas hydrophila SO2/2 was cloned in Escherichia coli C600-1 by using pIJ860, bifunctional plasmid, as a vector. The gene encodes for a temperature-labile serine protease (P2) with a molecular mass of approx. 68 kDa which is highly inhibited by PMSF. The gene was expressed in Streptomyces lividans 1326 by transforming protoplasts with the original clone pPA2. We were also able to transfer and express the prt P2 gene in Pseudomonas putida by mating experiments. The protein P2 was secreted into the periplasms of both P. putida and E. coli C600-1 being identical in properties to one of the proteases secreted into the culture supernatant by A. hydrophila SO2/2.     

Identification of the peptide bond cleaved during activation of human Clr

CNBr cleavage of unreduced proenzyme Clr yielded fragment CP2b, isolated by gel filtration and highpressure gel permeation chromatography. This fragment (˜ M_τ 55000) comprised at least 4 disulphidelinked peptides, which were separated by gel filtration after reduction and alkylation. Peptide CP2bRA4, overlapping the A- and B-chain regions in proenzyme Clr was digested by V8 staphylococcal protease, and the digest separated by reversed-phase HPLC. N-terminal sequence analysis of peptide CP2bRA4SP9 established that Clr activation involves the cleavage of a single Arg-Ile bond, located in the sequence: Gln-Arg-Gln-Arg-Ile-Ile-Gly-Gly     

Crystal structure of subtilisin complexed with its trapped substrateStreptomyces subtilisin inhibitor—Protein-protein interaction and evolution of serine proteinases and their proteinaceous inhibitors

The complex of a bacterial alkaline serine proteinase, subtilisin BPN’, with its proteinaceous inhibitorStreptomyces subtilisin inhibitor is unique in several respects, compared with other similar complexes containing serine proteinases of trypsin family. In addition to the usual antiparallelβ-sheet involving P₁-P₃ residues of the inhibitor, P₄-P₆ residues form antiparallelβ-sheet with a previously unnoticed chain segment (the ‘S4-6 site’) of subtilisin. The ‘S4-6 site’ does not exist in serine proteinases of trypsin family, whether of mammalian or microbial origin. Global induced-fit movement seems to occur on the ‘trapped substrate’Streptomyces subtilisin inhibitor: a channel-like structure in SSI remote from the contact region becomes about 2 Å wider upon complexing with subtilisin. Main role of the secondary contact region ofStreptomyces subtilisin inhibitor seems to support the reactive site loop (primary contact region). Steric homology for the two contact regions is so high between the inhibitors ofStreptomyces subtilisin inhibitor family and those of pancreatic secretory trypsin inhibitor-ovomucoid inhibitor family that it seems to favour a divergent evolution and to support the general notion as to the relationship of prokaryotic and eukaryotic genes put forwarded by Doolittle(Nature (London),272, 581, 1978).     

Nerve growth factor. A structural relationship between its proteolytic and leukocyte-chemotactic active sites

Summary High molecular weight mouse nerve growth factor(H M W-NGF), in addition to its effects on certain neural elements, is also chemotactic for human polymorphonuclear leukocytes. One of the subunits of H M W-NGF is a protease of the serine family and its active site contains a serine residue and a closely-neighboring histidine residue that are both essential for proteolysis. Elimination of enzyme activity by irreversibly blocking the single serine has no effect on leukotaxis, but blocking the histidine abolishes leukotaxis. These results suggest the possibility that part of the proteolytic active site of this enzyme may have evolved to perform more than one, completely different, biologic function — proteolysis as well as nonproteolytically mediated chemotaxis.Abbreviations HMW-NGF mouse submandibular gland nerve growth factor, purified as in Ref. 1 - DFP diisopropyl-phosphofluoridate - DIP-NGF diisopropyl-phosphoryl-NGF; phe-pro-arg-CH₂C1, D-phenylalanyl-L-propyl-L-argininyl chloromethyl ketone; TLCK, N-p-tosyl-L-lysine chloromethyl ketone - TAME N-p-tosyl L-arginine methyl ester - EDTA ethylenediamine tetraacetic acid     

Operation of the glycolate pathway in isolated bundle sheath strands of maize and Panicum maximum

