RESUMEN
Cardiovascular effects of subcutaneous administration of synthetic alpha-lactorphin, a tetrapeptide (Tyr-Gly-Leu-Phe) originally derived from milk alpha-lactalbumin, were studied in conscious spontaneously hypertensive rats (SHR) and in normotensive Wistar Kyoto rats (WKY) with continuous radiotelemetric monitoring. Alpha-lactorphin dose-dependently lowered blood pressure (BP) without affecting heart rate in SHR and WKY. The lowest dose which reduced BP was 10 microg/kg, and the maximal reductions in systolic and diastolic BP (by 23+/-4 and 17+/-4 mm Hg, respectively) were observed at 100 microg/kg dose in SHR. No further reductions were obtained at a higher dose of 1 mg/kg. There were no significant differences in the BP responses to alpha-lactorphin between SHR and WKY. Naloxone (1 and 3 mg/kg s.c.), a specific opioid receptor antagonist, abolished the alpha-lactorphin-induced reduction in BP and reversed it into a pressor response, which provides evidence for an involvement of opioid receptors in the depressor action of the tetrapeptide.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Presión Sanguínea/efectos de los fármacos , Hipertensión/tratamiento farmacológico , Oligopéptidos/farmacología , Animales , Bradiquinina/análogos & derivados , Bradiquinina/farmacología , Captopril/farmacología , Relación Dosis-Respuesta a Droga , Hipertensión/fisiopatología , Masculino , Naloxona/farmacología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptores Opioides/efectos de los fármacos , Receptores Opioides/fisiologíaRESUMEN
The aim of this study was to identify whey-derived peptides with angiotensin I-converting enzyme (ACE) inhibitory activity. The bovine whey proteins alpha-lactalbumin and beta-lactoglobulin were hydrolysed with pepsin, trypsin, chymotrypsin, pancreatin, elastase or carboxypeptidase alone and in combination. The total hydrolysates were fractionated in a two step ultrafiltration process, first with a 30 kDa membrane and then with a 1 kDa membrane. Inhibition of ACE was analysed spectrophotometrically. The peptides were isolated by chromatography and identified by mass and sequencing analysis. The most potent inhibitory peptides were synthesized by the 9-fluorenylmethoxycarbonyl solid phase method. Inhibition of ACE was observed after hydrolysis with trypsin alone, and with an enzyme combination containing pepsin, trypsin and chymotrypsin. Whey protein digests gave a 50% inhibition (IC50) of ACE activity at concentration ranges within 345-1733 micrograms/ml. The IC50 values for the 1-30 kDa fractions ranged from 485 to 1134 micrograms/ml and for the < 1 kDa fraction from 109 to 837 mg/ml. Several ACE-inhibitory peptides were isolated from the hydrolysates by reversed-phase chromatography, and the potencies of the purified peptide fractions had IC50 values of 77-1062 microM. The ACE-inhibitory peptides identified were alpha-lactalbumin fractions (50-52), (99-108) and (104-108) and beta-lactoglobulin fractions (22-25), (32-40), (81-83), (94-100), (106-111) and (142-146).
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Proteínas de la Leche/metabolismo , Fragmentos de Péptidos/farmacología , Animales , Carboxipeptidasas/metabolismo , Bovinos , Cromatografía Líquida de Alta Presión , Quimotripsina/metabolismo , Hidrólisis , Lactalbúmina/química , Lactalbúmina/metabolismo , Lactoglobulinas/química , Lactoglobulinas/metabolismo , Elastasa Pancreática/metabolismo , Pancreatina/metabolismo , Pepsina A/metabolismo , Fragmentos de Péptidos/aislamiento & purificación , Tripsina/metabolismo , Proteína de Suero de LecheRESUMEN
Bovine milk proteins alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) were hydrolysed with seven different proteolytic enzymes, and the effect of various hydrolysates on a genetically modified luminous Escherichia coli JM103 was tested in vitro with a bioluminescence assay for bacterial growth and metabolism. Undigested proteins did not inhibit the activity of tested E. coli JM103 at a concentration as high as 0.1 g ml-1. At the same concentrations, alpha-la hydrolysed with pepsin or trypsin and beta-lg hydrolysed with alcalase, pepsin or trypsin, showed a lower metabolic activity during the first 8 h of growth. The activity of E. coli JM103 in the presence of 25 mg ml-1 alpha-la or beta-lg hydrolysed with pepsin and trypsin was only 21% of the control after incubation for 6 h. The preliminary results indicated that ultrafiltration through 10 kDa and 1 kDa molecular mass cut-off membranes may be used to enrich bacteriostatic properties.
