The pyruvate dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex is shown to be a dimeric protein consisting of identical or very nearly identical polypeptide chains with a molecular weight 90 000 to 110 000. In reasonable agreement with data obtained by Reed and collaborators the native enzyme was found to be a species with a sedimention coefficient s°20, w= 9.8 S, a diffusion coefficent D°20, w= 4.4 × 10−7 cm2/sec, and a molecular weight of 200 000 to 220 000. Amino acid analyses and analyses of tryptic peptides showed that the minimum chemical molecular weight is around 100 000. Serine was found as an N‐terminal residue but with poor yields. One carboxyl terminal sequence…Arg‐Leu‐Ala‐CO2H per 105 daltons protein was shown by carboxypeptidase degradation. The protein could be dissociated by a variety of methods into only one species of subunit. The molecular weight of this subunit was found, by dodecylsulfate polycrylamide‐gel electrophoresis and sedimentation equilibrium in various solvents, to be 90 000 to 110 000.

The pyruvate dehydrogenase core complex from E. coli K-12, Since the pioneering experiments of Gunsalus and Hager (1) and of Koike, Reed, and Carroll (2), a-ketoacid dehydrogenase complexes from various sources have been extensively studied. Several years ago, we began to study the Escherichia coli pyruvate dehydrogenase complex from a genetic point of view (3), because it appeared that the molar ratio of the constituent polypeptide chains was far from unity. We therefore began to study the regulation of the synthesis of the enzyme complex, and it became clear during these studies (4-6) that one would be hampered in further such experiments without a precise knowledge about the composition of this multienzyme complex; thus, we turned our attention toward an elucidation of its structure (7,8,4 MATERIALS AND METHODSCells and Reagents. Pyruvate dehydrogenase complex was prepared from K-12 strain YMel and from the regulatory mutants K1-1 LR8-13 and K1-1 LR8-16, which synthesize the enzyme complex constitutively (6). Growth conditions, purification procedures, enzymc assays, and sources of reagents were described (8).Determination of Flavin-Adenine Dinucleotide (FAD). Our instrumentation was checked with FAD purchased from Boehringer iIannheim GmbH. The expected extinction coefficient (450 nm) of 1.13 cm2/mmol (9) was found after the substance was dried for 3 days under reduced pressure at 600 over P205. All spectra were measured in 0.05 M potassium phosphate, pH 7.5, with a Zeiss PMQJI spectrophotometer. Reduction and reoxidation of enzyme-bound FAD in the presence of 6.5 M urea was performed exactly as described by Massey, Hofmann, and Palmer (10). All protein concentrations for these spectral analyses were determined by the biuret reaction (11); the relation of dry weight to biuret assay has been reported$.Determination of Polypeptide Chain Ratio. Pure polypeptide chains were isolated by preparative dodecylsulfate-polyacrylamide gel electrophoresis (8). They were subjected to analytical electrophoresis under conditions essentially the same as those of Weber and Osborn (12). The gels contained 10% acrylamide, 0.135% methylene bisacrylamide, 0.05% sodium dodecylsulfate, and 0.05 M phosphate, pH 7.0. The gel columns were 60-mm high and had a diameter of 5 mm. Electrophoresis was for about 2.5 hr at 6 mA per column. The gels were stained for 2 hr with Commassie Brilliant Blue (12), and were immediately destained electrophoretically. The gels were then kept for 8 days in 7.5% acetic acid-5% methanol to remove all background stain. Stain intensity was measured with a Joyce-Loebl microdensitometer equipped with a red filter. Each column was measured twice; after the first tracing the column was rotated around its longitudinal axis by 900 and a second tracing was taken. The difference between the two measurements did not exceed 5% per band. Each point recorded in Fig. 1 is an average value of these two measurements. The areas below the tracings were cut out and weighed with a microbalance. (The density of the r...

