Bakers' yeast protease a purification and enzymatic and molecular properties

It has been previously demonstrated in our laboratory that uridine nucleosidase (EC 3.2.2.3) is inactivated by yeast protease A (EC 3.4.23.8). A complete purification procedure for protease A from bakers' yeast, which lacks the acidic activation step used by other workers, and the major properties of the enzyme are shown. The enzyme is homogeneous as judged by disc gel electrophoresis. Its molecular weight, calculated from both sodium dodecyl sulfate-disc gel electrophoresis and gel filtration experiments, is around 45,000. The protein does not possess quaternary structure. The isoelectric point is 4.1. Carbohydrate content is around 8%. Amino acids analysis and sulfur analysis reveal the presence of 1-SH group and two disulfide bridges. The free-SH group does not seem to be involved in catalysis. Amino terminal analysis shows that isoleucine is at the amino terminal position. The pH optima are 2.4 for the hydrolysis of azocasein and casein, and 3.3 for the hydrolysis of hemoglobin. The K_m value for hemoglobin is 1.7 × 10^?5m. The inhibition exerted by pepstatin on the proteolytic activity of protease A is pH dependent. Among various yest enzyme substrates only uridine nucleosidase is inactivated by protease A.     

A modular treatment of molecular traffic through the active site of cholinesterase

We present a model for the molecular traffic of ligands, substrates, and products through the active site of cholinesterases (ChEs). First, we describe a common treatment of the diffusion to a buried active site of cationic and neutral species. We then explain the specificity of ChEs for cationic ligands and substrates by introducing two additional components to this common treatment. The first module is a surface trap for cationic species at the entrance to the active-site gorge that operates through local, short-range electrostatic interactions and is independent of ionic strength. The second module is an ionic-strength-dependent steering mechanism generated by long-range electrostatic interactions arising from the overall distribution of charges in ChEs. Our calculations show that diffusion of charged ligands relative to neutral isosteric analogs is enhanced approximately 10-fold by the surface trap, while electrostatic steering contributes only a 1.5- to 2-fold rate enhancement at physiological salt concentration. We model clearance of cationic products from the active-site gorge as analogous to the escape of a particle from a one-dimensional well in the presence of a linear electrostatic potential. We evaluate the potential inside the gorge and provide evidence that while contributing to the steering of cationic species toward the active site, it does not appreciably retard their clearance. This optimal fine-tuning of global and local electrostatic interactions endows ChEs with maximum catalytic efficiency and specificity for a positively charged substrate, while at the same time not hindering clearance of the positively charged products.     

An immunomodulator used to protect young in the pouch of the Tammar wallaby, Macropus eugenii

Eugenin [pGluGlnAspTyr(SO(3))ValPheMetHisProPhe-NH(2)] has been isolated from the pouches of female Tammar wallabies (Macropus eugenii) carrying young in the early lactation period. The sequence of eugenin has been determined using a combination of positive and negative ion electrospray mass spectrometry. This compound bears some structural resemblance to the mammalian neuropeptide cholecystokinin 8 [AspTyr(SO(3))MetGlyTrpMetAspPhe-NH(2)] and to the amphibian caerulein peptides [caerulein: pGluGlnAspTyr(SO(3))ThrGlyTrpMetAspPhe-NH(2)]. Eugenin has been synthesized by a route which causes only minor hydrolysis of the sulfate group when the peptide is removed from the resin support. Biological activity tests with eugenin indicate that it contracts smooth muscle at a concentration of 10(-9) M, and enhances the proliferation of splenocytes at 10(-7) M, probably via activation of CCK(2) receptors. The activity of eugenin on splenocytes suggests that it is an immunomodulator peptide which plays a role in the protection of pouch young.     

Host-defence peptides of Australian anurans: structure, mechanism of action and evolutionary significance

Host-defence peptides secreted from the skin glands of Australian frogs and toads, are, with a few notable exceptions, different from those produced by anurans elsewhere. This review summarizes the current knowledge of the following classes of peptide isolated and characterized from Australian anurans: neuropeptides (including smooth muscle active peptides, and peptides that inhibit the production of nitric oxide from neuronal nitric oxide synthase), antimicrobial and anticancer active peptides, antifungal peptides and antimalarial peptides. Other topics covered include sex pheromones of anurans, and the application of peptide profiling to (i). recognize particular populations of anurans of the same species and to differentiate between species, and (ii). investigate evolutionary aspects of peptide formation.     