Asunto(s)
Escherichia coli/metabolismo , Lactalbúmina/metabolismo , Lactoglobulinas/metabolismo , Escherichia coli/crecimiento & desarrollo , Hidrólisis , Mediciones Luminiscentes , Pepsina A/metabolismo , Tripsina/metabolismo , UltrafiltraciónAsunto(s)
Endopeptidasas/metabolismo , Lactoglobulinas/metabolismo , Músculo Liso/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Secuencia de Aminoácidos , Inhibidores de la Enzima Convertidora de Angiotensina , Animales , Bovinos , Quimotripsina/metabolismo , Cobayas , Hidrólisis , Íleon/efectos de los fármacos , Lactoglobulinas/química , Datos de Secuencia Molecular , Contracción Muscular/efectos de los fármacos , Oligopéptidos/química , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Pepsina A/metabolismo , Fragmentos de Péptidos/química , Tripsina/metabolismoRESUMEN
The expulsion mechanism of xylitol 5-phosphate in Streptococcus mutans ATCC 25175 was studied using resting cells incubated in the presence of 14C-xylitol. The expulsion appeared to be a two-step process: xylitol 5-phosphate was first hydrolyzed to xylitol and inorganic phosphate, and the xylitol was subsequently expelled from the cells. The dephosphorylation step appeared to be energy-requiring and it was most likely associated with a phosphatase which was active on xylitol 5-phosphate. Two to three successive cultivations of the cells in the presence of 6% xylitol increased this enzyme activity 4.3-fold. These results are in accordance with the presence of an energy-dependent xylitol 5-phosphate cycle in S. mutans, which is regulated by exogenous xylitol.
Asunto(s)
Pentosafosfatos/farmacocinética , Streptococcus mutans/metabolismo , Arginina/farmacología , Arseniatos/farmacología , Radioisótopos de Carbono , Cromatografía por Intercambio Iónico , Metabolismo Energético , Fructosa/farmacología , Galactosafosfatos/metabolismo , Glucosa/farmacología , Glucofosfatos/metabolismo , Hidrólisis , Monoéster Fosfórico Hidrolasas/farmacocinética , Fluoruro de Sodio/farmacología , Streptococcus mutans/enzimología , Xilitol/metabolismoRESUMEN
The effect of successive cultivations in the presence of 6% xylitol on the uptake and expulsion of 14C-xylitol was studied using the cells of Streptococcus mutans 25175. Three sequential cultivations did not alter the growth inhibition percentage (approximately 50%) observed in the presence of 6% xylitol. The 14C-xylitol uptake experiments performed with growing and resting cells showed that both the uptake and the expulsion of xylitol were enhanced by xylitol-culturing. Both xylitol-cultured and resting control cells contained only one major labeled compound which was identified as 14C-xylitol 5-phosphate. The label subsequently was expelled from the cells as 14C-xylitol. These results indicate that S. mutans possesses an intracellular xylitol cycle and this cycle is regulated by adding xylitol to the growth medium.
Asunto(s)
Streptococcus mutans/metabolismo , Xilitol/metabolismo , Radioisótopos de Carbono/metabolismo , División Celular/efectos de los fármacos , Cromatografía en Capa Delgada , Medios de Cultivo , Farmacorresistencia Microbiana , Streptococcus mutans/efectos de los fármacos , Xilitol/farmacologíaRESUMEN
The effect of three successive cultures in the presence of 6% xylitol on the uptake of 14C-xylitol was studied using resting cells of Streptococcus sobrinus ATCC 27352 and Streptococcus mitis ATCC 36249. In the case of S. mitis, the three successive cultures did not alter the growth inhibition observed in the presence of xylitol. In the case of S. sobrinus, however, growth inhibition decreased. The 14C-xylitol uptake experiments also demonstrated that uptake of xylitol by S. sobrinus was decreased by culture in the presence of xylitol. Previous 14C-xylitol uptake levels were, however, re-established by culturing S. sobrinus in the presence of glucose alone. Culture in the presence of xylitol did not affect 14C-xylitol uptake in the case of S. mitis. These results show that S. sobrinus and S. mitis differ in the ways they handle of exogenous xylitol, and that uptake of xylitol by S. sobrinus could be reversibly regulated by addition xylitol to the growth medium.