The ubiquitin proteasome pathway is the predominant biological mechanism of myofibrillar protein (MF) degradation.  To test the hypothesis that amino acid and insulin act synergistically to regulate proteolysis, two experimental models were employed;  an <i>in vivo </i>study on growing calves and an <i>in vitro</i> C2C12 myotubes culture. Calves growing at 0.3kg/day, were constantly infused with glucose at a low (LDG) or high (HDG) dose (to stimulate insulin) with or without essential amino acids (EAA).  Glucose infusions increased plasma insulin and IGF-1 concentrations in a dose dependent manner (P<0.05).  HDG was associated with decreased plasma urea nitrogen and 3-MH concentrations and 3-MH:creatinine output (an index of MF degradation) (P < 0.05).  Glucose infusions down regulated the expression of 14-kDa E2 ubiquitin conjugating enzyme and C2 20 S proteasome sub unit, however EAA did not alter the effect of raised plasma insulin on muscle ubiquitin proteasome pathway suggesting that under the conditions employed, EAA do not act synergistically with insulin to decrease myofibrillar protein degradation, <i>in vivo.</i> In the <i>in vitro</i> experiments, amino acid deprivation (0.2 X physiological concentration amino acid;  PC AA) of myotubes for 8 h was associated with increased (P < 0.05) proteolysis (measured from TCA soluble ³H-tyrosine release in the medium), compared to controls (1.0 X PC AA).  Addition of insulin inhibited this increase (P < 0.05).  Rapamycin significantly increased proteolysis in 1.0 X PC AA media suggesting amino acid might regulate proteolysis through mTOR signalling pathway.  Reduced amino acid supply also increased 14-kDa E2 and C2 mRNA expression compared to controls (P < 0.05).  Increasing leucine concentration in 0.2 X PC AA basal media showed a dose dependent decrease in protein degradation and expression of 14-kDa E2, in the presence of insulin.  In conclusion, the results suggested that decreased availability of amino acids was associated with increased total proteolysis and that anti-catabolic effect of amino acid in C2C12 muscle cell cultures, was additive to that of insulin.

One of the most difficult problems in modern biochemistry is that of accurately predicting a protein's three dimensional structure from its sequence (the <I>protein folding problem</I>). This structure is essential for a proper understanding of how a protein functions. As experimental derivation of a protein's structure is far more time consuming than deriving a protein's sequence, prediction of structure from sequence is an important goal for many protein biochemists; several methods have been suggested for this. Given a protein of known structure of similar sequence to the protein you wish to model homology modelling is the method most likely to produce a fairly good model. In this work a tool was produced for examining the various stages of homology modelling and analysing how well various method for carrying out these stages perform. The tool produced consists of an object-oriented database of protein structures and testbed software written in a mixture of PROLOG and DAPLEX. Tests were carried out using this software to examine the predictivity of various guidelines suggested in the literature for the loop selection stage of cut and paste homology modelling. The results of these tests produced surprising new information on the relative importance of different factors which may be used to choose between candidate fragments for the variable regions of a protein being modelled. The results of the application of these automated modelling methods were then compared with a short series of modelling tests using human modellers in an attempt to measure how the usual modelling procedures using 'hand and eye' compare with automated measures. Finally the results of the tests carried out were used to guide the production of a model of a previously unmodelled serine proteinase.

Movements of calcium (Ca) across the maternal and fetal aspects of the human placenta were investigated using the isolated placental lobule dually perfused <I>in vitro.</I> Tissues uptakes and releases of calcium were measured and the effects on calcium movements by calcium-protein binding in the perfusion fluids, (associated with extracellular pathways and non-uniform perfusion), evaluated. The effects of ouabain, dinitrophenol (DNP), and cooling on calcium movements were measured and compared to movements of Na⁺ and K⁺. These indicated the presence of active transport of calcium but no evidence was obtained for Ca²⁺/Na⁺ exchange. Cyclic adenosine 3', 5' -monophosphate (cAMP) levels in dually perfused tissue were measured following microwave fixation. This technique was used to measure changes in tissue cAMP production following exposure to forskolin, 3-iso-butyl-l-ethyl-xanthine (IBMX), and various fragments of both bovine parathyroid hormone (bPTH) and human parathyroid hormone-related peptide (hPTHrP). Rises in cAMP were produced by exposure to bPTH(1-34) but not hPTHrP(1-34), hPTHrP(67-86) or hPTHrP (107-138). It is concluded that calcium is actively transported across the placenta but there is no major contribution via a Ca²⁺/Na⁺ exchanger. The patterns of calcium uptake as a function of perfusate calcium concentration support the evidence of other workers that extracellular pathways are present in the syncytiotrophoblast. A significant amount of passive movement of calcium may therefore take place across the perfused placenta. The 1 to 34 region of the PTH molecule stimulates the production of cAMP by the trophoblast, but there is no indication that this has any effect on transplacental Ca transport.