Ascorbic acid enhances endothelial nitric-oxide synthase activity by increasing intracellular tetrahydrobiopterin

Ascorbic acid enhances NO bioactivity in patients with vascular disease through unclear mechanism(s). We investigated the role of intracellular ascorbic acid in endothelium-derived NO bioactivity. Incubation of porcine aortic endothelial cells (PAECs) with ascorbic acid produced time- and dose-dependent intracellular ascorbic acid accumulation that enhanced NO bioactivity by 70% measured as A23187-induced cGMP accumulation. This effect was due to enhanced NO production because ascorbate stimulated both PAEC nitrogen oxide (NO(2)(-) + NO(3)(-)) production and l-arginine to l-citrulline conversion by 59 and 72%, respectively, without altering the cGMP response to authentic NO. Ascorbic acid also stimulated the catalytic activity of eNOS derived from either PAEC membrane fractions or baculovirus-infected Sf9 cells. Ascorbic acid enhanced bovine eNOS V(max) by approximately 50% without altering the K(m) for l-arginine. The effect of ascorbate was tetrahydrobiopterin (BH(4))-dependent, because ascorbate was ineffective with BH(4) concentrations >10 microm or in PAECs treated with sepiapterin to increase intracellular BH(4). The effect of ascorbic acid was also specific because A23187-stimulated cGMP accumulation in PAECs was insensitive to intracellular glutathione manipulation and only ascorbic acid, not glutathione, increased the intracellular concentration of BH(4). These data suggest that ascorbic acid enhances NO bioactivity in a BH(4)-dependent manner by increasing intracellular BH(4) content.     

Clustering of T cell ligands on artificial APC membranes influences T cell activation and protein kinase C theta translocation to the T cell plasma membrane

T cell activation is associated with active clustering of relevant molecules in membrane microdomains defined as the supramolecular activation cluster. The contact area between these regions on the surface of T cells and APC is defined as the immunological synapse. It has been recently shown that preclustering of MHC-peptide complexes in membrane microdomains on the APC surface affects the efficiency of immune synapse formation and the related T cell activation. Disruption of such clusters may reduce the efficiency of stimulation. We describe here an entirely artificial system for Ag-specific, ex vivo stimulation of human polyclonal T cells (artificial APC (aAPC)). aAPC are based on artificial membrane bilayers containing discrete membrane microdomains encompassing T cell ligands (i.e., appropriate MHC-peptide complexes in association with costimulatory molecules). We show here that preclustering of T cell ligands triggered a degree of T cell activation significantly higher than the one achieved when we used either soluble tetramers or aAPC in which MHC-peptide complexes were uniformly distributed within artificial bilayer membranes. This increased efficiency in stimulation was mirrored by increased translocation from the cytoplasm to the membrane of protein kinase theta, a T cell signaling molecule that colocalizes with the TCR within the supramolecular activation cluster, thus indicating efficient engagement of T cell activation pathways. Engineered aAPC may have immediate application for basic and clinical immunology studies pertaining to modulation of T cells ex vivo.     

Cell proliferation, apoptosis, NF-kappaB expression, enzyme, protein, and weight changes in livers of burned rats

Thermal injury has been shown to alter gut epithelium and heart myocyte homeostasis by inducing programmed cell death. The effect of thermal injury on hepatocyte apoptosis and proliferation, however, has not been established. The purpose of this study was to determine whether a large thermal injury increases liver cell apoptosis and proliferation and whether these changes were associated with alterations in hepatic nuclear factor kappaB (NF-kappaB) expression and changes in liver enzymes and amount of protein. Sprague-Dawley rats received a 40% total body surface area scald burn or sham burn. Rats were killed and livers were harvested at 1, 2, 5, and 7 days after burn. Liver cell apoptosis was determined by terminal deoxyuridine nick end labeling (TUNEL) assay and cell proliferation by immunohistochemistry for proliferating cell nuclear antigen. Hepatic NF-kappaB expression was determined by Western blot, and total hepatic protein content was determined by protein assay. Protein concentration decreased after burn compared with sham controls (P < 0.05). Liver cell apoptosis, proliferation, and NF-kappaB expression in hepatocytes increased in burned rats compared with controls (P < 0.05). It was concluded that thermal injury induces hepatic cell apoptosis and proliferation associated with an increase in hepatic NF-kappaB expression and a decrease in hepatic protein concentration.     

Solid-phase synthesis of the native sequence of horse heart cytochrome c-(66-104)-nonatriacontapeptide and of a C-terminal carboxyamide analog selectively modified at Met-80

The peptide corresponding to the (66-104) sequence of horse heart cytochrome c and its carboxyamide analog, selectively modified at the critical Met80 residue, have been synthesized by stepwise solid-phase methods on PAM and BHA resins respectively. The correctness of the growing peptide chain as well as the homogeneity of the final products have been monitored by several analytical methods including quantitative Edman degradation. After HF cleavage both peptides were purified by semipreparative HPLC. The overall yields were 24% for the native (66-104) and 10% for the carboxyamide analog. The homogeneity of the purified synthetic peptides have been determined by different criteria including HPLC, amino acid composition, Edman degradation, electrophoresis, and tryptic peptide mapping. The synthetic fragments have been utilized for preliminary semisynthesis experiments with the native [Hse greater than 65] (1-65)H heme-sequence.