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Mycoplasma laidlawii possesses a single glutamate dehydrogenase (GDH) with dual coenzyme specificity [specificity for nicotinamide adenine dinucleotide (H) and nicotinamide adenine dinucleotide phosphate (H) ]. A purification procedure is reported which results in an enzyme preparation with a specific activity of 79.5 units/mg and which displays only one significant protein band after gel electrophoresis. This one band was determined, by activity staining, to have all of the GDH nucleotide specificities. The molecular weight of the enzyme is 250,000 + 10%, and it has a subunit size of about 48,000. The enzyme exhibits measurable activity with aspartate and pyruvate but is inactive with eight other possible substrates. Purine nucleotides do not affect the activity. The Km for reduced nicotinamide adenine dinucleotide was 1.8 x 10-4 M. The optimal substrate concentrations and pH optimum for each of the respective GDH activities are also reported. MATERIALS AND METHODS Chemicals. a-Ketoglutaric acid, NAD, NADPH, ribonuclease (all A grade) and deoxyribonuclease (B grade) were obtained from Calbiochem. Phenol reagent (Folin-Ciocalteau) was from Fisher Scientific Co. Chemicals for acrylamide gel electrophoresis were all products of Matheson, Coleman, and Bell. Phenazine methosulfate, MTT tetrazolium [3(4,5 dimethyl thiazolyl-2)-2, 5-diphenyl tetrazolium bromide], tris(hydroxymethyl) aminomethane (Tris), NADP (Sigma grade), and NADH (grade 111) were obtained from Sigma Chemical Co. Sepharose 6B and diethylaminoethyl (DEAE) Sephadex A-50 were obtained from Pharmacia Fine Chemicals, Inc. Ammonium sulfate, A.R. was from Mallinckrodt Chemical Works. DEAE cellulose DE.11 was obtained from Reeve Angel, Inc. Crude extract preparation. M. laidlawii, strain A, culture and osmotic lysis procedure were as described previously (4). The organisms were extracted once with 10-3 M ethylenediaminetetraacetic acid (EDTA), pH 7.5, followed by two more extractions with demineralized water. The extracts were then combined and subjected to the enzyme fractionation procedure described later.

we combined circular dichroism, optical rotatory dispersion, and infrared data to give evidence for more ß (pleated sheet) structure than a structure in whole membranes of Mycoplasma laidlawii, strain A. The circular dichroism data exhibit a typical -helical pattern. However, when this pattern is subjected to computer analysis, using a curve-fitting program, the best fit to the membrane experimental curve is obtained with 56.0% ß, 30.1% a, and only 13.2% coil. Calculation of the Moffit-Yang a0 and ba parameters from optical rotatory dispersion data yields 30-45 % ß, 26 % a, and 30-45 % coil. Inc V circular dichroism and optical rotatory dispersion spectra of various cellular membranes have generally been interpreted to have shapes indicating substantial amounts of a helix and showing no evidence of ß structure. While circular dichroism spectra may not give evidence for ß structure, it would be difficult to disprove the presence of substantial amounts of beta structure on the basis of such spectra. Gratzer et al. (1968), Hammes and Schullery (1968), and Stevens et al.(1968) have shown that the circular dichroism bands and optical rotatory dispersion maxima of certain ß structures can have much lower intensity and be displaced to the red of the commonly used reference poly-L-lysine in the ß conformation. Thus it is possible that the circular dichroism minimum of the ß structure could actually be placed at a position normally assigned to the negative ( --) transition of -helical peptides (222-225 µ).Wallach et al. (1969) and Graham and Wallach (1969) have recently used this argument to account for evidence of ß structure in their infrared spectra of mitochondrial membranes in the dry-film state. They do this in spite of the fact that several authors (Urry et al., 1967; Stein and Fleisher, 1967; Wrigglesworth and Packer, 1968; Urry and Ji, 1968) have assumed that ß structure is lacking on the basis of optical rotatory dispersion and circular dichroism measurements.

The kinetics of sodium dodecyl sulfate solubilization of aqueous suspensions of Mycoplasma laidlawii membranes have been investigated by light scattering in a stopped-flow apparatus. There was evidence of direct interaction between the membranes and sodium dodecyl sulfate micelles above the critical micelle concentration, although of lower order kinetically than with monomeric dodecyl sulfate anions below the critical micelle concentration. The activation energy remained the same in either case, about 10 kcal/mol. Static light-scattering studies at higher resolution showed that the solubilized membranes are in the form of small aggregates.Since the isolation of "structural protein" from various membrane sources (1), the use of the anionic detergent sodium dodecyl sulfate (SDS) for membrane solubilization has been an established method for the investigation of membrane substructure. There has recently been a flurry of interest in the "miniproteins" (molecular weight about 5,000), discovered in SDS-solubilized membranes from mammals (2, 3). Many other peptide fractions are detected by SDS-acrylamide gel and SDS-Sepharose 6B columns (2,4-7).Few membrane peptides have been purified from SDS solutions to date. We have performed kinetic studies of the interaction between aqueous membrane suspensions and SDS in order to better understand the mechanism of SDS action. W"le have chosen to work with Mycoplasma because of their special advantages (8, 9), and the extensive work with SDS disaggregation of these membranes (8-13).The present study with stopped-flow and light-scattering apparatus brings to light the following information about the SDS solubilization of M. laidlawii (strain A) membranes. (a) There is evidence for direct interaction between SDS micelles and membranes above the critical micelle concentration (cmc) for the detergent. (b) Although a higher order of interaction occurs with monomeric SDS anions below the cmc, the activation energy for the solubilization remains the same, about 10 kcal/mole, indicating that a similar rate-determining step occurs in the interaction of membranes with either micelles or monomeric detergent anions. (c) The membranes disaggregate to an average molecular weight of slightly more than 70,000 when solubilized in SDS at room temperature. (d) Heat treatment of the solubilized membranes causes a considerable decrease in light-scattering ability. Thus, solubilized membranes are in the form of small aggregates, perhaps dimers to tetramers. MATERIALS AND METHODS MembranesMembranes were prepared from Mycoplasma laidlawii (strain A) by osmotic lysis as described (14). The washed membranes were suspended in twice-distilled, demineralized water. The suspension was diluted 1:10 with 2-chloroethanol and absorbance was measured at 280 nm in a Beckman DB spectrophotometer. An extinction coefficient of 1.82 for 1 mg/ml of membrane protein in a 1-cm cell was used (14). The pH of a 1 mg/ml aqueous membrane suspension was typically 7.2. As reported (14), M. laidlawii membranes do not c...

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