Hepatic microsomal cytochrome P-450 has previously been postulated to be an important factor in determining the rates of hepatic haem biosynthesis and biodegradation. The basis for this proposal is that the haem moiety of cytochrome P-450 appears to be in equilibrium between binding to apocytochrome P-450 and existing in some form in the central hepatic pool of haem concerned with the regulation of the haem metabolic pathways. Consequently, any change in the levels of hepatic cytochrome P-450 would be anticipated to affect the pathways of hepatic haem biosynthesis and biodegradation. At the onset of this project, relatively few chemical agents were known to destroy cytochrome P-450 (either by degradation of the haem moiety of, or dissociation of the haem moiety from hepatic microsomal cytochrome P-450) and to affect hepatic haem biosynthesis and/or haem biodegradation (e.g. AIA, Cs₂ and various metals). We thus attempted to further establish the relationship between the ability of compounds to affect hepatic cytochrome P-450 and to affect hepatic haem metabolism in vivo, using the three anaesthetic agents, fluroxene, halothane and trichloroethylene. During the preparation of this thesis, several other chemicals have been found which destroy cytochrome P-450 and affect hepatic haem metabolism (e.g. norethisterone, morphine). In addition to the above, it has been attempted to clarify the roles of the degradation of different forms of cytochrome P-450 and of the different mechanisms of destruction of cytochrome P-450 in the control of hepatic haem metabolism. The three anaesthetic agents, fluroxene, halothane and trichloroethylene were chosen for study since they destroy cytochrome P-450 by apparently different mechanisms. Both fluroxene and trichloroethylene specifically degrade the haem moiety of different forms of cytochrome P-450, but fluroxene converts the haem moiety of cytochrome P-450 to an N-substituted porphyrin, while TCE apparently degrades the haem into uncoloured products. In contrast, halothane appears to degrade the haem of cytochrome P-450 to uncoloured products as well as to facilitate the dissociation of haem from intact cytochrome P-450.

The role of the carboxy terminal tail of the chicken gonadotropin-releasing hormone receptor was determined by testing the activity of a series of chicken gonadotropinreleasing hormone receptors with progressive deletions in their carboxyl terminus. The 55 amino acid carboxy terminal tail of the chicken gonadotropin-releasing hormone receptor was progressively truncated, resulting in cS320STOP, cR330STOP, cS337STOP, cS346STOP, cT35ISTOP, cD356STOP, cS366STOP and cC375STOP truncated mutants, which were all tested in parallel with the wild type chicken gonadotropin-releasing hormone receptor. Truncation of the entire carboxy terminal tail from the chicken gonadotropin-releasing hormone receptor, cS320STOP abolished gonadotropin-releasing hormone binding and gonadotropin-releasing hormone-induced inositol phosphate production. The loss of gonadotropin-releasing hormone binding by the cS320STOP-truncated mutant suggests that this receptor is possibly not expressed on the cell membrane, which might be due to improper receptor folding by cS320STOP. The carboxy terminal tail of the chicken gonadotropin-releasing hormone receptor might therefore be required for proper folding of newly formed chicken gonadotropin-releasing hormone receptors and expression of these receptors on the cell membrane. The cR330STOP mutant had a maximal gonadotropin-releasing hormone binding of ~12%, which is the lowest receptor expression detected. The amino acid region between P³¹⁹ and L³²⁹ might therefore play a role in receptor expression. Progressive increase in the carboxy terminal tail from L³²⁹ resulted in progressive increase in the receptor expression. Maximal gonadotropin-releasing hormone binding levels reached wild type levels at truncation of the cGnRHR at S³⁶⁶. These results indicate that the first 45 amino region, ie. between P³¹⁹ and S³⁶⁶ of the chicken gonadotropin-releasing hormone receptor carboxy terminal tail contains elements that promote receptor expression. Gonadotropin-releasing hormone-induced inositol phosphate production was enhanced for all the truncated receptors except cR330STOP and cS337STOP, though all the truncated receptors had coupling efficiency values larger than the wild type chicken gonadotropin-releasing hormone receptor. This enhanced inositol phosphate production might be due to an increased coupling efficiency between the truncated chicken gonadotropin-releasing hormone receptors and the aq111-type G-protein. However, none of the truncated chicken gonadotropin-releasing hormone receptors have left-shifted EC50 values, indicating that coupling efficiency did not increase. Alternatively, a loss or retardation in receptor desensitization and/ or internalization for the truncated chicken gonadotropin-releasing hormone receptor mutants might be responsible for the enhanced gonadotropin-releasing hormone-induced inositol phosphate production by the truncated chicken gonadotropin-releasing hormone receptors. The chicken gonadotropin-releasing hormone receptor has a highly conserved cysteine residue in position 328 that might be palmitoylated. Replacing this cysteine in the chicken gonadotropin-releasing hormone receptor with an alanine [cC328A] increased receptor expression 2 fold, reduced maximal inositol phosphate production to ~69% and severely impaired coupling efficiency