The rate of breakdown of a range of rumen bacteria by rumen protozoa in whole rumen fluid ranged from 11.5%/h for <I>Butyrivibrio fibrisolvens</I> SH13 to 2.7%/h for <I>Eubacterium ruminantium </I>2388. When the rates of breakdown of the bacteria were measured using hen's egg white lysozyme, an N-acetylmuramidase, they ranged from 10.1%/h to 1.1%/h. There was correlation between levels of activity in lysozyme and rumen fluid, with R<SUP>2</SUP>=0.52 (P>0.01). The rates of breakdown of the rumen bacteria using mutanolysin, also an N-acetylmuramidase<I> </I>were also measured. They ranged from 16.0 to 1.2%/h, and the R<SUP>2</SUP> value was 0.81. When the same bacteria were incubated with an N-acetylglucosaminidase, the rates of breakdown ranged from 3.9 to 0.27%/h, and there was no correlation with the activity in rumen fluid. This implied that the principal bacteriolytic activity in rumen fluid is similar to lysozyme (EC 3.2.1.17). Protozoal lysozyme was partially purified from <I>Entodinium caudatum</I> using a combination of cation exchange and gel filtration chromatography. The partially-purified enzyme resembled other lysozymes in that it was a basic protein which degraded <I>Micrococcus lysodeikticus</I> cell walls and had a high isoelectric point of pI9. It displayed optimal activity at pH 6.5, ionic strength of 0.05 M and at a temperature of 40 °C. It had an apparent affinity constant (K<I><SUB>app</SUB></I>) of 388 mg <I>M. lysodeikticus</I> lysed/I. It had an apparent molecular mass of 14 kDa, from SDS-PAGE, and its N-terminal amino acid sequence slightly resembled that of a distinct class of lysozymes found in <I>Streptomyces</I> species, the fungus <I>Chalaropsis </I>and the protozoan parasite <I>Entamoeba histolytica</I>. Thus the major type of bacteriolytic activity in rumen fluid was found to be a lysozyme-like enzyme which was partially purified and characterised. Further characterisation of this enzyme could provide important information that would be useful in developing a means of preventing wasteful breakdown of bacterial protein in the rumen.

No description available.

The four cyanogen bromide fragments of yeast 3-phosphoglycerate kinase (PGK) have been isolated and characterised. After digestion with proteolytic enzymes and specific cleavage reagents, the resuting peptides were purified by various methods and sequenced using the manual dansyl-Edman technique and the Beckman 890C liquid phase sequencer. The entire sequence of yeast PGK (415 residues) has been determined using a combination of amino acid sequence data and nucleotide sequence data. Nucleotide sequence data were supplied by Dr. A. Kingsman, University of Oxford. The yeast PGK sequence data have been fitted tothe 2.S electron density map and the nucleotide binding site has been fully characterised. The fitting of sequence data to the electron density map permitted identification of additional electron density which is probably attributable to the triose phosphate substrate. This binding site has also been characterised. The construction of the 1g:1cm model of yeast PGK permitted interpretation of chemical modification, NMR, hydrodynamic and kinetic data from a structural point of veiw, thereby allowing a catalytic mechanism to be proposed. This mechanism involves a major conformational change, triggered by the breaking of a salt-bridge between glutamate 190 and histidine 388 concommittant with the formation of the ternary enzyme-substrates complex. The conformational change brings the two substrates into close proximity, thereby permitting the in-line, direct, associative,phosphoryl transfer reaction to take place. The hydrodynamic properties of yeast PGK were examined in order to determine conditions under which PGK adopted its closed , catalytically active conformation. The solubility of yeast PGK in organic solvents commonly used as crystallising media was examined and experiments performed which were designed to crystallise a) the closed conformation of yeast PGK, and b) the substrate-free form of yeast PGK. No crystals have yet been observed in these experiments.

No description available.

No description available.

No description available.

No description available.