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University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 A Xerox Education Company 72-21,030 WHITE, Rodney Cecil, 1943- STUDIES ON CELL WALL METABOLISM OF THE GREEN ALGA CHLORELLA FYRENOIDOSA. The Ohio State University, Ph.D., 1972 Biochemistry University Microfilms, A XE ^OXCompany f Ann Arbor, Michigan THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. STUDIES ON CELL WALL METABOLISM OF THE GREEN ALGA CHLORELLA PYRENOIDOSA DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Rodney Cecil White, B.A. The Ohio State University 1972 Approved by ^ d v is or Department of Biochemistry PLEASE NOTE: Some pages may have indistinct print. Filmed as received. University Microfilms, A Xerox Education Company ACKNOWLEDGEMENT I wish to extend my appreciation to the entire faculty of biochem­ istry who always readily assisted me in the form of advice or use of equipm ent. Very special thanks are extended to my advisor, Dr. George A. Barber, whose interest and valuable consultations made this research p ossible. Financial support was provided by an N .S.F. Traineeship, Special thanks are algo given to my wife who always showed great fortitude and love throughout this work. VITA 1943 ...................................................................... Born-Houlton, Maine 1965 ......................................................................B.A., Ricker College, Houlton, Maine 1968-1972 ..........................................................N.S.F. Trainee, Dept, of Biochemistry, The Ohio State University, Columbus, Ohio PUBLICATIONS The Synthesis of 7(Adenosine 51 -Pyrophosphoryl) -D-Sedoheptulose by an Enzyme System from the Green Alga Chlorella Pyrenoidosa. R. C. White and G. A. Barber, Biochim. Biophys. Acta, In Press. An Acidic Polysaccharide from the Cell Wall of Chlorella Pyr enoidosa. R.C, White and G. A. Barber, Biochim. Biophys, Acta, In Press. TABLE OF CONTENTS P ag e ACKNOWLEDGEMENT................................................................................................ii VITA .................................................................................................................................... iii LIST OF TABLES ................................ v LIST OF FIGURES ............................ vi INTRODUCTION........................................................................................................... 1 LITERATURE REVIEW ........................................................................................... 3 Synchronous Culture of Chlorella Enzyme Patterns in Synchronous Chlorella Cells Sugar Nucleotide Metabolism in Chlorella Chlorella Cell Walls METHODS AND MATERIALS..................................................................................17 Culture of Alga Paper Chromatography and Electrophoresis Gas-liquid Chromatography Gel Filtration and Ion Exchange Chromatography C hem icals Analytical Methods Analytical Enzymes Experimental Procedures and R esults .............................................................24 Specific Activity of Enzymes that Catalyze the formation of Sugar Nucleotides in Synchronous Chlorella Cultures ‘The Synthesis of 7(Adenosine 51-Pyrophosphoryl)-D-Sedohep- tulose by an Enzyme System from Chlorella pyr enoidosa Attempted Biosynthesis of Chlorella Cell Wall Polysaccharides Studies on Chlorella pyr enoidosa Cell Wall Polysaccharides DISCUSSION......................................................................................................................90 BIBLIOGRAPHY.............................................................................................................97 IV LIST OF TABLES Table P ag e 1. E nzym es Studied in Synchronous C u ltu re ....................................6 2. Specific Activities of Enzymes..................................................... 33 3. Ratio of Adenosine/Phosphate/Sugar .....................................43 4. Effects of TPP and MgCl2 ..............................................................46 5. Results of Differentially Labeled l4C-Glucose-6- Phosphate Experiments.....................................................................50 6. Apparent and V Values .................................................... 52 M m ax 7. Molar Ratios and Yields of Monosaccharides .................... 80 8. Chromatography and Electrophoresis of Aldobiuronic A c i d s ..........................................................................................................88 v LIST OP PIGURES Optical Absorbancy and Cell Numbers of the Syn­ chronous Culture ............................................................................... 26 Proposed Sequence of Reactions ............................................. 37 Structure of 7(Adenosine 51-Pyrophosphoryl)-D-Sedo- h e p tu lo s e ................................................................................. 41 Lineweaver-Burk P lo ts .............................................................. 54 Extraction Procedure for Cell Wall Polysaccharides . 63 Elution Profile from the DEAE-Sephadex Column. 66 Fractionation Effected by the Bio-Gel P-300 Column. 69 Elution Pattern from the Sepharose 6B Column . 71 Sedimentation Pattern of the Acidic Polysaccharide . 75 Plot of Rayleigh Interference Pattern D ata ..................... 77 Results of L-Fucose Dehydrogenase Reactions . ... 84 Elution Profile of Aldobiuronic Acids from Ag-lX8 Column ; . 87 INTRODUCTION The plant cell wall, which functions predominantly to give struc­ tural rigidity to the cell, consists mainly of polysaccharides. The types of polysaccharides which are present are strain specific; how­ ever, most contain a high percentage of cellulose. Most of the varia­ tion encountered is in a group of polysaccharides often referred to as hemicelluloses or pectic substances. These mixtures of polysacchar­ ides are usually obtained from the wall in an impure form, i.e., small amounts of other biological material such as lipids and proteins are always present. The physical appearance and the chemical composi­ tion of many plant cell walls have been defined by a variety of techni­ ques, especially be electron microscopy (1), Although cell walls are reasonably well characterized, and are synthesized and deposited in large quantities, the means by which plants carry out this synthesis has remained essentially unsolved. This is the problem to which the research in this thesis was directed. The unicellular green alga, Chlorella pyr enoidosa, which has a cell wall like that of many higher plants (2), was utilized in these stu­ dies, Chlorella cells are thought to be particularly useful for the stu­ dy df this problem because of a special characteristic in their growth cycle. The cell in the process of division forms 2, 4, 8, to as m any as 32 autospores which synthesize new cell walls in a relatively short period of time within the mother cell (3). By using the technique of synchronous culture, it was felt that enzyme preparations could be ob­ tained at the period of autospore wall formation and would contain an enriched quantity of wall-synthesizing enzymes. Three aspects of the cell wall in synchronous Chlorella pyr enoi­ dosa were studied: (a) specific activities of enzymes which produce sugar nucleotides, (presumably cell wall precursors), (b) chemical composition of the cell wall, and (c) biosynthesis of cell wall poly­ saccharides. LITERATURE REVIEW Synchronous Culture of Chlorella In theory, the best m aterial to use for studying the physiological and biochemical events of a cell, especially those age-dependent events, would be a single cell. Since this