Graduation date: 1994

Thesis (Ph. D.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains ix, 89 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 82-88).

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The endolysosomal system is a multifunctional system and is involved in catabolism, antigen presentation and regulation of hormones. The descriptions of, and functions assigned to organelles within the system are often applied using different criteria. Further, the properties of the hydrolases within the system, and the organelles that house them are usually studied in isolation from one another. Considering that the endolysosomal system may be extremely dynamic, an understanding of the system as an integrated whole is a necessary first step. Thus, a review of the endolysosomal system was undertaken. It was determined that the enzymes within the endolysosomal system are probably subject to 'organelle-dependent' regulation, i.e. the organelles create the appropriate luminal conditions for these enzymes to work. It is also likely that the effectors of these luminal conditions are regulated in a manner that is related to GTPase networks that control much of the cell's functions. The organisation of the endolysosomal system may be hierarchical, with proteases being downstream effectors of a system that is regulated at the whole body level. The main endoprotease class within the endolysosomal system are cysteine proteases. A literature review suggested that these enzymes may not be redox regulated within the endolysosomal system. However, the lysosomal endoprotease cathepsin B has been implicated in many pathologies where it is operating in pH and redox conditions different from the endolysosomal system. To study this, cathepsin B was isolated from bovine livers using a novel procedure that exploits the ability of tertiary butanol to temporarily inhibit protease interactions in tissue homogenates. This would prevent artefactual, as well as protease-inhibitor interactions. This novel procedure resulted in increased yields of cathepsin B. Cathepsin D, an aspartic protease, was isolated using established methods. In order to test the hypothesis that cathepsin B may be redox regulated in vivo, cathepsin B activity and stability were measured in cysteine and/or cystine-containing redox buffers. Cathepsin B activity in cysteine-containing buffers was similar at pH 6.0 and pH 7.0, over all thiol concentrations tested. In contrast, the stability of the enzyme was greater at pH 6.0 than at pH 7.0. This suggests that the enzyme's operational pH in vivo may be < pH 7.0. The activity of the enzyme was depressed in glutathione-containing buffers. When assessed in cysteine:cystine redox buffers (pH 6.0 - 7.0) cathepsin B was active over a broad redox potential range, suggesting that cathepsin B activity may not be redox regulated. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2003.

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Proteolytic processes are often mediated by highly orchestrated cascades, through which protease enzymes function coordinately to ensure a stepwise activation. This thesis presents experimental data which supports and complements the previously postulated mechanism of KLK (kallikrein-related protease) activation through proteolytic cascades. Further examination of the seminal KLK cascade has revealed several of its key (patho) physiological roles in human reproductive system. Multiple members of the seminal KLK cascade, in particular KLK14, were shown to play a pivotal role in regulating semen liquefaction. The cascade was further shown to be tightly regulated through a series of highly orchestrated feedback loops, to prevent deleterious effects due to aberrant protease activation. Accordingly, a strong association was observed between the expression level of several seminal KLKs, delayed liquefaction, and other markers of semen quality, including semen hyperviscosity. Furthermore, a strong association was found between delayed liquefaction and abnormal sperm motility. Therefore, dysregulated KLK expressions and/or activities were proposed as an underlying cause of male subfertility. Finally, this thesis has provided initial insights into a novel potential function of multiple members of the seminal KLK cascade in activation of the key immune-deviating agent, TGFβ1, in seminal plasma. TGFβ1 activation is postulated to be mediated directly through complete fragmentation or indirectly through partial cleavage and conformational changes of the LAP propeptide motif of the latent TGFβ1. KLK- mediate proteolytic cleavage of the TGFβ1 binding protein, LTBP1, is also suggested as a potential physiological mechanism for release of the membrane-bound latent TGFβ1. Overall, the data provided here may suggest a common regulatory mechanism, involved co-temporally in the two key processes of semen liquefaction and immune-suppression. This might be critical in protecting motile sperms following their release from semen coagulum. Understanding KLK-mediated proteolytic events in seminal plasma can shed light not only on the physiological role of this family of enzymes, but also on some of causes of male subfertility. Accordingly, therapeutic induction of this cascade may be utilized to supplement the current clinical treatment of male subfertility. Conversely, targeted inhibition of key components of the cascade may have potential pharmaceutical utility as a novel topical contraceptive strategy.

Cathepsin B was the most active cysteine proteinase in the Pacific whiting (Merluccius productus) fish fillet, and cathepsin L in surimi when the activities of the most active cysteine proteinases (cathepsin L, B, and H) were compared. Cathepsin L showed maximum activity at 55°C in both fish fillet and surimi, indicating its function in myosin degradation during conventional cooking of fish fillet and surimi. Washing during surimi processing removed cathepsin B and H but not cathepsin L. Autolytic analysis of surimi proteins showed that the myosin was the primary target, while actin and myosin light chain showed limited hydrolysis during 2 hr incubation. When purified Pacific whiting proteinase was incubated with various component of fish muscle, proteinase was capable of hydrolyzing purified myofibrils myosin, and native and heat-denatured collagen. The degradation pattern of myofibrils by the proteinase was the same as the autolytic pattern of surimi. Inhibition by the food-grade proteinase inhibitors varied with the catalytic type of proteinase. Beef plasma protein (BPP) had a higher percentage of papain inhibitors, followed by whey protein concentrate (WPC), potato powder (PP), and egg white (EW). On the other hand, EW had a higher percentage of trypsin inhibitors followed by BPP, PP, and WPC. EW inhibited trypsin activity completely at levels as low as 1%. WPC inhibited the autolytic activity of fresh surimi. Bovine serum albumin (BSA) was not effective as WPC. WPC can be used as an inhibitor for the Pacific whiting surimi, but high concentration is required. A limited number of inhibitory components were found, as the components in food-grade inhibitors were characterized by inhibitory activity staining. Both EW and PP showed more serine proteinase inhibitors than cysteine proteinase inhibitors. PP showed one cysteine inhibitory component while EW did not show any. BSA in both WPC and BPP acts as an nonspecific competitive inhibitor and reduces the enzyme activity. An unidentified high molecular weight protein (HMP) found in WPC, BPP, and BSA functions as an alternative substrate for papain while it functions as true inhibitor for trypsin. / Graduation date: 1995

Thesis (Ph. D.)--University of Adelaide, Dept. of Biochemistry, 1984. / Includes bibliographical references (leaves 118-130).