l-Glycerol 3-Phosphate Dehydrogenase

Effects of the substrate on the initial reaction velocity of nicotinamide adenine dinucleotide-linked L-glycerol 3-phosphate dehydrogenase purified from rabbit liver were studied. The true Michaelis constants for all substrates were determined in 0.1 M tris(hydroxymethyl)aminomethane-HCl buffer (pH 7.5) and were found to be 0.68, 0.19, 0.018, and 0.004 mM for L-glycerol 3-phosphate, nicotinamide adenine dinucleotide, dihydroxyacetone phosphate, and reduced nicotinamide adenine dinucleotide, respectively. The true Michaelis constants measured in 0.1 M glycine-NaOH buffer (pH 10.0) were found to be 0.065 and 0.03 XnM for L-glycerol 3-phosphate and nicotinamide adenine dinucleotide, respectively. High concentration of all four substrates in the reaction mixture was found to be to some extent inhibitory. At low concentrations of L-glycerol 3-phosphate or nicotinamide adenine dinucleotide, a plot of initial reaction velocity as a function of L-glycerol 3-phosphate or nicotinamide adenine dinucleotide concentration is sigmoidal. No sigmoidicity is seen in plotting of initial reaction velocity as a function of either dihydroxyacetone phosphate or reduced nicotinamide adenine dinucleotide under the conditions used. However, a plot of initial reaction velocity as a function of dihydroxyacetone phosphate concentration is sigmoidal in the presence of L-glycerol 3-phosphate but is not sigmoidal in the presence of nicotinamide adenine dinucleotide. On the other hand, a plot of initial reaction velocity as a function of reduced nicotinamide adenine dinucleotide concentration is sigmoidal in the presence of nicotinamide adenine dinucleotide but is not sigmoidal in the presence of L-glycerol 3phosphate. The results suggest that there are two allosteric sites, one for L-glycerol j-phosphate and the other for nicotinamide adenine dinucleotide. The binding of L-glycerol 3-phosphate to its allosteric site lowers the Michaelis constant for L-glycerol 3-phosphate but increases the Michaelis constant for dihydroxyacetone phosphate. Similarly, the binding of nicotinamide adenine dinucleotide to its

