Isolation and characterization of a metal ion-dependent alkaline protease from a halotolerant Bacillus aquimaris VITP4.

A halotolerant bacterium Bacillus acquimaris VITP4 was used for the production of extracellular protease. Fractional precipitation using ammonium chloride was used to obtain the enzyme. The protease exhibited optimum activity at pH 8.0 and 40°C and retained 50% of its optimal proteolytic activity even in the presence of 4 M NaCl, suggesting that it is halotolerant. The molecular mass of protease, as revealed by SDS-PAGE was found to be 34 kDa and the homogeneity of the enzyme was confirmed by gelatin zymography and reverse-phase HPLC. Upon purification, the specific activity of th enzyme increased from 533 U/mg to 1719 U/mg. Protease inhibitors like phenyl methane sulphonyl fluoride and 2-mercaptoethanol did not affect the activity of the enzyme, but EDTA inhibited the activity, indicating the requirement of metal ions for activity. Cu­­­2+, Ni2+ and Mn2+ enhanced the enzyme activity, but Zn2+, Hg2+ and Fe2+ decreased the activity, while Mg2+, Ca2+ and K+ had no effect on the enzyme activity. The protease was quite stable in the presence of cationic (CTAB), anionic (SDS) and neutral detergents (Triton X-100 and Tween-20) and exhibited antimicrobial activity against selected bacterial and fungal strains. The stability characteristics and broad spectrum antimicrobial activity indicated the potential use of this protease in industrial applications.

Avaliação morfológica de diferentes técnicas de desepitelização da membrana amniótica humana / Morphological assessment of different amniotic membrane epithelial denuding techniques

OBJETIVO: Avaliar as características morfológicas da membrana amniótica desepitelizada por diferentes técnicas. MÉTODOS: A membrana amniótica humana foi coletada no momento do parto, fixada em concentrações crescentes de glicerol (0-50 por cento em DMEM) e preservada a 80°C até a hora de ser usada. O estudo consistiu de 4 grupos: epitélio intacto (controle) e membranas desepitelizadas pela tripsina (2 mg/mL a 1:250), dispase (1,2 U/mL em solução salina balanceada de Hank livre de Mg2+ e Ca2+) e ácido etilenodiaminotetra-acético (EDTA), 0,02 por cento). As amostras foram submetidas à análise por microscopia eletrônica (de varredura e de transmissão). RESULTADOS: A microscopia eletrônica de varredura mostrou epitélio intacto no grupo controle e sua ausência nas membranas amnióticas desepitelizadas pela tripsina e pela dispase. Naquelas tratadas com o ácido etilenodiaminotetra-acético, havia áreas com e sem epitélio. Quando avaliadas pela microscopia eletrônica de transmissão, o epitélio estava intacto e firmemente aderido à membrana basal através de hemidesmossomos nos grupos controle e em parte do ácido etilenodiaminotetra-acético. Havia apenas fibras colágenas nas membranas tratadas com dispase e tripsina. CONCLUSÕES: O tratamento da membrana amniótica com tripsina e dispase pode causar completa retirada do epitélio e da membrana basal, ao passo que o ácido etileno- diaminotetra-acético pode preservar áreas com epitélio intacto e parcialmente destruir a membrana basal em outras.

PURPOSE: To evaluate the morphological features of the amniotic membrane denuded by different techniques. METHODS: Human amniotic membrane was collected at the time of delivery, fixed in increasing concentrations of glycerol (0-50 percent in DMEM) and preserved at -80°C until the time of use. The study consisted of 4 groups: intact epithelium (control) and denuded by trypsin (2 mg/mL at 1:250), dispase (1.2 U/mL in Mg2+ and Ca2+ free Hank's balanced salt solution) or ethylenediaminetetraacetic acid (EDTA), 0.02 percent. Specimens were submitted to electron (scanning and transmission) microscopy analysis. RESULTS: Scanning electron microscopy disclosed intact epithelium in the control group and its absence in the amniotic membranes denuded by trypsin and dispase. In those denuded by ethylenediaminetetraacetic acid there were areas with and without epithelium. When assessed by transmission electron microscopy, the epithelium was intact and firmly adhered to the basement membrane by hemidesmossomes in controls and in parts of ethylenediaminetetraacetic acid group. There were only collagen fibers in the dispase- and trypsin-treated groups. CONCLUSIONS: Trypsin and dispase treatment of the amniotic membrane may cause complete denuding of the epithelium and basement membrane whereas ethylenediaminetetraacetic acid may leave some intact epithelium-areas and partially destroy the basement membrane in others.

Susceptibility of lactase to luminal proteases in developing rat intestine.

BACKGROUND: Postnatal development of rat intestine is associated with a decline in brush-border lactase activity. This phenomenon is similar to the adulthood hypolactasia in humans. However, the mechanism underlying this process is not understood. METHODS: The effect of luminal proteases from adult rat intestine on the intestinal lactase activity in animals aged 7, 14, 21 and 30 days was studied in in vitro experiments. Lactase levels were estimated using enzyme assays and Western blot analysis. RESULTS: Incubation of purified brush borders with increasing concentrations of luminal proteases reduced the lactase activity in intestine of 7-day-old rats, but not in that of adult animals. Western blot analysis revealed low signal of the 220-kDa lactase protein in 7-day-old animals, but not that of older weaned animals. CONCLUSIONS: Our findings suggest that luminal proteases may be responsible for the maturational decline in intestinal lactase activity.

