1. Multiple forms of soluble esterase activity have been resolved in tissue extracts and sera from 11 animal species by polyacrylamide gel electrophoresis. The esterase patterns observed differ markedly between species, and suggest that the differences could be used to identify species and to study genetic variations within a species.
2. The distribution of these multiple forms in the tissues of the various animals is tissue specific in most cases, yet underlying species characteristics can be recognized. This illustrates the effectiveness of the zymogram method in the characterization of tissue individuality (Markert and Hunter, 1959).
3. Reflectance densitometry was used in this study to obtain a semiquantitative estimate of the relative activities of the multiple esterase forms in soluble tissue extracts and sera.
4. By carrying out substrate and inhibitor specificity studies on the gel, these heteromorphs have been characterized into four main classes: carboxylesterases, arylesterases, acetylesterases and cholinesterases. This method of classification is somewhat artificial, however, and a more conclusive classification would be obtained from a detailed kinetic study on the purified enzymes.
5. Each of the esterase groups may be considered as an isoenzyme system in terms of the definition recommended by the Standing Committee on Enzymes.
6. A total of 24 multiple forms of esterolytic activity were observed in guinea pig tissue extracts and serum, and the occurrence of these individual forms in the different tissues interrelated. The soluble carboxylesterases existed as 10 separate species, cholinesterases 5, acetylesterases 5, and arylesterases 4. Further differentiation of the multiple forms in some of these major classes was achieved on the basis of the physicochemical and developmental parameters utilized. This treatment would appear to implicate at least 12 structural genes in the biosynthesis of the soluble cavian esterases; a multiplicity of control which is considerably in excess of previous estimations for mammalian sources. Detailed genetic studies would be required, however, to establish this.
7. Thirty-three multiple forms of soluble esterase activity have been resolved from rat tissue extracts and serum and subsequently classified as 15 carboxylesterases, 10 cholinesterases, 3 acetylesterases and 5 arylesterases. The developmental and physicochemical properties of these heteromorphs provide evidence for further differentiation within the carboxylesterase, arylesterase and cholinesterase groups. This treatment would appear to implicate at least 13 structural genes in the biosynthesis of soluble rat esterases.
8. Soluble porcine and duck tissue esterases have been separated into their multiple forms and classified. Sixteen multiple forms of pig esterase activity (6 carboxylesterases, 1 cholinesterase, 5 acetylesterases and 4 arylesterases) and 19 forms of duck esterase activity (5 carboxylesterases, 2 cholinesterases, 4 acetylesterases and 8 arylesterases) were observed. Further differentiation of esterase activity was achieved from an investigation into the developmental and physicochemical properties of these enzymes.
9. The multiplicity of soluble esterases from other mammalian sources was also investigated. Horse tissue esterases existed as 22 multiple forms (16 carboxylesterases, 3 cholinesterases and 3 arylesterases); sheep tissue esterases as 25 forms (10 carboxylesterases, 7 cholinesterases, 5 acetylesterases and 3 arylesterases); ox tissue esterases as 20 forms (8 carboxylesterases, 3 cholinesterases, 5 acetylesterases and 4 arylesterases); and possum tissue esterases as 25 forms (10 carboxylesterases, 7 cholinesterases, 5 acetylesterases and 3 arylesterases).
10. Further animal tissue esterases were investigated so as to complete a representative cross section of phylogeny. Lizard (reptile) tissue esterases existed as 12 multiple forms (1 carboxylesterase, 6 cholinesterases, 5 arylesterases); frog (amphibian) esterases as 8 forms of activity (a carboxylesterase, 6 cholinesterases and 1 arylesterase); and catfish (bony fish) esterases as 8 forms (3 carboxylesterases and 5 arylesterases).
11. Evidence is presented in this thesis for further differentiation of activity within the isoenzymic divisions giving 3 groups of arylesterases, 3 of cholinesterases and 5 groups of carboxylesterases. While these sub-groupings achieve a preliminary degree of order in a very complex field and reveal interesting phylogenetic differences, they are based on limited data and will have to be substantiated by more intensive investigations into the molecular properties of esterase multiple forms.
12. This study has not established anything concerning the function of esterases, nor has it contributed a great deal towards a knowledge of the exact nature of esterase multiplicity. It has, however, provided an understanding concerning the number and biochemical characteristics (species distribution, tissue distribution, developmental behaviour and some physicochemical properties)of the many esterase-active proteins found in biological material and serves as a basis for further investigations.
All of the research work recorded in this thesis has been published, or is in the process of being published. Publications include -
1. "The Developmental Multiplicity and Isoenzyme Status of Cavian Esterases" by R.S. Holmes and C.J. Masters, Biochim. Biophys. Acta, 132 (1967) 379. doi:10.1016/0005-2744(67)90157-X
2. "The Developmental Multiplicity and Isoenzyme Status of Rat Esterases" by R.S. Holmes and C.J. Masters, Biochim. Biophys Acta (1967) In the Press. doi:10.1016/0005-2744(67)90080-0
3. "A Comparative Study of the Multiplicity of Mammalian Esterases " by R.S. Holmes and C.J. Masters, Biochim. Biophys. Acta (1967) (submitted for publication). doi:10.1016/0005-2744(68)90169-1
4. "The Developmental Multiplicity of Esterases" by R.S. Holmes and C.J. Masters, Aust. J. Science, 29 (1966) 83.
5. "Rat Esterase Multiplicity" by R.S. Holmes and C.J. Masters Aust. J. Science (1967) In the Press.
Future publications will include -
1. "The Ontology of Pig and Duck Esterases".
2. "A Comparative Study of the Multiplicity of Vertebrate Esterases". doi:10.1016/0010-406X(68)90004-2