to 30% relative to the wild type levels. This finding indicates that C³²⁸ might be palmitoylated and is required for receptor coupling. In conclusion, the ammo terminal region of the chicken gonadotropin-releasing hormone receptor carboxy terminal tail increases receptor expression, either by affecting the transport of newly synthesized chicken gonadotropin-releasing hormone receptors to the plasma membrane and/or the proper folding of this receptor. The intracellular carboxy terminal tail of the chicken gonadotropin-releasing hormone receptor might play a negative role in G-protein coupling. However, the enhanced inositol phosphate production from the truncated chicken gonadotropin-releasing hormone receptors could be due to reduced internalization and/ or desensitization of the carboxy terminal truncated receptors. Point-mutation of C³²⁸ to A resulted in decreased coupling suggesting that C³²⁸ may be a palmitoylation site and might play a role in coupling or desensitization.

The objective of this study was to investigate the cell- and species-specific regulation of the α2(1) pro collagen gene by analysing trans-acting factor interactions within the proximal promoter of the gene and to identify the genes coding for these trans-acting factors. α2(1) procollagen gene expression was examined in a number of diff erentiate<;l cell lines and shown to differ significantly between normal fibroblasts (WI-38, FG₀), transformed fibroblasts (CT-1, SVWI-38), HT1080 fibrosarcoma, HepG2 hepatocellular carcinoma, L77 lymphoblasts and breast cancer epithelial cells (MDA-MB-231, ZR-75-2). These differences were due to changes in transcription of the α2(1) procollagen gene as shown by Northern blot analysis and nuclear runon transcription experiments . Analysis of DNA-protein interactions with the proximal α2(1) procollagen promoter showed the presence of at least two DNA-protein complexes (complexes I and III) in collagen producing cell lines, while cells where collagen synthesis did not occur contained a third DNA-protein complex (complex II). α2(1) procollagen gene expression was therefore shown to be associated with the presence of complexes I and III while repression of the gene was associated with the presence of complexes I and II and the partial or complete absence of complex III. Complex I is a ubiquitous factor which binds the inverted CCAAT box located between -92 and -80 (G/CBE) with an apparent Kd of 2.9nM. Complexes II and III both bind an adjacent DNA sequence between -78 and -67 (the CME) with Kd values of 4.2 and 3.5nM respectively. While the CCAA T boxes in the human and mouse promoters are identical, a 3bp mismatch was detected in the CME. This mismatch abolished the formation of complex II and III on the mouse promoter, even though mouse cells contained complex II proteins. The difference in the CME binding site between rodent and human promoters implied species-specific regulation of the α2(1) procollagen gene. Transfection of human and mouse proximal α2(1) procollagen promoter/CAT constructs into human cells (CT-1) indicated that the human promoter had higher activity than the mouse promoter, whilst the two promoters had equivalent activities in rodent cells. These promoter activities may be accounted for by the differences in trans-acting factor binding to the two promoters. Complex I formation was competed out by the mouse CBF and NF-Y consensus oligonucleotides, while the mouse anti-CBF-B antibody resulted in a supershifted complex I. These results indicate that complex I is a member of the heterologous CCAAT-binding proteins and possibly related to or similar to the mouse CBF. The treatment of nuclear extracts with calf intestinal phosphatase resulted in a loss of complex I formation on the human and CBF binding to the mouse promoters. The Ser/Thr phosphatase, PP2A, specifically inhibited complexes II and ill formation. Nuclear extracts from CT-1 and U937 cell lines treated with the kinase inhibitor, staurosporin, was accompanied by a loss in DNA-protein interaction. This inhibition of DNA-binding activity was not observed using the tyrosine kinase inhibitor, genistein, and the PP2A phosphatase inhibitor, okadaic acid. Staurosporin also had a significant inhibitory effect on α2(1) procollagen promoter activity in CT-1 cells transfected with the human proximal α2(1) procollagen promoter and on steady state collagen mRNA levels. These results indicate that phosphorylation is required for the binding of trans-acting factors to the proximal α2(1) procollagen promoter and in transcriptional regulation of this gene. In support of the suggestion that phosphorylation events play a role in transcriptional regulation of the α2(1) procollagen gene, CT-1 cells treated with the protein kinase C activator, PMA, showed a significant reduction in α2(1) procollagen mRNA levels. A lambda gt11 expression library was screened to obtain cDNA's encoding proteins that bind the CME in the human α2(1) proximal promoter. A cDNA clone of 958 bp with a predicted open reading frame of 116 amino acids (12.5kD) was obtained. No significant DNA or polypeptide sequence homologies existed in the databank, indicating the possibility of a novel trans-acting factor. Binding of this fusion protein was specific for the CME as observed in South Western blotting and gel shift assays using competitor DNA sequences. Northern blot analysis detected a mRNA transcript of approximately 4kb predominantly in cells where α2(1) procollagen expression is repressed.

