Previous studies have shown that ability to perform anaerobic activities is markedly reduced following prolonged exercise. It has been hypothesized that such changes are the result of reduced glycolytic capability. This study examined the changes in selected glycolytic enzyme activities in the leg muscles of men before, immediately after and 24 hours after prolonged exertion. Muscle biopsies obtained from the vastus lateralis were assayed for glycogen, total phosphorylase, lactate dehydrogenase (LDH), phosphofructokinase (PFK), and alpha glycerophosphate dehydrogenase ((GPDH). Muscle glycogen content was round to decrease significantly (P <.0l) from rest (116 mmoles/kg) Co post exercise (66 mmoles/kg), but 24 hours later it was only 15% lower than the pre exercise value. Phosphorylase and LDH increased significantly (+12.9% and +9.4%, respectively) as a result of the exercise. After 24 hours of rest phosphorylase returned to the pre exercise level, but LDH showed an additional increase of 6.2% in activity above the post exercise value. These data fail to support the hypothesis that prolonged severe exercise inhibits anaerobic capacity by reducing essential glycolytic enzymes.Supported by a grant for NIH (R0l AM17083-01).

Gaucher disease (GD) is caused by an inherited deficiency of the human lysosomal enzyme acid β-glucosidase (GBA, EC 3.2.1.45). Absence of functional enzyme results in lysosomal glycolipid accumulation. This disorder primarily affects organs of the reticuloendothelial system and disease severity ranges from mild hepatosplenomegaly to extreme neurological degeneration. Disease symptoms have been shown to be greatly ameliorated by enzyme replacement therapy (ERT). Limitations to therapy include the high cost of current ERT and its inability to treat neurological symptoms. In the present study I sought to produce a GBA-fusion enzyme in an economical manner that can be used to treat neurological GD. I explored the use of Pichia pastoris as an economical recombinant protein expression system for production of human GBA. In addition, I synthesized a protein transduction domain (PTD)-GBA fusion protein for its potential to be used as a neurotherapeutic. The results show that GBA-PTD4 can be expressed and purified from P. pastoris. Hydrophobic interaction chromatography and gel filtration chromatography were successful in purifying GBA-PTD4. Further optimization of expression and purification techniques is required for effective large scale production of recombinant enzyme. / Graduate

The object of this study has been to characterise barley mutants which lack the functional nitrate reductase (NR) enzyme system. In the long term it is hoped that such studies will lead to improved nitrate utilisation and ultimately to improved quality barley protein. The progeny of nine chlorate resistant selections, in the barley cultivars Mavis Mink and Golden Promise, were studied. Four (R9201, R11301, R12202 and R12801) lacked NADH-NR and FMNH2-NR activities, the rest had the NR+ phenotype. None of the four were nitrate uptake mutants since they all possesed wild type or greater levels of nitrate. R9201, R11301 and the previously characterised R9401 (Bright et al, 1983) were not molybdenum uptake or Mo-accumulation mutants. R9201, R11301 and R12202 lacked xanthine dehydrogenase (XDH) (an enzyme which contains the same molybdenum-containing cofactor, MoCo) activity suggesting that these lines have defective MoCo's, whilst R12801 possessed XDH activity indicating that it might have defective apoprotein subunits. These four lines are similar to R9401 since they lack "NR activity and are unlike other previously selected ' barley NR mutants (Kleinhofs ' et al, 1980) which are leaky and possess up to 5% of the wild type (cv. Steptoe) in vitro NR activity. R9201 and R11301, like R9401, were all caused by single recessive nuclear gene mutations. The MoCo mutants, R9201, R9401, R11301 and R12202, could be divided into two groups on the basis of i) allelism, ii) presence or absence of wild type levels of dimeric NR and iii) the ability of their extracted MoCo's to reconstitute NADPH-NR in an extract of N. crassa nitl mutant (which supplies NR monomers) in the presence of excess molybdate. R11301 is not allelic to R9201, whilst R9201 and R9401 are allelic. R9401 is also thought to be allelic to Az 34, a barley MoCo mutant isolated and characterised by Kleinhofs et al (1980). Az 34 has been designated nar2a and it is proposed that the allelic R9401 and R9201 should be classified as nar2b and nar2c respectively. It is possible that R11301 is either allelic with one of the other barley MoCo lines, nar3, nar4 or it is defective in a different MoCo gene. The same is also true for R12202. R11301 was shown to possess inactive NR dimer at wild type concentrations, whilst R9201, R9401 and R12202 had little or no NR (inactive dimer) present under these conditions. R12801 possessed no dimer. The MoCo extracted from R11301 was able to reconstitute the same level of NADPH-NR in the Hill extract as MoCo extracted from-the wild types. R9201, R9401 and R12202 lacked this ability.

