Geltosky, John Edward (1974) A study of some of the enzymes involved in the synthesis and use of tryosine in Drosophila. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-10142005-092513
Tyrosine is an integral component in scierotization and melanization, two developmentally controlled processes which are necessary for synthesis and pigmentation of the insect cuticle. Studies were undertaken to investigate the means by which tyrosine arises and, also, the nature of the reaction whereby tyrosine is hydroxylated to yield dopa, the subsequent step in both of the above mentioned processes in Drosophila melanogaster.
It was found that phenylalanine is rapidly converted to tyrosine in vivo and the enzyme(s) responsible for the catalysis is readily extractable. The in vitro conversion is dependent upon the presence of tetrahydropteridine (DMPH4), which is a cofactor in mammalian phenylalanine hydroxylase systems. Other common biological reductants are ineffective in this respect. The concentration of hydroxylase in the animal is developmentally regulated, the level building up during early third instar and peaking at puparium formation. Detectable quantities of enzyme are not found in pupae. The activity of the crude enzyme responds to the concentration of enzyme, substrate and DMPH4 in a linear fashion (up to the point of saturation).
Tyrosine may give rise to dopa via two distinct enzymatic means: tyrosinase or tyrosine hydroxylase. Drosophila are known to possess a very active tyrosinase system; however, this molecule also contains a dopa oxidase function which rapidly dehydrogenates dopa to yield dopaquinone, an irreversible step in melanization. In the absence of suitable regulation, this system does not allow for accumulation of dopa which is also necessary for sclerotization.
Crude extracts are capable of supporting the conversion of tyrosine to dopa in a DMPH4 stimulated reaction. A high concentration of pteridine also serves to arrest dopa oxidase activity in vitro. DMPH4 is an essential cofactor for mammalian tyrosine hydroxylase activity. The rates of accrual and utilization of dopa (by dopa oxidase) in crude extracts indicate that there are at least two synthetic pathways by which dopa arises. The ratio of the abilities to hydroxylate tyrosine and oxidize dopa varies as a function of the age of the animal, the highest ratio observed in early third instar larvae and the lowest at puparium formation.
The activity responsible for catalyzing the hydroxylation of tyrosine behaves in a manner similar to tyrosinase under a number of conditions. Pure tyrosinase, which is derived by sucrose gradient centrifugation, catalyzes a DMPH4 stimulated hydroxylation reaction. Multiple forms of tyrosinase are derived in sucrose gradients and heterogeneity with respect to substrate utilization is observed in these species, a situation which may have significance in a consideration of tyrosinase's involvement in supplying dopa for sclerotization. This possibility is discussed.
Since DMPH4 plays such a crucial role in these reactions (stimulation of hydroxylation and inhibition of dopa oxidase activity), the nature of these involvements was investigated. There are some indications that the pteridine is not functioning to stimulate the hydroxylation reaction by serving as a non-specific reductant (reductants, such as, ascorbate and DPNH are known to stimulate the hydroxylase activity of pure tyrosinase), but may be serving in a more direct fashion with the enzymatic mechanism. The nature of this interaction is discussed.
Banded tyrosinase, using dopa as a substrate, catalyzes the synthesis of an unidentified compound. This reaction is stimulated by DMPH4. The nature of this material and its significance to aromatic amino acid metabolism are discussed.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||18 September 1973|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||14 Oct 2005|
|Last Modified:||26 Dec 2012 03:05|
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