Endothelial cells take up and degrade both low density lipoproteins and low density lipoproteins which have been modified by acetylation (AcLDL). In this study, receptors that may be involved in the uptake of these lipoproteins were characterized. The cells used were aortic endothelial cells obtained from a bovine foetus, with subsequent cloning (A₃Cl₂). A cell culture system which closely resembled the in vivo monolayer was established, by growing the cells on gelatin-coated Petri dishes. Endothelial cell and lipoprotein interactions were examined by incubating the cells with ¹²⁵I-labelled lipoproteins under various conditions. The main findings were the following: The receptor affinity of bovine aortic endothelial cells was higher for AcLDL than that for LDL. The half-maximal rates of degradation, obtained from degradation saturation curves which were linearized using the Scatchard method, were about 20 μg protein/ml for LDL and about 2 μg protein/ml for AcLDL. Analyses of binding data were not accurate due to the large amount of non-saturable material bound. However, the bulk of the lipoproteins was taken up and degraded via the saturable process. Competition studies demonstrated that there were two distinct receptors for LDL and AcLDL on the endothelial cells. AcLDL did not compete with LDL for the LDL receptor, and conversely LDL did not compete with AcLDL for the AcLDL receptor. The receptor activities for LDL and AcLDL were examined as a function of culture age. Sparse cultures incubated at low lipoprotein concentrations (10-20 μg protein/ml) had a higher receptor activity for LDL than for AcLDL. In contrast, confluent cultures, catabolized more AcLDL than LDL. In comparing sparse to confluent cell cultures, the rate of ¹²⁵1- labelled LDL degradation decreased about twice, while the degradation rates of ¹²⁵I-labelled AcLDL increased about three times. Whereas the LDL receptor could be regulated, the AcLDL receptor was not as susceptible to regulation. Up-regulation was measured by pre-incubation of the cells with lipoprotein-deficient serum medium (LPDS-medium) for 48 h. Using degradation data, the LDL receptor was up-regulated about 4-fold, whereas the AcLDL receptor was not up-regulated under these circumstances. Down-regulation by incubating the cells with 25-hydroxycholesterol for 24 h resulted in a 96 % decrease in the LDL receptor activity and only a 30 % decrease in the AcLDL receptor activity. Furthermore, both LDL and AcLDL could down-regulate the LDL receptor, but neither could down-regulate the AcLDL receptor. Upon exposing endothelial cells for 72 h to either LDL or AcLDL, it was found that the total amount of cellular cholesterol increased (by about 50 %). However, the increase of total cholesterol was largely in the form of free cholesterol. This is in contrast to macrophages, where the increase in total cholesterol upon exposure to AcLDL is largely in the form of cholesteryl esters.

