Date of Award

5-1988

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

Abstract

Many studies of the protein interactions that compose the microtrabecular lattice have been done in dilute solutions. However, solutions containing inert polymers such as polyethylene glycol (PEG) more effectively mimic the crowded interior of the cell. Therefore, the interactions of D-glyceraldehyde-3-phosphate ketolisomerase (TPI, EC 5.3.1.1), D-phosphoglycerate 2,3-phosphomutase (PGM, EC 5.4.2.1), ATP:3-phospho-D-glycerate 1-phosphotransferase (PGK, EC 2.7.2.3), 2-phospho-D-glycerate hydro-lyase (enolase, EC 4.2.1.11), D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase (aldolase, 4.1.2.13), D-glyceraldehyde-3-phosphate:NAD$\sp{+}$ oxidoreductase (GAPDH, EC 5.3.1.1), D-glucose-6-phosphate ketol-isomerase (GPI, EC 5.3.1.9), (S)-lactate:NAD+ oxidoreductase (LDH, EC 1.1.1.27), and ATP:Pyruvate O$\sp2$-phosphotransferase (PK, EC 2.7.1.40) with each other, with ATP:D-fructose-6-phosphate 1-phosphotransferase (PFK, EC 2.7.1.11) and with F-actin have been studied in the presence of PEG. Rabbit muscle glycolytic enzymes, either purified or as in myogen, were centrifuged in the presence or absence of F-actin and/or PEG and/or KCl for 35 minutes at 145,000 x g. The supernate and pellet were then assayed.

In the absence of PEG and F-actin, the enzymes did not pellet. PEG and/or F-actin increased and KCl decreased the pelleting of all enzymes studied. A significant increase in pelleting with the addition of PEG and/or F-actin along with a decrease with the addition of KCl indicates the presence of an ionic interaction which is enhanced by PEG. For all of the enzymes tested, an ionic interaction with F-actin that was enhanced by the presence of PEG was evident. GPI, aldolase, GAPDH, PK, and LDH also were tested for the specificity of this interaction by using TPI as a control. All five enzymes demonstrated a specific interaction with F-actin, which for GPI, GAPDH, and PK was enhanced by PEG. More myogen than purified enolase, GPI, aldolase, GAPDH, PK, and LDH pelleted under several conditions. Greater pelleting in samples prepared with myogen than in those prepared with purified enzyme suggests an interaction of the enzyme with the proteins of myogen. Therefore, purified enolase, GAPDH, aldolase, GPI, and LDH were also tested for pelleting with other purified enzymes in the presence of PEG. Aldolase pelleted the most with PK and LDH; GPI, enolase, and GAPDH pelleted the most with PFK; and LDH showed no differences in pelleting with the various enzymes.

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