Effects of the substrate on the initial reaction velocity of nicotinamide adenine dinucleotide-linked L-glycerol 3-phosphate dehydrogenase purified from rabbit liver were studied. The true Michaelis constants for all substrates were determined in 0.1 M tris(hydroxymethyl)aminomethane-HCl buffer (pH 7.5) and were found to be 0.68, 0.19, 0.018, and 0.004 mM for L-glycerol 3-phosphate, nicotinamide adenine dinucleotide, dihydroxyacetone phosphate, and reduced nicotinamide adenine dinucleotide, respectively. The true Michaelis constants measured in 0.1 M glycine-NaOH buffer (pH 10.0) were found to be 0.065 and 0.03 XnM for L-glycerol 3-phosphate and nicotinamide adenine dinucleotide, respectively.
High concentration of all four substrates in the reaction mixture was found to be to some extent inhibitory.
At low concentrations of L-glycerol 3-phosphate or nicotinamide adenine dinucleotide, a plot of initial reaction velocity as a function of L-glycerol 3-phosphate or nicotinamide adenine dinucleotide concentration is sigmoidal. No sigmoidicity is seen in plotting of initial reaction velocity as a function of either dihydroxyacetone phosphate or reduced nicotinamide adenine dinucleotide under the conditions used. However, a plot of initial reaction velocity as a function of dihydroxyacetone phosphate concentration is sigmoidal in the presence of L-glycerol 3-phosphate but is not sigmoidal in the presence of nicotinamide adenine dinucleotide. On the other hand, a plot of initial reaction velocity as a function of reduced nicotinamide adenine dinucleotide concentration is sigmoidal in the presence of nicotinamide adenine dinucleotide but is not sigmoidal in the presence of L-glycerol 3phosphate.
The results suggest that there are two allosteric sites, one for L-glycerol j-phosphate and the other for nicotinamide adenine dinucleotide. The work by Meyerhof (l), Green (a), and von Euler et al. (3,4) has established that there are two types of glycerol-3-P dehydrogenases in mammalian systems which are capable of oxidizing L-glycerol-3-P.
Later, Raranowski (5) isolated and crystallized one of the enzymes, NAD-linked glycerol-3-P dehydrogenase (L-glycerol 3-phosphate:NAD oxidoreductase EC. 1. 1.1.8) from rabbit skeletal muscle. Since then, many investigators have made a study of this enzyme from rabbit skeletal muscle (6-lo), while a few studies of this enzyme in other tissues and species have been published (11)(12)(13)(14). NO detailed work with respect to the hepatic NAD-linked enzyme has been published, however.
Attempts were, therefore, made to study in detail the physicochemical and catalytic properties of this enzyme for a better understanding of physiological functions of this enzyme and of the significance of the glycerol-3-P cycle in carbohydrate and lipid metabolism.
This paper presents the effects of the substrate on the catalytic properties of hepatic NADlinked enzyme from the rabbit.  The rabbits were maintained on Purina rabbit chow and tap water ad libitum until use.
,Vethods-The concentrations of the substrates, NADf, NADH, glycerol-3-P, or dihydroxyacetone-P, were determined enzymatically based on the fact that at pH 10 the reaction strongly favors glycerol-3-P oxidation and NADH formation and that at pH 7.5 the reaction favors NAB+ and glycerol-3-P formation.
In the presence of one of the substrates at high concentration, the other substrate at low concentration can be determined at pH 10.0 or 7.5. Calculation was made by using 6.22 x lo3 or-1 cm-1 as the molar extinction coefficient of NADH at 340 nm.
The dehydrogenase activity was determined spectrophotometrically by measuring t'he rate of formation or disappearance of NADH at 340 nm, which was accompanied by the oxidation of glycerol-3-P or the reduction of dihydroxyacetone-P, respectively. A Gary 15 recording spectrophotometer was used to measure the rate of change in absorbance.
All measurements were carried out at room temperature.
The reaction mixture used for the measurement of t,he rate of oxidation of glycerol-3-P consisted of 0.1 RI glycine-Na0H buffer, 2.5 mM hydrazine, 10 rnM glycerol-3-P, 0.5 rnbf NAD+, and a proper amount of enzyme to produce linearity for a period of approximately 45 s (Fig. 1). The final pH and final volume were 10.0 and 1.0 ml, respectively. This procedure was routinely used for the determination of the activity during the purification and for the estimation of unit of an enzyme solution.
The rate of oxidation of glycerol-3-P was also measured in 0.1 RI Tris-HCl buffer, 10 mM glycerol-3-P, 0.5 IIIM NADf, and the proper amount of enzyme in 1.0 ml of the reaction mixture at pH 7.5 (Fig. 1). The routine assay mixture for the rate of reduction of dihydroxyacetone-P consisted of 0.1 M Tris-HCl buffer, 0.4 mM dihydroxyacetone-P, 0.1 mM NADH, and proper amount of enzyme to produce linearity for approximately 45 s (Fig. 1). The final pH and final volume were 7.5 and 1.0 ml, respectively.
A 150.fold purified enzyme preparation' was used in this study.
Under the experimental conditions used, the initial reaction velocity was found to be 1 The detailed purification procedure will be published elsewhere. proportional to the amount of enzyme added to each reaction mixture as shown in Fig. 2.
Biuret reagent was routinely used for the determination of protein concentration, and crystalline bovine serum albumin was used as standard.   Fig. 3. Results indicate that the K, for glycerol-3-P is dependent upon the second substrate, NAW, concentration. In Fig. 4 Fig. 5. It is of interest to note that a plot of initial velocity as a function of NAD+ concentration is not hyperbolic (Fig. 4).  14. Inhibition of the rate of reduction of dihydroxyace-Effect of High Substraie Concentration on Initial Velocity-During the determination of Michaelis constants it was noted that high substrate concentrations elicited an inhibitory effect as shown in Figs. 12 and 13. An additive effect is apparent when both glycerol-3-P and NAD+ concentrations are high (Fig.  13). It is possible that the inhibitory effect caused by higher substrate concentration may result from high ionic strength introduced by the addition of the substrates.