Cancer remains one of the major causes of morbidity and mortality globally. Many novel and innovative approaches have been employed to develop new chemotherapeutic strategies, of which targeted therapies aim to identify a molecular lesion or dysregulated pathway that cancer cells are dependent on. Research in our laboratory and others identified the nuclear import protein, Karyopherin β1 (KPNB1), to be overexpressed in various cancers and that inhibiting its expression blocks the proliferation of cancer cells. However, little is known about the potential role of KPNB1 in other cancer cell phenotypes and inflammatory signalling pathways. The aim of this study was to investigate the anticancer and anti-inflammatory effects of inhibiting nuclear import via KPNB1 and to characterise the in vivo effect of the small molecule inhibitor of nuclear import, INI-43, on tumour formation. Using siRNA and a small molecule inhibitor, INI-43, to inhibit KPNB1 we found that cervical cancer cell migration and invasion was significantly reduced. The reduced motility of cancer cells was associated with a decrease in MMP-2 and -9 expression and an increase in TIMP-1 and -2 expression following INI-43 treatment. This corresponded with a decrease in MMP-9 gelatinase activity in KPNB1-inhibited cervical cancer cells. Extended periods of KPNB1 inhibition lead to decreased proliferation and apoptosis. These changes in cancer cell biology when KPNB1 is inhibited may in part be due to its function as a nuclear transporter of transcription factors associated with cancer cell proliferation, migration and invasion. We therefore investigated the effects of KPNB1 inhibition on the nuclear localisation and transcriptional activity of key transcription factors; NFkB and AP-1, both having been implicated in many of the hallmarks of cancer. Immunofluorescent analysis and nuclear/cytoplasmic fractionation assays showed that KPNB1 inhibition blocked the nuclear localisation of NFkB. Electromobility shift assays confirmed a reduced NFkB binding to an NFkB DNA-binding sequence in the nuclear extract of KPNB1-inhibited cells. Luciferase reporter assays containing NFkB and/or AP-1 consensus binding sites showed reduced transcriptional activity for both transcription factors following KPNB1 inhibition. Associated with these changes in NFkB and AP-1 activity was reduced inflammatory cytokines; IL-6, IL-1β, TNF-α and GM-CSF target gene expression. To further characterise the role of INI-43 as a potential chemotherapeutic, the effects on tumour growth and development were investigated in an ectopic xenograft mouse model. INI-43 treatment significantly reduced tumour growth in mice and associated with the redistribution and reduction in KPNB1 levels. INI-43 treated tumours also showed altered morphological features including; better tissue differentiation and reduced inflammatory stromal infiltration, as well as reduced Ki-67 expression. The expression of extracellular matrix components and the cytoskeletal structure of cancer cells was analysed to further investigate the role of KPNB1 inhibition in tumour development. Inhibition of KPNB1 in cancer cells caused reduced expression of both collagen type IV and MMP-9. The redistribution of B-catenin and F-actin suggested that INI-43 treatment caused a loss of mesenchymal features required for tumour progression. The nuclear transport system has been of particular interest in recent years for the development of targeted anticancer drugs. However, most studies have focused on nuclear export inhibitors with little known on the potential of nuclear import inhibitors as anticancer drugs. This study provides evidence that inhibiting the nuclear import protein, KPNB1, has anti-inflammatory and anticancer effects and shows promise as an anticancer approach requiring further investigation.

Artemisinin, a natural product and its derivatives are potent antimalarial compounds, which have shown anticancer activity. In this study, we further characterized a novel artemisinin derivative namely EXP57EA which was previously designed and synthesized by the Chemistry Department at the University Of Cape Town. We determined the effect of EXP57EA on a panel of cancer cell lines, characterized the mode of cell death and also performed preliminary investigations of the signaling pathways that trigger the mode of cell death. Dihydroartemisinin (DHA), EXP57EA and cisplatin were screened on a selected panel of cancer cell lines: 3 esophageal cancer cell lines WHCO1, WHCO5, KYSE150; one breast cancer cell line MDA-MB-231 and one cervical cancer cell line SiHa. The 3-[4, 5-dimethylthiazol-2-yl]-2, 