An alternative use for an amperometric enzyme electrode is as an anodic half-cell of a fuel cell. A biofuel cell based upon the oxidation of methanol by the quinoprotein alcohol dehydrogenase was developed.

No description available.

No description available.

Aspects of cholinesterase distribution were studied in both the central nervous system (CNS) and blood in relation to the- central cholinergic deficit in senile dementia of the Alzheimer type (SDAT). In the MAT neocortex, the - greatest loss of cholinergic-related acetylcholinesterase (AChE) activity was from the upper cortical layers. previous reports indicate that the majority of neuropathological, neurochemical and morphological changes occur in the lower cortical layers, suggesting that the 'cýolinergic deficit in SDAT may not be directly associated with the primary disease processes of this disorder. Furthermore, the study of-AChE molecular forms revealed that in both MAT and demented-Parkinson's disease subjects, the loss of neocortical AChE activity'is due'to a selective loss of the G4 . form of the enzyme. Hence, th6 cortical cholinergic changes in MAT way not'be uniquely associated with Alzheimer-type neuropathological changes. A similar extensive loss of the G4 form of AChE occured in the cholinergically-denervated rat hippocan-pus, indicating that this form is probably associated with cholinergic axonal processes and suggests that these structures degenerate in demented Alzheimer-type an d Parkinsonian cases. In contrast to AChE, ' the., physiological function of butyry1cholinesterase (BChE) is unknown. However, the different intracortical and inter-regional distributions. of AChE and BChE in the CNS, along vith the observation that despite reduced levels of ch6linergic activity in SDAT, BChE was unaltered, all suggest that BChE is not intimately associated with central cholinergic neurotransmission. In the blood, measurement of cholinesterase activity in MAT revealed normal levels of erythrocyte AChE and plasm BChE and elevated plasma AChE activity. The increased plasm AChE activity may reflect increased leakage, through the blood-brain barrier -and/or increased release from degenerating'cholinergic structures. %bilst these results confirm the involvement of the central cholinergic system in SDAT, they also, however, suggest that these chang es may be secondary to more fundamental pathological processes.

No description available.

No description available.

Cette recherche comprend l'etude de la bioacatalyse de la chlorophyllase partiellement purifiee a partir d'algue (Phaeodactylum tricornutum) dans differents milieux organiques incluant les solvants miscibles et non miscibles a l'eau et les systemes micellaires tertiaires. Les quantites optimales d'eau et de solvant, choisi en fonction de son degre d'hydrophobicite et incluant l'acetone, l'ethanol, le propanol, le butanol, le pentanol, l'hexanol, le toluene, le pentane, l'hexane, l'heptane et l'octane, necessaire a la catalyse de l'effet hydrolytique de la chlorophyllase, ont ete mesurees. Les valeurs d $V sb{ rm max}$ obtenues mesurant l'activite hydrolytique de la chlorophyllase sont plus elevees chez les solvants miscibles a l'eau (log P 0.8). L'activite hydrolytique de la chlorophyllase decroit d'approximativement 12 fois lorsque le nombre de carbone pour les alcools, passent de 2 a 5. Certains resultats a partir du FTIR ("Fourier transform infrared spectroscopy") tendent a montrer que le phytol pourrait agir comme un donneur d'electron, ainsi, si un compose nucleophilique approprie est ajoute a un syteme biphasique, la solubilite de la chlorophylle dans l'hexane augmentera et l'activite hydrolytique de la chlorophyllase augmentera. En milieu biphasique (hexane/eau), l'addition de solvants polaires tels que l'acetone, l'ethanol, et le propanol augmente l'activite de la chlorophyllase et ce seulement a certaines concentrations. La mesure optimale de l'activite hydrolytique de la chlorophyllase en milieu micellaire tertiaire (tampon/hexane/surfactant) utilisant les polysorbates et les Spans ainsi que differentes chai nes hydrophobiques comme surfactant indique que les concentrations necessaires de surfactants sont dependantes a la fois de la chai ne hydrophobique et du groupement polaire. Toutefois, l'utilisation du Span 85 s'est avere plus approprie et les valeurs de $V sb{ rm max}$ obtenues mesurant l'activite enzymatique de la chlorophyllase sont 288 fois