Graduation date: 1978

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

Graduation date: 1984

Chapter 1 General introduction Section 1 contains a brief review of the structure and function of different muscle fibre types in teleost fish. Section 2 concerns the structure and function of fish liver including the classification of different liver cell types, the ultrastructure of fish hepatocytes and liver metabolism Section 3 The concepts of evolutionary adaptation and physiological acclimation are explained and an outline is given of some of the underlying cellular and molecular mechanisms. Chapter 2 Seasonal influences on the biochemical composition of tissues in dab (Limanda limanda L.) Seasonal changes in the biochemical composition of liver and skeletal muscle have been investigated in juvenile dab (Limanda limanda L.) from the wild. Chapter 3 Seasonal influences on the ultrastructure of myotomal muscles of dab CLimanda limanda L.) Seasonal influences on the ultrastructure of myotomal muscles of the immature dab (Limanda limanda L.) have been studied. Fish specimens were taken from the same population of animals as in chapter 2. The fractional volume (%) of each fibre type occupied by mitochondria, myofibrils and lipid droplets was determined using a point counting morphormetric method. Chapter 4 Seasonal influenes on the ultrastructure of hepatocytes of dab (Limanda limanda L.) The dab (Limanda limanda L.) exhibited marked seasonal alterations in the ultrastructure of hepatocytes during the year. Endoplasmic reticulum (ER) was more dilated and Golgi complex were highly developed in the hepatocytes of winter- adapted dabs. Mitochondrial profiles were also found to be well organized in the hepatocytes of winter caught animals. The hepatocytes in the summer-adapted dabs were characterised by containing enormous lipid droplets whereas those in the autumn-adapted fish were featured by numerous lipopigment-like granules. Different forms of hepatic glycogen and triglyceride were observed in the hepatocytes of dab (Limanda limanda L.) throughout the year. In the autumn, the hepatic glycogen and triglyceride existed in a form of loosely packed homogeneous masses throughout the cytoplasm whereas a more densely packed form for both glycogen and triglyceride was observed during the winter. Chapter 5 General discussion The major findings in the present study are discussed in relation to seasonal influences on tissue composition and ultrastructure of myotomal muscle fibres and hepatocytes in the juvenile dab (Limanda limanda L.). Some suggestions for further works are given.

Chapter 1. General Introduction The general Introduction begins with a brief description of the natural history of the plaice. This is followed by a review of the literature describing the development of teleost embryos and larvae. The pattern of innervation with development and the differentiation of muscle fibres are both discussed in detail, as are larval locomotion and patterns of swimming behaviour. The remainder of the general introduction concentrates upon the phenotypic plasticity of muscle and the mechanisms by which this may be achieved. Teleost muscle in particular has been found to show an extremely plastic response to changes in ambient temperature. The contractile protein composition, ultrastructure and contraction properties of fish muscle are all influenced by temperature. However most research has concentrated primarily upon adult teleosts and fewer studies have examined the effects of temperature on muscle in larval and juvenile fish. Experiments describing the effect of temperature upon muscle structure and contraction both in the larval and adult stages are discussed at the end of the Introduction. Chapter 3. The influence of temperature on somitogenesis and organogenesis in embryos of the plaice, Pleuronectes platessa L. The development of plaice embryos was examined at two temperatures, 8° and 12°. Temperature influenced the rate of embryonic development but did not affect the order of appearance of morphological characters. The rate of somitogenesis was observed in embryos reared at 5° and 12°. Chapter 4. Muscle development in plaice, Pleuronectes platessa L. The effects of temperature on muscle fibre ultrastructure were examined using larvae reared at 5, 8, 10, 12 and 15°. Two types of muscle fibre could be distinguished in the newly hatched larvae; beneath the skin was a single, superficial layer of smaller diameter muscle fibres, which completely surrounded the larger diameter inner muscle fibres. Larvae reared at 15° only survived for a few days and had significantly more myotomal muscle fibres of larger average cross-sectional area than those hatching at 5-10°. Chapter 5. Electrophoretic analysis of the myofibrillar components of red and white muscle fibres from adult plaice, Pleuronectes platessa L. The myofibrillar proteins of the two main fibre types were identified using various electrophoretic techniques, including SDS PAGE, IEF PAGE, NEPHGE PAGE and alkaii-urea gels. The myosin light chain composition of each of the four histochemically identified fibre types was determined. Chapter 6. Developmental transitions in myosin sub-unit composition from larval inner to adult fast muscle in plaice, Pleuronectes platessa L. The myosin sub-unit composition of plaice deep white muscle fibres was examined at different stages of development. The myosin heavy chain component of the inner muscle fibres was, however, found to be different in group 1 juveniles and adult deep white fibres. Chapter 7. General Discussion. The major findings of the study are discussed in relation to other experiments examining muscle development and differentiation in teleosts. The influence of temperature on the structure and development of plaice myotomal muscle is discussed, with particular reference to larval survival. Possible mechanisms by which muscle development in teleosts may be regulated are described together with suggestions for further work.

Antifreeze proteins (AFPs) protect marine teleosts from freezing in icy seawater by binding to nascent ice crystals and preventing their growth. It has been suggested that the gene dosage for AFPs in fish reflects the degree of exposure to harsh winter climates. The starry flounder, _Platichthys stellatus_, has been chosen to examine this relationship because it inhabits a range of the Pacific coast from California to the Arctic. This flatfish is presumed to produce type I AFP, which is an alanine-rich, amphipathic alpha-helix. Genomic DNA from four starry flounder was Southern blotted and probed with a cDNA of a winter flounder liver AFP. The hybridization signal was consistent with a gene family of approximately 40 copies. Blots of DNA from other starry flounder indicate that California fish have far fewer gene copies whereas Alaska fish have far more. This analysis is complicated by the fact that there are three different type I AFP isoforms. The first is expressed in the liver and secreted into circulation, the second is a larger hyperactive dimer also thought to be expressed in the liver, and the third is expressed in peripheral tissues. To evaluate the contribution of these latter two isoforms to the overall gene signal on Southern blots, hybridization probes for the three isoforms were isolated from starry flounder DNA by genomic cloning. Two clones revealed linkage of genes for different isoforms, and this was confirmed by genomic Southern blotting, where hybridization patterns indicated that the majority of genes were present in tandem repeats. The sequence and diversity of all three isoforms was sampled in the starry flounder genome by PCR. All coding sequences derived for the skin and liver isoforms were consistent with the proposed structure-function relationships for this AFP, where the flat hydrophobic side of the helix is conserved for ice binding. There was greater sequence diversity in the skin and hyperactive isoforms than in the liver isoform, suggesting that the latter evolved recently from one of the other two. The genomic PCR primers are currently being used to sample isoform diversity in related right-eyed flounders to test this hypothesis. / Thesis (Master, Biochemistry) -- Queen's University, 2009-01-30 13:38:08.346