5- diphenyltetrazolium bromide (MTT) assay, and analysis with GraphPad prism software were used to calculate IC₅₀ values. EXP57EA displayed toxicity in the panel of cancer cell lines studied, and had lower IC₅₀ values (IC₅₀ values were ranging from 15.8 μM to 25.1 μM) than DHA and cisplatin. DHA was only active in two cells lines: WHCO1 (21.3 μM) and WHCO5 (77.3 μM), IC₅₀ values of cisplatin were ranging from 31.2 μM to 108.1 μM. EXP57EA was further investigated to understand the mode of cell death activated in the panel of cancer cell lines. The results showed that EXP57EA did not induce apoptosis in any of the cell lines studied, whereas DHA induced apoptosis, based on the PARP cleavage assay. In contrast, treatment with EXP57EA induced the appearance of vacuoles in treated cells compared to untreated cells, which was suggestive of autophagy. Autophagy was monitored by analyzing the expression level of two autophagy markers, Beclin1 and LC3-II by western blot. It was observed that EXP57EA treatment caused changes in the expression levels of both Beclin1 and LC3-II. We showed that EXP57EA induced elevated levels of autophagy, based on an increase in the flux of autophagy in the treated cells, since the lysosomal inhibitors ammonium chloride (NH₄Cl) and chloroquine substantially blocked LC3-II turnover in WHCO1 (confirmed previous result in our laboratory) and SiHa cancer cell lines. Furthermore, we also showed that treatment with EXP57EA resulted in increased expression of CHOP (by Real-Time PCR), and activated the PERK/eIF2α pathway, since treatment of WHCO1 cells with EXP57EA stimulated phosphorylation of eIF2α, suggesting that ER stress might be involved in mediating EXP57EA-induced cell death. Our results also suggested that EXP57EA activated the JNK pathway since treatment of WHCO1 and WHCO5 cells with EXP57EA stimulated phosphorylation of cjun and resulted in elevated levels of total c-jun. These results suggested the JNK pathway might also be involved in EXP57EA-induced cell death. However, the proposed involvement of the PERK/eIF2α pathway and the JNK pathway in EXP57EA-mediated autophagy is of a preliminary nature, and further work will have to be done to confirm the involvement of these pathways. This study showed that EXP57EA may have potential as an anticancer drug lead.

Cervical cancer is one of the most common female cancers in Africa, both in terms of incidence and mortality, and is disproportionately prevalent in developing nations due to a lack of adequate access to healthcare. While new vaccine technologies are rapidly reducing the incidence of Human Papilloma Virus (HPV) infection, the primary causative agent of cervical cancer, new cases continue to accumulate in the developing world. Beyond the role of HPV in the early stages of cancer development, the molecular aetiology of this disease is poorly understood. Frequent exposure to seminal fluid (SF), the liquid component of semen, has been proposed as a potential driver of oncogenesis in cervical cancers and has been shown to exacerbate some aspects of cervical cancers. While some of the cellular signaling pathways responsible for these phenomena have been identified, much remains to be elucidated. We hypothesized that TBX2 and TBX3, two highly homologous transcription factors frequently implicated in other cancers, may be responsible for mediating some of the effects of SF on cervical cancer cells. We established that TBX3 protein is significantly overexpressed in both primary cervical adenocarcinomas and squamous cell carcinomas compared to normal tissue. SF was shown to increase expression of both TBX2 and TBX3 mRNA in HeLa and CaSki, but not C-33 A, cervical cancer cell lines. Furthermore, SF upregulated TBX3 protein expression in both of these cell lines. In contrast, TBX2 protein was undetectable in these cell lines. In addition, our results showed that SF treatment of HeLa cells increases the expression of the known TBX3 target gene, p21CIP1/WAF1 (p21), while having no effect on PTEN expression. Transient knockdown of TBX3 resulted in decreased p21 expression in SF-treated cells suggesting that SF upregulation of p21 is dependent on TBX3. This is the first study to investigate TBX3 protein expression in primary cervical tissues and SF regulation of TBX3. However, further research is required in order to elucidate the role of SF-induced TBX3 in cervical cancer development. The identification of the role of TBX3 in cervical cancer development could aid in the development of more effective treatments for cervical cancers and could potentially impact sexual health policy recommendations for women with cervical cancer.

