Purification and characterization of an irreversible serine protease inhibitor from skin secretions of Bufo andrewsi

Amphibian skin secretions contain many bioactive compounds. In the present work, an irreversible serine protease inhibitor, termed baserpin, was purified for the first time from the skin secretions of toad Bufo andrewsi by Successive ion-exchange and gelfiltration chromatography. Baserpin is a single chain glycoprotein, with an apparent molecular weight of about 60 kDa in SDS-PAGE. Baserpin is an irreversible inhibitor and effectively inhibits the catalytic activity of trypsin, chymotrypsin and elastase. SDS-stable baserpin-trypsin complex could be seen in SDS-PAGE indicates that it possibly belongs to the serpin superfamily. According to the association rates determined, baserpin is a potent inhibitor of bovine trypsin (4.6 X 10(6) M-1 S-1), bovine chymotrypsin (8.9 X 10(6) M-1 s(-1)) and porcine elastase (6.8 X 10(6) M-1 s(-1)), whereas it shows no inhibitory effect on thrombin. The N-terminal sequence of baserpin is HTQYPDILIAKPXDK, which shows no similarity with other known serine protease inhibitors. (c) 2005 Elsevier Ltd. All rights reserved.

Frog albumin is expressed in skin and characterized as a novel potent trypsin inhibitor

A novel potent trypsin inhibitor was purified and characterized from frog Bombina maxima skin. A full-length cDNA encoding the protein was obtained from a cDNA library constructed from the skin. Sequence analysis established that the protein actually comprises three conserved albumin domains. B. maxima serum albumin was subsequently purified, and its coding cDNA was further obtained by PCR-based cloning from the frog liver. Only two amino acid variations were found in the albumin sequences from the skin and the serum. However, the skin protein is distinct from the serum protein by binding of a haem b (0.95 mol/mol protein). Different from bovine serum albumin, B. maxima albumin potently inhibited trypsin. It bound tightly with trypsin in a 1: 1 molar ratio. The equilibrium dissociation constants (K-D) obtained for the skin and the serum proteins were 1.92 x 10(-9) M and 1.55 x 10(-9) M, respectively. B. maxima albumin formed a noncovalent complex with trypsin through an exposed loop formed by a disulfide bond (Cys(53)-Cys(62)), which comprises the scissile bond Arg(58)(P-1)-His(59)(P-1'). No inhibitory effects on thrombin, chymotrypsin, elastase, and subtilisin were observed under the assay conditions. Immunohistochemical study showed that B. maxima albumin is widely distributed around the membranes of epithelial layer cells and within the stratum spongiosum of dermis in the skin, suggesting that it plays important roles in skin physiological functions, such as water economy, metabolite exchange, and osmoregulation.

A small trypsin inhibitor from the frog of Odorrana grahami

A novel peptide inhibitor (OGTI) of serine protease with a molecular weight of 1949.8, was purified from the skin secretion of the frog, Odorrana grahami. Of the tested serine proteases, OGTI only inhibited the hydrolysis activity of trypsin on synthetic chromogenic substrate. This precursor deduced from the cDNA sequence is composed of 70 amino acid residues. The mature OGTI contains 17 amino acid residues including a six-residue loop disulfided by two half-cysteines (AVNIPFKVHFRCKAAFC). In addition to its unique six-residue loop, the overall structure and precursor of OGTI are different from those of other serine protease inhibitors. It is also one of the smallest serine protease inhibitors ever found. (C) 2008 Elsevier Masson SAS. All rights reserved.

A serine proteinase inhibitor from frog eggs with bacteriostatic activity

By Sephadex G-50 gel filtration, Resource Q anionic exchange and C4 reversed phase liquid high performance liquid chromatography, a proteinase inhibitor protein (Ranaserpin) was identified and purified from the eggs of the odour frog, Rana grahami. The protein displayed a single band adjacent to the molecular weight marker of 14.4 kDa analyzed by SDS-PAGE. The inhibitor protein homogeneity and its molecular weight were confirmed again by MALDI-TOF mass spectrometry analysis. The MALDI-TOF mass spectrum analysis gave this inhibitor protein an m/z of 14422.26 that was matched well with the result from SDS-PAGE. This protein is a serine proteinase inhibitor targeting multiple proteinases including trypsin, elastase, and subtilisin. Ranaserpin inhibited the proteolytic activities of trypsin, elastase, and subtilisin. It has an inhibitory constant (K-i) of 6.2 x 10(-8) M, 2.7 x 10(-7) M and 2.2 x 10(-8) M for trypsin, elastase, and subtilisin, respectively. This serine proteinase inhibitor exhibited bacteriostatic effect on Gram-positive bacteria Bacillus subtilis (ATCC 6633). It was suggested that ranaserpin might act as a defensive role in resistance to invasion of pests or pathogens. This is the first report of serine proteinase inhibitor and its direct defensive role from amphibian eggs. (C) 2007 Elsevier Inc. All rights reserved.

