RESUMEN
We previously found a novel chymotrypsin-like protease in honeybee, designated as HCLPase. The recombinant enzyme expressed in insect cells was produced and compared to that in Escherichia coli. Both enzymes showed equivalent molecular size and specificity. However, HCLPase produced in insect cells showed higher specific activity. The C-terminal cleavage sites of HCLPase were phenylalanine, leucine, and tyrosine residues.
Asunto(s)
Quimasas/química , Expresión Génica , Proteínas de Insectos/química , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Abejas , Bovinos , Quimasas/antagonistas & inhibidores , Quimasas/genética , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Insectos/antagonistas & inhibidores , Proteínas de Insectos/genética , Cinética , Leucina/química , Oligopéptidos/química , Fenilalanina/química , Inhibidores de Proteasas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Albúmina Sérica Bovina/química , Células Sf9 , Spodoptera , Especificidad por Sustrato , Tirosina/químicaRESUMEN
Royal jelly (RJ) intake lowers serum cholesterol levels in animals and humans, but the active component in RJ that lowers serum cholesterol level and its molecular mechanism are unclear. In this study, we set out to identify the bile acid-binding protein contained in RJ, because dietary bile acid-binding proteins including soybean protein and its peptide are effective in ameliorating hypercholesterolemia. Using a cholic acid-conjugated column, we separated some bile acid-binding proteins from RJ and identified the major RJ protein 1 (MRJP1), MRJP2, and MRJP3 as novel bile acid-binding proteins from RJ, based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Purified MRJP1, which is the most abundant protein of the bile acid-binding proteins in RJ, exhibited taurocholate-binding activity in vitro. The micellar solubility of cholesterol was significantly decreased in the presence of MRJP1 compared with casein in vitro. Liver bile acids levels were significantly increased, and cholesterol 7α-hydroxylase (CYP7A1) mRNA and protein tended to increase by MRJP1 feeding compared with the control. CYP7A1 mRNA and protein levels were significantly increased by MRJP1 tryptic hydrolysate treatment compared with that of casein tryptic hydrolysate in hepatocytes. MRJP1 hypocholesterolemic effect has been investigated in rats. The cholesterol-lowering action induced by MRJP1 occurs because MRJP1 interacts with bile acids induces a significant increase in fecal bile acids excretion and a tendency to increase in fecal cholesterol excretion and also enhances the hepatic cholesterol catabolism. We have identified, for the first time, a novel hypocholesterolemic protein, MRJP1, in RJ. Interestingly, MRJP1 exhibits greater hypocholesterolemic activity than the medicine ß-sitosterol in rats.
Asunto(s)
Ácidos Grasos/química , Glicoproteínas/química , Proteínas de Insectos/química , Animales , Western Blotting , Células CACO-2 , Caseínas/farmacología , Colesterol/sangre , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Glicoproteínas/farmacología , Células Hep G2 , Humanos , Proteínas de Insectos/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratas , Sitoesteroles/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
A flow-injection spectrophotometric method has been developed for the simultaneous determination of selenium(IV) and (IV + VI) at nanogram per milliliter levels. It is based on the catalytic effect of selenium(IV) on the photooxidative coupling of p-hydrazinobenzenesulfonic acid (HBS) with N-(1-naphthyl)ethylenediamine (NED) to form an azo dye (lambda(max) = 538nm). In this reaction, bromide acted as an activator for the catalysis of selenium(IV) and an reducer for selenium(VI) to selenium(IV) in an acidic medium which allowed the determination of selenium(IV + VI). A sample solution, being split by Y-piece into two portions, passed through the low-temperature coil (4m, 25 degrees C) and the high-temperature coil (20m, 100 degrees C). By monitoring the absorbance of the dye produced in the two portions, selenium(IV) and (IV + VI) in the range of 0.2-6ngml(-1) were determined simultaneously. The relative standard deviations for 3ngml(-1) selenium(IV) and (VI) (n = 10) were 1.2 and 1.3%, respectively. There were few interfering ions in the selenium determination. The proposed method was applied to the determination of selenium(IV) and (VI) in natural water samples.
RESUMEN
A flow-injection chemiluminescent (CL) method is proposed for the successive determination of nanogram levels of vanadium(IV) and total vanadium. The method is based on the catalytic effect of vanadium(IV) on the oxidation of purpurogallin by periodate to produce light emission at 4 degrees C. The presence of hydrogen carbonate enhanced the light emission arising from the vanadium(IV)-catalyzed reaction. Since vanadium(V) did not catalyze the CL reaction of purpurogallin, vanadium(V) was determined after being reduced to vanadium(IV) by using an on-line silver-reducing column. Calibration curves for vanadium(IV) and (V) were linear in the range 0.1-10 ng ml(-1) with sampling rate of about 50 h(-1). The limit of detection for signal-to-noise ratio of 2 was 0.05 ng ml(-1) and the relative standard deviations were 1.4 and 1.6% for ten determinations of 2.0 ng ml(-1) vanadium(IV) and (V), respectively. Interferences from metal ions could be eliminated by the use of O,O'-bis(2-aminoethyl)ethyleneglycol- N,N,N',N'-tetraacetic acid and diphosphate as masking agents. The proposed method was successfully applied to the determination of vanadium(IV) and total vanadium in fresh water samples.