The molecular basis of alpha thalassaemia in the so-called 'Cape Coloured' population of the Western Cape was investigated. Restriction endonuclease digestion, Southern blotting and hybridisation with alpha and zeta globin-specific probes were used to investigate the incidence of the the various alpha thalassaemia determinants and their disorders. Results indicate that one determinant in this population results from the deletion of a single alpha globin gene on the short arm of chromosome 16. In individuals homozygous or heterozygous for this deletion, digestion with restriction endonuclease Bam H1 shows the presence of a shorter 10,5kb alpha globin-specific fragment as opposed to the 14kb fragment found in normal individuals. Individuals with both alpha globin genes deleted on the same chromosome i.e. the genotype --/aa, were detected and their alpha thalassaemia determinant characterised by: 1. a family study 2. quantification of the alpha/gamma glob in gene ratio, and 3. mapping with the zeta globin probe since the deletion extends into the zeta locus. The --/ alpha thalassaemia determinant was found to be of Southeast-Asian origin. A non-deletion form of alpha thalassaemia was also detected in which the alpha globin restriction map appeared to be normal. This condition may have resulted from a point mutation within the alpha ilobin gene region which affects transcription or RNA processing. The DNA of infants born with detectable levels of Hb Bart's in their cord blood was investigated in order to estimate the frequency of the single and double gene deletions in this population. The results indicate that infants with Hb Bart's in the 4 - 8% range predominantly have the genotype -a/-a. Using the data obtained the incidence of the heterozygote was calculated according to the Hardy-Weinberg equation. The calculated incidence of the heterozygote (-a/aa) was found to be 16,9%.

In an attempt to understand the roles played by the ubiquitin-dependent and calpain pathways in protein degradation in chicken skeletal muscles, biochemical studies were conducted on components of these two systems as well as their potential endogenous and exogenous substrates. ATP- and ubiquitin-dependent breakdown of endogenous proteins (measured by tyrosine release) or exogenous proteins (measured by the appearance of trichloroacetic acid-soluble radiolabel after incubation with 125I-lysozyme) took place in muscle extracts; the specific activities of these processes were significantly lower than those detected in rabbit reticulocytes. Conjugation of ubiquitin to a subset of endogenous proteins was detected by incubating muscle extracts (fraction II: depleted of ubiquitin by DEAE-cellulose chromatography) with 125I-ubiquitin and Mg2+-ATP, followed by analysis of the radiolabelled conjugates by one-dimensional polyacrylamide gel electrophoresis in the presence of SOS, and autoradiography. Discrete conjugates were formed with apparent molecular weights between 30 -100 000, as well as a large number of undifferentiated entities of higher molecular weights. Conjugation of ubiquitin to the exogenous protein lysozyme was detected only when fresh, as opposed to previously frozen fraction II preparations were assayed: three bands were obtained, as opposed to the six ubiquitin conjugates formed by reticulocyte extracts. The muscle system catalyzed the ubiquitination of partially purified myofibrillar proteins, principally myosin and possibly actin. Fractionation of the ubiquitin-activating enzymes into El and E2 on the one hand, and E3 on the other, permitted mixing experiments to be conducted by means of conjugation assays, and confirmed the low content of E3 in muscle as opposed to reticulocytes. Fraction II from muscle displayed ubiquitin conjugate-degrading activity but again this was less active than in reticulocytes. A number of other proteolytic activities, independent of ubiquitin, were also present. Isopeptidases, active on 125I-ubiquitin conjugates were strongly inhibited by sulphydryl alkylating agents such as N-ethylmaleimide. The overall picture of the ubiquitin pathway in muscle is one where many proteins may be converted into long-lived conjugates but not in all cases requiring the action of E3: some E3-dependent protein degradation undoubtably does occur in this physiologically basal system. Formation of a ubiquitin conjugate of the ubiquitinactivating enzyme (E1) and some of the ubiquitin carrier proteins (E2 's) was detected during incubations of 125Iubiquitin and ATP lasting 2 hr or longer. Because treatment of such systems with NaOH, even at early times during the incubations, greatly enhanced the appearance of the same entities, the phenomenon appeared to be one of auto-, rather than E3-mediated ubiquitination. The bonds involved had properties compatible with their being peptidic in nature, and their formation occurred from ubiquitin thiolesters bound to E1 and E2. The protease inhibitor and alkylating agent, TLCK, when pre-incubated with fraction II for 2 hr before the addition of 125I-ubiquitin and ATP, greatly enhanced the subsequent auto-ubiquitination of E1 in the absence of NaOH treatment, and caused the inhibition of its adenylate-forming and thiolester-transferring activities: thus ubiquitin transfer to E2's and further to other acceptors was markedly impaired. such an inactivation of El by TLCK may, in a manner analogous to that described in the thermolabile ts85 mutants (Finley et al., 1984), be the basis of the action of this agent to block the cell cycle in late G2 or early M phase (Schnebli & Haemmerli, 1974). TLCK-induced inactivating auto-ubiquitination of El may be an important tool for the study of ubiquit-independent processes which (apart from possible intrinsic protease activity), all appear to require the activity of this enzyme. The number of calpain species existing in chicken skeletal muscle is controversial with only one (Ishiura et al., 1978) or three (Wolfe et al., 1985) species having been reported. When extracts of chicken skeletal muscle were applied to a DEAE-cellulose column and the bound protein eluted in a linear salt gradient, two calpain activities, separated from their endogenous inhibitors (calpastatins), were detected. The first eluting activity, "calpain I", was active at low ca2+ concentrations, was heat-labile and had a lower apparent molecular weight on gel filtration when compared with the later eluting activity which appeared to be a typical calpain II species. "Calpain I". was not an autolytic product of calpain II but appeared to be derived from a more heat-stable calpain I species. A proportion (up to 14%) of the calpains in crude muscle extracts was bound to membrane fractions in the presence of ca2+; this could be removed by EGTA treatment. In addition, membrane-bound fractions examined by 9el filtration contained calpain· forms of an apparent molecular · weight lower than that of calpain which had not been membrane-associated. Membrane binding of the calpains (especially of calpain II), may be important in physiological activation.