Effect of Substrate Concentration on Initial Reaction Velocity-
This possibility was checked and was ruled out, although much higher salt concentration or ionic strength was reported to inhibit a dialyzed Y.-P. Lee and J. E. Craine 7621 enzyme preparation of the NAD-linked glycerol-3-P dehydrogenase from rabbit skeletal muscle (17).
Effect of Product on Initial Reaction Velocity-The kinetics of the product inhibit,ion was studied for differentiating possible enzymatic reaction mechanisms. Inhibition of the rate of reduction of dihydroxyacetone-P by various amounts of gIycerol-  Fig. 14. The reduction of dihydroxyacetone-P is inhibited noncompetitively by low glycerol-3-P concentration, whereas a high concentration causes neither competitive nor noncompetitive inhibition according to the method of Dixon (18) (Fig. 14A). Rased on the double re-ciproca1 plot, a high glycerol-3-P concentrat'ion elicits a mixed type inhibition, and at low concentrations the inhibition is noncompetitive (Fig. 14B). Similar data, as shown in Fig. 15, were obtained with NAD+ as an inhibitor of KADH oxidation.

3-P is illustrated in
NADf at low concentrations was found to be a noncompetitive inhibitor of the reduction of dihydroxyacetone-P, and at high concentrations it caused a mixed type inhibition with respect to NADH.
Further studies show that a plot of initial reaction velocity as a function of dihydroxyacetone-P concentration is sigmoidal in the presence of glycerol-3-P but is not sigmoidal in the presence of NAD+ (Fig. 16). NAD+ (4 mM) elicits an inhibition which is comparable to that caused by 2 rnxf glycerol-3-P under the same conditions.
Interestingly, no sigmoidicity is seen in the presence of such an amount of NAD+.
On the other hand, a plot of initial reaction velocity as a function of NADH concentration is sigmoidal in the presence of 0.8 mz NAD+ but is not sigmoidal (  in the presence of 5 mM glycerol-3-P which elicits a greater inhibition (Fig. 17). Dihydroxyacetone-P was found to be a competitive inhibitor of glycerol-3-P oxidation with respect to glycerol-3-P (Fig. 18). Likewise, NADH inhibits glycerol-3-P oxidation competitively with respect to NAD+ (Fig. 19). No biphasic slopes are seen in either case which differs from the effects of glycerol-3-P or NAD+ as seen in Fig. 14A or 15B. The inhibition constants (K;) of these compounds were found to be 0.021 rnnf and 3.5 FM for dihydroxyacetone-P and NADH, respectively.
Each inhibition constant is very close to its Michaelis constant when it serves as the substrate, as shown in Table I.

DISCUSSION
The catalytic properties of NAD-linked glycerol-3-P hydrogenase from rabbit skeletal muscle have been studied by quite a number of investigators (8,(19)(20)(21). However, no worker has noted that the muscle enzyme is one of the allosteric enzymes. Studies on the nature of catalytic site and structure of the muscle enzyme have also been carried out by several workers (7,9,10,22). On the basis of the present study it is evident that there are some differences in the catalytic properties between the muscle and liver enzyme so far as we have examined. Glycerol-3-P is not only one of the substrates, but also one of t'he modifiers of the hepatic NAD-linked glycerol-3-P dehydrogenase.
Glycerol-3-P acts on the one hand as a positive effector for the oxidation of glycerol-3-P and on the other hand as negative effector of the reduction of dihydroxyacetone-P. Similarly, NAD+ is a positive effector of the reduction of NAD+ and a negative effector of the oxidation of NADH.
It is possible that dihydroacetone-P or NADH does not bind to the allosteric site for glycerol-3-P or NAD', respectively, since the inhibition of the oxidation of glycerol-3-P by dihydroxyacetone-P is purely competitive.
Or, the affinity of the binding of dihydroxyacetone-P or NADH to the allosteric site of glycerol-3-P or NAD+ must be much lower than that of glycerol-3-P or NADf.
It is also possible that dihydroxyacetone-P or NADH has its own allosteric sit'e which has very high affinity to dihydroxyacetone-P or NADH.
The fluorometric methods may be able to answer this question.
This possibility will be investigated.