Graduation date: 1941

Thesis (M. Med. Sc.)--University of Hong Kong, 2008. / Includes bibliographical references (p. 58-64)

Orientador : Julia Prado Franceschi / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-14T00:31:18Z (GMT). No. of bitstreams: 1 Donato_JoseLuiz_M.pdf: 3688292 bytes, checksum: 2e7a0147e00d44423d6b315b1646bccc (MD5) Previous issue date: 1991 / Resumo: Peçonhas ofídicas são constituídas principalmente de proteínas. Muitas destas proteínas tem características enzimáticas como as fosfolipases, proteases, esterases, L-aminoacido oxidase, entre outras. Inicialmente, comparamos as atividades enzimáticas e biológicas da peçonha de Bothrops Lanceolatus em relação as atividades de sete espécie de bothrops brasileiras (B-alternatus, B. erythromelas, B. insularis, B. jararaca, B. jararacussu, B moojeni e B. neuwiedi). Determinados assim, que a peçonha Bothroes lanceolatus apresenta altas atividades caseinolitica, fosfolipasica, esterasica, sobre o TAME e hemorragica, Além disso, apesar de não exibir atividade coagulante sobre plasma humano citratado, esta peçonha coagula o fibrinogenio o que sugere a presença de enzima (s) tipo trombina. Verificamos que, entre as peçonhas estudadas, a atividade TAME-esterasica não está diretamente relacionada com a atividade coagulante sobre plasma. O isolamento da fração esterasica da peçonha de B.lanceolatus foi feito utilizando-se cromatografias de exclusão e de troca iônica. Em cada etapa do isolamento, as amostras foram ensaiadas em relação as atividades TAME-esterasica, caseinolitica, fosfolipasica e coagulante sobre plasma e fibrinogênio humanos. Ao Final deste processo, foi obtida uma fração (F-II-1a ) com atividade esterasica 14,5 vezes maior do que a peçonha total. O rendimento protéico foi de 4%, com uma recuperação de 58% da atividade enzimática. Utilizando-se eletroforese em gel de poliacrilamida-SDS, constatamos que a proteínas presente na fração F-II-1a apresenta uma única cadeia polipeptídica, com peso molecular estimado em 38.100. Em testes de imunoeletroforese e imunodifusão, esta fração reagiu com o soro especifico como com o polivalente promovendo a formação de apenas uma linha precipitação. Estes resultados sugerem elevado grau de pureza da proteína presente na fração F-II-1a Utilizando o TAME como substrato, a fração F-II-1a apresentou os valores de 8,5 X 10 ¿4 M e 38,55 umoles/min/mg para km e velocidade máxima, respectivamente. A atividade TAME esterasica foi fortemente influenciada pelo pH do meio de reação, sendo o valor de 8,05 encontrado como o pH ótimo de hidrolise.Esta atividade não foi afetada pela EDTA 10 mM, ao passo que foi totalmente inibida por PMSF 5mM. A fração F-II-1a apresentou baixa atividade coagulante sobre plasma e fibrinogênio humanos, bem como, uma discreta atividade proterminado que a fração F-II-1a apresenta atividade A'alfa¿ e B 'beta¿ fibrinogenolitica. A cadeia A 'alfa¿ foi rapidamente hidrolisada(10min de reação), enquanto que a B¿beta¿ só foi hidrolisada em tempo prolongados de reação . Esta fração não teve efeito sobre a cadeia 'alfa¿, mesmo após 4h de hidrolise / Mestrado / Bioquimica / Mestre em Ciências Biológicas

Serine proteases are known to perform a wide range of functions essential to life; however there has to be some form of control mechanism in place. One of the many control mechanisms is their specific inhibition by protein proteinase inhibitors. Proteinase inhibitors in plants, present in their seeds, participate in defense mechanisms and their production is induced by herbivory or wounding. Plant proteinase inhibitors have been reported to inhibit a variety of serine proteinases, including enzymes of the blood coagulation cascade. In this study, various indigenous seed extracts were screened for potential serine proteinase inhibition. Acacia schweinfurthii was selected as a potential inhibitor that inhibited trypsin and factor X. The AS inhibitor was successfully purified to homogeneity by precipitating with 80 percent (v/v) acetone and the sequential chromatographic steps including ion-exchange chromatography, size exclusion chromatography, affinity purification on a trypsin-agarose column and RP-HPLC. Reducing SDS-PAGE conditions revealed an inhibitor of two polypeptide chains A and B of approximate molecular weights 16 and 10 kDa, respectively, and under non-reducing conditions, 25 kDa was observed. The inhibitor was shown to inhibit trypsin, chymotrypsin and factor X indicating the dynamic nature of the reactive site. An enzyme: inhibitor ratio of 1:1, and a Ki of 3.45nM was determined for the AS inhibitor on trypsin, and the inhibitor also weakly inhibit chymotrypsin. AS inhibitor and STI inhibited factor X with a Ki values of 13.7nM and 77.5μM respectively. Amino acid analysis revealed Mmin values of the A- and B- chain of 15,000 and 7,800, respectively. The effect of seed extracts on the activated partial thrombin time (APTT) and prothrombin time (PT) was tested. No prolongation of the PT was obtained. For the crude extracts of AK and AS, IC200 values of 4.6 and 189.62 μg/mL, were respectively obtained. For the purified fractions of STI, AS and AK, IC200 values of 51.5, 114.31 and 893.8 μg/ml were observed, respectively. Keywords: proteinase inhibitors, Acacia species, trypsin inhibitor, FX inhibitor.