Isolation, expression and characterization of a novel dual serine protease inhibitor, OH-TCI, from king cobra venom

Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibito

A horsefly saliva antigen 5-like protein containing RTS motif is an angiogenesis inhibitor

The Ag5 proteins are the most abundant and immunogenic proteins in the venom secretory ducts of stinging insects. An antigen 5-like protein (named tabRTS) composed of 221 amino acid residues was purified and characterized from the salivary glands of the horsefly, Tabanus yao (Diptera, Tabanidae). Its cDNA was cloned from the cDNA library of the horsefly's salivary gland. TabRTS containing the SCP domain (Sc7 family of extracellular protein domain) was found in insect antigen 5 proteins. More interestingly, there is an Arg-Thr-Ser (RTS) disintegrin motif at the C-terminus of tabRTS. The RTS motif is positioned in a loop bracketed by cysteine residues as those found in RTS-disintegrins of Crotalidae and Viperidae snake venoms, which act as angiogenesis inhibitors. Endothelial Cell Tube formation assay in vitro and chicken chorioallantoic membrane (CAM) angiogenesis assay in vivo were performed as to investigate the effect of tabRTS on angiogenesis. It was found that tabRTS could significantly inhibit angiogenesis in vitro and in vivo. Anti-alpha(1)beta(1) monoclonal antibody could dose-dependently inhibit the anti-angiogenic activity of tabRTS. This result indicated that tabRTS possibly targets the alpha(1)beta(1) integrin to exert the anti-angiogenic activity as snake venom RTS-/KTS-disintegrins do. The current work revealed the first angiogenesis inhibitor protein containing RTS motif from invertebrates, a possible novel type of RTS-disintegrin. (C) 2009 Elsevier Ltd. All rights reserved.

Studies on phenolase enzyme in prawns. II. The inhibitory action on the enzyme by certain chemical agents

The effect of certain chemical agents on dopa oxidation by phenolases has been examined. Sulphur containing amino carboxylic acids are inhibitory agents for dopa oxidation. Tyrosine, a substrate for the enzyme also acts as an inhibitor for dopa oxidation by the enzyme. The possible mode of action has been discussed. The function of diethyl dithiocarbamate in suppressing the display of enzyme activity has been detailed and its behaviour has been compared to the other chemical agents studied.

Oxidation of residual lipids in fish protein concentrates and its effect on the nutritional quality of the protein

The paper reviews the work reported on the changes in the nutritive value of fish protein concentrates (FPC) during, storage, with special emphasis on the effects of the interactions between oxidised residual lipids and proteins of the FPC. Theories on the oxidised lipid-protein interactions are reviewed and the nutritional significance of these reactions is discussed.

Effect of oxidation of dietary fish lipids on the quality of proteins in diet

The paper reports results of a study on the effect of oxidised fish oil in the diet on the quality of dietary fish proteins. With increased oxidation of oil, digestibility and utilization of the protein registered a decrease. Oxidation of fish oil beyond a stage was found to affect the intake of diet itself and rats lost weight rapidly.

Effect of nitrite, ammonia, and temperature on P. monodon larvae

P. monodon larvae were studied for the effects of temperature, ammonia, and nitrite on survival. Toxicity levels of nitrite were found to vary with larval stage. Larvae could tolerate ammonia up to about 10 ppm, with the effect more clearly shown by the zoea stage. Survival and growth were not significantly affected by temperature, although moulting was enhanced at temperatures higher than 29 C. Larvae of P. monodon have lower tolerance toward nitrite and ammonia compared to postlarvae. Although high survival was obtained at low levels of nitrite and ammonia, it is still necessary to know their effects on metabolism, in order to examine possible biochemical parameters for diagnosing sublethal toxicity or stress.