Enzima inativadora de bradicinina liberada de fígado preservado ex-vivo / Bradykinin-inactivating enzyme is released from the ex-vivo stored liver

Objetivo - Ofígado inativa quantidades consideráveis de bradicinina; a principal enzima hepática cinino-inativadora (BIE, bradykinin inativating endopeptidase) hidrolisa especificamente a ligaçao Phe-Ser do nonapeptídio e foi caracterizada como sendo a oligoendopeptidase EC 3.4. 24.15. No transplante ortotópico de fígado existe correlaçao entre aumento da concentraçao de aminoácidos no líquido de preservaçao (conseqUência de proteólise) e falência do enxerto. O objetivo deste trabalho é verificar se ocorre liberaçao da BIE de fígados preservados ex-vivo no líquido Braun-Collins ou em soluçao de Krebs-Henseleit bicarbonato (Krebs). Método. Fígado de ratos Wistar (180-220g) foram exsangüinados e a pós remoçao foram preservados em líquido Braun Collins ou em soluçao Krebs, a 4 graus Celsius. Foram retiradas alíquotas do líquido de preservaçao nos tempos 0, 4, 8 e 24 horas, para dosagem de ALT, AST, DHL e BIE. A atividade fluorimétrica da BIE foi ensaiada com o substrato Abz-RPPGFSPFRQ-EDDnp (análogo sintético da bradicinina) e sua presença confirmada por immunoblotting, revelado com anticorpo específico anti-EC 3.4.24.15. Resultados. A liberaçao de ALT, AST, DHL e BIE é significativa no período 8-24 hs. Nas alíquotas de 24 hs, em relaçao ao tempo zero, a concentraçao das quatro enzimas aumentou, respectivamente, no líquido Braun Collins, 8, 7, 19 e 10 vezes e, na soluçao de Krebs, 21, 17, 27 e 21 vezes; a relaçao ALT/DHL foi sempre inferior a um. Conclusao. Ocorre liberaçao de BIE durante a preservaçao ex-vivo do fígado, o que poderá servir como indicaçao da condiçao de preservaçao do enxerto; diminuiçao da capacidade cinino-inativadora do fígado poderá afetar sua reatividade vascular.

Isolation and characterization of brain-specific transglutaminases from rat

The relevance of transglutaminases with neural function and several disorders has been emphasized recently. Especially, many polypeptides associated with neurodegenerative diseases are suggested to be putative transglutaminase substrates such as beta amyloid protein of Alzheimer's disease, microtubule-associated proteins and neurofilaments, etc. In addition, the CAG repeated gene products with probable polyglutamine tract, putative transglutaminase substrates, were identified in several neurodegenerative disorders. However, the identity of the brain transglutaminase has not been confirmed, because of enzymic stability and low activity. In the present experiment, we have isolated brain-specific transglutaminases, designated as TGase NI and TGase NII, which are different from other types of transglutaminases in respects of molecular weights (mw. 45 kDa, 29 kDa respectively), substrate affinity, elution profile on ion-exchange chromatography, sensitivity to proteases and ethanol, and immunological properties. The enzymes were localized specifically in the brain tissues but not in the liver tissue. And neural cells such as pheochromocytoma cell, glioma cell, primary neuronal and glial cells were shown to be enriched with TGase NI and TGase NII. The possible biological roles of the enzymes were discussed not only on the aspect of crosslinking activity but also of signal transducing capacity of the enzyme in the brain.

Isolation and partial characterization of Lachesis muta melanocephala coagulant proteinase: biochemical parameters of the venom

The coagulant proteinase of L. m. melanocephala was purified by DEAE Sephadex A-50 followed by agmatine CH- Sepharose and gel filtration on Sephadex G-100. The enzyme exhibited m,any of the properties adscribed to -mudasa-, the coagulant proteinase from L. m. stenophirs venom. Its molecular weight by gel filtration was 35 kDa and its specific clotting activity was 702 NIH/mg of protein. A 32 fold increase in the clotting was obtained by purification. The coagulant proteinase exibited esterolytic activities toward lysine and arginine esters as well as amidolytic. Significant differences are observed when compared with the activities of -mudasa-, the former is less esterolytic, although its activity toward TLEME is higher. Significant differences in the activities are also observed when the venom from the Pacific and Atlantic L. muta populations (corresponding to the subspecies L.m. melanocefala and L.m. Stenophyrs) are compared toward the same substrates. The Pacific type is less amidolytic and more esterolytic toward BAME and BAEE, although toward the lysine and tyrosine esters no significant differences are observed. The venon from the Pacific populations is more coagulant and less proteolytic than the venom from the Atlantic population. Analytical isoelectric focusing of both populations of venom revealed important differences in the number and intensity of the protein bands. The results here given further substantiate the taxonomical differentiation already given to the Pacific and Atlantic Costa Rican population of L. muta.