published_or_final_version / Dentistry / Master / Master of Dental Surgery

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Proteolytic degradation of fish flesh occurring at elevated temperatures is the primary limitation for the commercial utilization of arrowtooth flounder (ATF). Characterization of the autolytic activity of ATF muscle incubated at various pHs and temperatures indicated the involvement of heat-activated proteinases active at acidic and alkaline pHs. Further characterization of the proteinase extract from fish muscle indicated the proteinase was more active at acidic pH than at alkaline pH in hydrolysis of Z-Phe-Arg-NMec and all types of protein substrates tested. Based on molecular weight and hydrolytic properties, activity peak separated on size exclusion chromatography, or activity bands observed on activity-stained substrate gels were presumed to be cathepsin L or like. A muscle proteinase showing similar hydrolytic properties to a proteinase extract was purified to electrophoretic homogeneity and subsequently confirmed by kinetic studies to be cathepsin L. Therefore, the results clearly indicated that cathepsin L is primarily responsible for autolytic activity of ATF muscle and surimi at the elevated temperatures. Gelation of fish myofibrillar proteins, mainly myosin, is an important process for surimi production. Elucidation of the gelation mechanism and the effect of proteolysis on myosin provide information regarding protein interactions that improve ATF product quality. Heat-induced changes in physicochemical properties of myosin, free of endogenous proteinases, indicated myosin gelation consisted of two processes, denaturation and aggregation. ATF myosin was shown to be extremely sensitive to heat, resulting in denaturation at a lower temperature than other fish myosins. Denaturation began at 25°C and was initiated by the unfolding of the α-helical region. Following denaturation was the exposure of the hydrophobic and sulfhydryl residues, which were subsequently involved in aggregation and the gelation process. Changes in dynamic properties indicated ATF myosin formed a gel in three different stages, as shown by the first increase in gel rigidity at 28°C, followed by a decrease at 35°C and a second increase at 42°C. A model system using ATF myosin and papain was developed to investigate how proteolysis affects the heat-induced gelation of fish myosin. The addition of papain decreased the onset temperature and the rate at which G' developed during heating. DSC thermograms indicated papain significantly decreased the enthalpy required to induce myosin denaturation with no significant changes in the onset or the maximum temperature. Thermal denaturation kinetics indicated a decrease in both the activation energy of the denaturation process and the denaturation rate of myosin. Although myosin gels could be formed, structural disruption caused by proteolysis, i.e., reduction in molecular size and loss in structural domain, resulted in lowering of the gelling ability of myosin and rigidity of the formed gels. / Graduation date: 2000

No description available.

Cathepsin D is a lysosomal acid proteinase that exists as different isoforms. This enzyme is found in most cell types, and macrophages have particularly high levels of this enzyme. Cathepsin D has been implicated in some disease related inflammatory processes that have also been associated with increased macrophage infiltration. The existance of isoforms of cathepsin D suggested the possibility that individual forms of this enzyme may have immunologically unique characteristics. Monoclonal antibodies to cathepsin D could aid in the study of the immunological characteristics of individual isoforms of this enzyme as well as provide a means by which to screen a variety of sample types for cathepsin D levels. In this study we generated a panel of monoclonal anti-cathepsin D antibodies and used these to screen isoelectrically separated cathepsin D samples for unique immunoreactivity patterns. We also used these antibodies to measure changes in the levels of intra- and extracellular cathepsin D that accompany monocyte to macrophage differentiation, and changes that occur in response to the treatment of melanoma cells with tumor necrosis factor (TNF) and interferon. We found that there are unique immunological characteristics associated with individual isoforms of human liver cathepsin D as well as cathepsin D from different cell types. We also observed increases in levels of this enzyme as monocytic cells differentiate to macrophage-like cells. This indicates that cathepsin may be involved in some of the proteolytic processes mediated by macrophages. Different inducers of differentiation resulted in different cathepsin 1D immunoreactivity patterns, suggesting that there may be isoform specificity dependent on the inducer. Cathepsin D levels also increase in neoplastic cells treated with TNF and/or interferon indicating that cathepsin D may play some role in the mechanism of action of the tumor inhibitory effects of these agents. The use of monoclonal antibodies provides assays for cathepsin D which can be used with a variety of sample types and further provides enhanced specificity and sensitivity relative to other assay systems used in the past.

No description available.