Operation of the glycolate pathway in isolated bundle sheath (BS) strands of two C₄ species was demonstrated from ¹⁴C incorporation into two intermediates, glycine and serine, under conditions favourable for photorespiratory activity. Isolated BS strands fixing ¹⁴CO₂ under light at physiological rates incorporate respectively 3% (Zea mays L., cv. INRA 258) and 7% (Panicum maximum Jacq.) of total ¹⁴C fixed into glycine + serine, at low bicarbonate levels (less than the Km for CO₂ fixation, 0.8 mM). Higher bicarbonate concentrations depressed the percentage of incorporation into the two amino acids. No labelling was observed in the absence of added glutamate. Oxygen was required for glycine + serine labelling, since ¹⁴C incorporation into glycine was largely depressed by argon flushing, and labelling of the two amino acids was nearly suppressed by the addition of the strong reductant, dithionite, especially in maize. Two inhibitors of the glycolate pathway were tested. With α-hydroxypyridine-methanesulfonic acid, an inhibitor of glycolate oxidase, labelling of glycine and serine remained minimal whereas glycolate was accumulated. Isoniazid, an inhibitor of the transformation of glycine to serine induced a 50% increased labelling of glycine in maize BS, and a large decrease in serine labelling. In Panicum, the increase in [¹⁴C]-glycine was 90%. These results suggest that the pathway glycolate → glycine → serine operates in these plants. However, leakage of metabolites occurs in BS cells, especially in maize and a large part of newly formed glycolate, glycine and serine is exported out of the cells. Operation of ribulose-1,5-bisphosphate oxygenase activity in competition with ribulose-1,5-bisphosphate carboxylase is demonstrated by the lowering of total ¹⁴CO₂ fixation when O₂ is increased at low bicarbonate concentration. An interesting feature observed in maize BS, at low bicarbonate concentration, was an increase in ribulose-1,5-bisphosphate labelling when the O₂ level was decreased. This was accompanied by an increase in CO₂ fixation. This could indicate an increased rate in synthesis of ribulose-1,5-bisphosphate (which accumulated) due to a stimulation of ATP synthesis by cyclic photophosphorylation under anaerobic conditions.     

Protease activities in non-embryogenic and embryogenic carrot cell strains during callus growth and embryo formation

Embryogenic and non-embryogenic cell strains of Daucus carota L. were examined for their protease activity using a wide range of chromogenic synthetic peptides as substrates. High arginine-specific activity was present in all strains, but no protease activity "specific" for embryogenic or non-embryogenic strains could be detected with the substrates tested. The specific protease activity was 5–10 times higher in the non-embryogenic as compared to the embryogenic strain for most tested substrates, and this difference was not due to release of proteases in the latter. All strains showed a decrease in protease activity when cultured in media without 2,4-dichlorophenoxyacetic acid, but the embryos had high protease activity in comparison with the nondifferentiated cell aggregates. In the latter aggregates, hydrolyzing activity towards three of the substrates (H-D-Phe-Pip-Arg- p -nitroanilide, Suc-Ala-Pro-Phe- p -nitro-anilide and Bz-Phe-Val-Arg- p -nitroanilide) was absent, whereas the embryos were able to hydrolyze them.     

An intramolecular linkage involving isodityrosine in extensin

We isolated isodityrosine, a diphenyl ether linked amino acid, from cell wall hydrolysates and from two tryptic peptides of extensin. Determination of the molecular weights, net charges and composition of the peptides indicated that isodityrosine (IDT) can form a short intramolecular linkage in sequences consisting of:

     

Mode of high temperature injury to wheat during grain development

High temperature stress adversely affects wheat growth in many important production regions, but the mode of injury is unclear. Wheat ( Triticum aestivum L. cv. Newton) was grown under controlled conditions to determine the relative magnitude and sequences of responses of source and sink processes to high temperature stress during grain development. Regimes of 25°C day/15°C night, 30°C day/20°C night, and 35°C day/25°C night from 5 days after anthesis to maturity differentially affected source and sink processes. High temperatures accelerated the normal decline in viable leaf blade area and photosynthetic activities per unit leaf area. Electron transport, as measured by Hill reaction activity, declined earlier and faster than other photosynthetic processes at the optimum temperature of 25/15 °C and at elevated temperatures. Changes in RUBP carboxylase activities were similar in direction but smaller in magnitude than changes in photosynthesic rate. Increased protease activity during senscence was markedly accentuated by high temperature stress. Specific protease activity increased 4-fold at 25/15 °C and 28-fold at 35/25 °C from 0 to 21 days after initiation of temperature treatments. Grain-filling rate decreased from the lowest to the highest temperature, but the change was smaller than the decrease in grain-filling duration at the same temperatures. We concluded that a major effect of high temperature is acceleration of senescence, including cessation of vegetative and reproductive growth, deterioration of photosynthetic activities, and degradation of proteinaceous constituents.