The dihydrolipoamide transacetylase component of the Escherichia coli K-12 pyruvate dehydrogenase complex has been shown to consist of identical or very nearly identical polypeptide chains with a molecular weight of 80000 (f 5 O/J. Amino acid and tryptic peptide analyses gave a minimum chemical molecular weight of about 80 000. Carboxypeptidase degradation revealed one C-terminal sequence . . . Arg-Arg-(Va1,Leu)-Met-CO,H per 80000 molecular weight protein. An N-terminal amino acid was not found. Dodecylsulfate polyacrylamide gel electrophoresis of the transacetylase subunit (either native, performic acid oxidized, or carboxymethylated) showed one protein band moving as a species with molecular weight of 80000.These data are at variance with the results of Reed and associates who had found that the transacetylase component of the E. coli Crookes pyruvate dehydrogenase complex consists of identical 36000 molecular weight polypeptide chains. It has been found that in strain W945 T I of E . coli K-12, the 80000 molecular weight transacetylase subunit is cleaved during the purification procedure, yielding a major fragment with a molecular weight of about 38000. It is possible that this cleavage explains the inconsistency between the transacetylase subunit molecular weights .It has been confirmed that the dihydrolipoamide dehydrogenase consists of subunits with molecular weight 56 000.A new, relatively rapid purification procedure for the pyruvate dehydrogenase complex is described; it allows the preparation of 200-300 mg enzyme complex, more than goo/,, pure, within one week.The pyruvate dehydrogenase complex catalyzes the overall reaction : Pyruvate + NAD+ + CoA + Acetyl-CoA + CO, + NADH + H+. (1) The complex consists of three enzyme components, pyruvate dehydrogenase (El), dihydrolipoamide transacetylase (E2), and dihydrolipoamide dehydrogenase (E3). These components participate in reaction (1) by the following sequence of reactions [1,21: .____

It is shown that very likely the Escherichia coli K-12 pyruvate dehydrogenase complex is not a unique entity in that the amount of pyruvate dehydrogenase component it contains can vary. A complex can be obtained from which "excess" pyruvate dehydrogenase component is removed and this complex, called core complex, has an entirely reproducible polypeptide chain composition. The core complex has a molecular weight of 3750000 3: 200000.

Cytosolic glutathione S-transferase (GST) activity is confined to the proximal convoluted and straight tubules. Damage to these parts of the nephron should result in leakage of GST into the urinary space. Lactate dehydrogenase (LDH), in contrast, is more generally distributed along the nephron. Measurement of both enzyme activities could therefore be expected to discriminate between different localizations of nephrotoxicity. To test this hypothesis, we determined both enzyme activities in 24 h urine samples from 10-12 female Sprague-Dawley rats, each treated with single i.p. injections of puromycin aminonucleoside (PAN, 130 mg/kg), Na2 CrO4 10, 20, 30 mg/kg), mercuric chloride (HgCl2, 0.5, 0.75, 1.0 mg/kg), folic acid (125, 350, 375 mg/kg), ethyleneimine (0.5, 2.0, 5.0 microliters/kg). Bovine serum albumin (BSA) was injected by the same method, twice daily on 3 consecutive days (2.5, 7.14 g/kg). The results obtained indicate a characteristic dose- and time-dependent pattern of excreted enzyme activities for each of the tested compounds. In both models with primarily glomerular damage, proximal tubular parts were also affected, as could be demonstrated by increased urinary GST and histopathological changes. Damage, mainly to the S1/S2 segment by 20 or 30 mg Na2 CrO4/kg, resulted in moderate to marked increases in LDH excretion, while GST was only moderately elevated at 30 mg/kg. Extreme increases in GST and LDH output were measured after predominant S3 segment damage after 0.75 and 1.0 mg HgCl2/kg. The distally active compounds, folic acid and ethyleneimine, did not increase GST excretion at lower doses. At the high doses, a small rise in GST excretion indicated some, probably secondary, proximal tubular involvement, which correlated with the histopathological findings in these groups.