An inhibitor of c-Jun N-terminal kinases (CEP-11004) counteracts the anti-HIV-1 action of trichosanthin

Trichosanthin (TCS) is a type I ribosome-inactivating protein possessing multiple biological and pharmacological activities. One of its major actions is inhibition of human immunodeficiency virus (HIV) replication. The mechanism is still not clear. It is

D77, one benzoic acid derivative, functions as a novel anti-HIV-1 inhibitor targeting the interaction between integrase and cellular LEDGF/p75

Integration of viral-DNA into host chromosome mediated by the viral protein HIV-1 integrase (IN) is an essential step in the HIV-1 life cycle. In this process, Lens epithelium-derived growth factor (LEDGF/p75) is discovered to function as a cellular co-fa

Sifuvirtide, a potent HIV fusion inhibitor peptide

Enfuvirtide (ENF) is currently the only FDA approved HIV fusion inhibitor in clinical use. Searching for more drugs in this category with higher efficacy and lower toxicity seems to be a logical next step. In line with this objective, a synthetic peptide

A novel serine protease inhibitor from Bungarus fasciatus venom

By Sephadex G-50 gel filtration, cation-exchange CM-Sephadex C-25 chromatography and reversed phase high-performance liquid chromatography (HPLC), a novel serine protease inhibitor named bungaruskunin was purified and characterized from venom of Bungarus fasciatus. Its cDNA was also cloned from the cDNA library of B. fasciatus venomous glands. The predicted precursor is composed of 83 amino acid (aa) residues including a 24-aa signal peptide and a 59-aa mature bungaruskunin. Bungaruskunin showed maximal similarity (64%) with the predicted serine protease inhibitor blackelin deduced from the cDNA sequence of the red-bellied black snake Pseudechis porphyriacus. Bungaruskunin is a Kunitz protease inhibitor with a conserved Kunitz domain and could exert inhibitory activity against trypsin, chymotrypsin, and elastase. By screening the cDNA library, two new B chains of beta-bungarotoxin are also identified. The overall structures of bungaruskunin and beta -bungarotoxin B chains are similar; especially they have highly conserved signal peptide sequences. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor. (c) 2007 Elsevier Inc. All rights reserved.

Nutrient salts in the United Arab Emirates waters (the Arabian Gulf and the Gulf of Oman)

This paper deals with the levels and distributions of nutrient salts in the United Arab Emirates waters. Water samples were collected bimonthly during 1994-1995 from the marine environment of the United Arab Emirates, which extends more than 800km along the Arabian Gulf and the Gulf of Oman. Concentrations of ammonium, nitrite, nitrate, phosphate, silicate, as well as total concentrations of total dissolved nitrogen, phosphorus, and silicon in the area were scattered in the ranges: (ND-6.32; mean: 0.84 µg-at N/l), ND-3.02; mean: 0.42 µg-at N/l), (ND-10.88; mean: 1.18 µg-at N/1), (ND-4.22; mean: 0.62 µg-at P/l), (1.14-28.80; mean: 6.52 µg-at Si/l), (1.52-39.58; mean: 12.28 µg-at N/l), (0.40-4.98; mean: 1.07 µg-at P/l), and (2.77-44.74; mean: 13.02 Si/l) respectively. Of inorganic nitrogen species, ammonium was the highest in the Arabian Gulf waters and nitrate was the highest at the Gulf of Oman. The dissolved inorganic nitrogen total species, phosphate and silicate amounted to 16.4, 47.6, 56.5% respectively, of the concentrations of nitrogen, phosphorus and silicon in the Arabian Gulf and 22.6, 64.4, 44.9% respectively, in the Gulf of Oman, indicating that more than 80% of nitrogen was present in organic forms. Distributions of nutrient in the two regions were higher in the summer season and lower in the winter season due to the oxidation of organic materials. Regional distributions revealed higher values for nitrite (1.3 times), nitrate (2.8 times), phosphate (2.2 times), total dissolved nitrogen (1.3 times), total dissolved phosphorus (1.6 times), and total dissolved silicon (1.3 times) in the Gulf of Oman compared to the Arabian Gulf, indicating more oligotrophic conditions at the Arabian Gulf Whereas no distinct patterns of distribution were observed in the Arabian Gulf waters, an increase in the seaward direction was measured at the Gulf of Oman. Vertical distributions indicated a general increase with depth in the two regions. The mean ratios for total concentrations of phosphorus, nitrogen, and silicon in the Arabian Gulf (1: 11.6: 12.6) and the Gulf of Oman (1: 10.1: 11.8) were lower than the Redfield ratio.