Two manuscripts based on the work reported in this thesis were submitted and accepted for publication, the abstracts of which follow: SOLUBLE AND PARTICULATE FORMS OF MUSCLE ALKALINE PROTEINASE SHOW DIFFERENTIAL SENSITIVITY TO ENDOGENOUS INHIBITOR(S): Firhaad Ismail and Wieland Gevers, (Biochemistry International, In Press). Membrane-free washed myofibrils derived from rat skeletal muscle homogenates contained a chymostatin-sensitive protease(s) which acted on associated myofibrillar proteins, at an optimum pH of 8.5, much less rapidly at low ionic strength (insoluble myofilaments) than at high salt concentrations (solubilized proteins). When the myofibrillar fraction was added to the particle-free cytosol prepared from the muscle extracts, proteins of the cytosol were also degraded, but the activity in this case was much more pronounced at low ionic strength. This was because inhibitor(s) of the proteinase present in the cytosol fraction were only effective at high ionic strength when all the myofibrillar (and associated) proteins were in solution. The protease was separated from the bulk of the myofibrillar proteins by gel chromatography at high ionic strength. On dialysis against a low-salt buffer, part of the enzyme was precipitated. The putative cytosolic inhibitor(s) were again only effective on the soluble enzyme at high ionic strength. A HIGH MOLECULAR WEIGHT CYSTEINE ENDOPEPTIDASE FROM RAT SKELETAL MUSCLE: Firhaad Ismail and Wieland Gevers, (Biochim. Biophys. Acta, In Press). A cytosolic enzyme of high molecular weight (about 500000), which attacks native or denatured proteins (inter alia casein, globin and hexokinase) was purified about 1000-fold from mixed rat skeletal muscles, including muscles freed of mast cells by prior treatment of the animals with the degranulator, compound 48/80. Peptides of varying size were generated from radio-actively labelled globin, but no free amino acids were formed; free tyrosine was also not released from azocasein. The pH optimum was 7.5 and the presence of an essential cysteine group was suggested because dithiothreitol (1 mM) stimulated the activity and N-ethylmaleimide (5 mM) and p-chloromercuriphenylsulphonic acid (1 mM) were inhibitors. The activity was markedly inhibited by Zn²⁺ but not by leupeptin, chymostatin or pepstatin. The enzyme was stabilized by ATP, at concentrations as low as 0.1 mM, against inactivation at 42°C. The endopeptidase was clearly separated on gel chromatography from another large protease, also sensitive to Zn²⁺, but with marked aminopeptidase activity and the properties of Hydrolase H. The activity levels of the protease, assayed after chromatography on Sepharose 68 of high-speed supernatant fractions, did not vary significantly in skeletal muscle samples which were derived from denervated, starved, diabetic or hyperthyroid animals, in all of which the abnormal physiological states expressed themselves as enhanced rates of tyrosine release by incubated soleus and extensor digitorum longus muscles. Nevertheless, the enzyme described here may be part of an ATP-dependent, multi-component proteolytic system similar to that already known to be present in reticulocytes.