Isolierung und Charakterisierung eines membrangebundenen Pyruvatdehydrogenase-Komplexes aus dem phototrophen Bakterium Rhodospirillum rubrum /Isolation and Characterization of a Membrane-Bound Pyruvate Dehydrogenase Complex from the Phototrophic Bacterium Rhodospirillum rubrum

1977 ◽  
Vol 32 (5-6) ◽  
pp. 351-361 ◽  
Author(s):  
Rosemarie Lüderitz ◽  
Jobst-Heinrich Klemme
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Metal Ion ◽  
Rhodospirillum Rubrum ◽  
Coenzyme A ◽  
Specific Activity ◽  
Pyruvate Dehydrogenase Complex ◽  
Thiamine Pyrophosphate ◽  
Enzyme Complex ◽  
Isolation And Characterization ◽  
Dehydrogenase Complex

Abstract The pyruvate dehydrogenase complex from the photosynthetic bacterium Rhododospirillum rubrum was associated with the membrane fraction both in heterotrophically and photosynthetically grown cells. The complex was separated from the membranes and partially purified by precipitation with MgSO4 and gelfiltration through Sepharose 4B. The purified complex had a specific activity of 1.5 - 2 μmol/min-mg protein and contained the following partial activities: pyruvate dehydrogenase (EC 1.2.4.1), dihydrolipoamide transacetylase (EC 2.3.1.12) and dihydrolipoamide dehydrogenase (EC 1.6.4.3). Contrary to other bacterial pyruvate dehydrogenase complexes, the enzyme complex from R . rubrum revealed no cooperativity between pyruvate binding sites. The kinetic constants [Km) for the overall reaction were (in mм) : 0.14 (pyruvate), 0.07 (NAD) and 0.025 (coenzyme A). The Km for thiamine pyrophosphate was dependent on the nature and the concentration of the divalent metal ion (Mn or Mg) present in the reaction mixture, the values ranging from 0.5 to 3 μm . NADH was a potent inhibitor (Ki = 5 μm) of the enzyme complex and the dihydrolipo­ amide dehydrogenase. The inhibition was competitive with respect to NAD. In addition to its rapid inhibitory effect, NADH also inactivated the enzyme. Cysteine partially protected the enzyme complex against NADH-inactivation. Acetyl-coenzyme A also inhibited the overall reaction (Ki = 40 μм) . The inhibition was dependent on the concentration of coenzyme A, but independent of the concentration of pyruvate. Sugar phosphates, phosphoenolpyruvate, citric acid cycle intermediates and nucleosidephosphates (1 mм) had no pronounced effect on the overall reaction.

scholarly journals Cross-linking and 1H n.m.r. spectroscopy of the pyruvate dehydrogenase complex of Escherichia coli

1982 ◽  
Vol 205 (2) ◽  
pp. 389-396 ◽  
Author(s):  
Leonard C. Packman ◽  
Richard N. Perham ◽  
Gordon C. K. Roberts
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
The Other ◽  
Random Coil ◽  
Cross Linking ◽  
Enzyme Complex ◽  
Cross Links ◽  
Dehydrogenase Complex ◽  
Lipoyl Domain

The pyruvate dehydrogenase complex of Escherichia coli was treated with o-phenylene bismaleimide in the presence of the substrate pyruvate, producing almost complete cross-linking of the lipoate acetyltransferase polypeptide chains as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This took place without effect on the catalytic activities of the other two component enzymes and with little evidence of cross-links being formed with other types of protein subunit. Limited proteolysis with trypsin indicated that the cross-links were largely confined to the lipoyl domains of the lipoate acetyltransferase component of the same enzyme particle. This intramolecular cross-linking had no effect on the very sharp resonances observed in the 1H n.m.r. spectrum of the enzyme complex, which derive from regions of highly mobile polypeptide chain in the lipoyl domains. Comparison of the spin–spin relaxation times, T2, with the measured linewidths supported the idea that the highly mobile region is best characterized as a random coil. Intensity measurements in spin-echo spectra showed that it comprises a significant proportion (probably not less than one-third) of a lipoyl domain and is thus much more than a small hinge region, but there was insufficient intensity in the resonances to account for the whole lipoyl domain. On the other hand, no evidence was found in the 1H n.m.r. spectrum for a substantial structured region around the lipoyl-lysine residues that was free to move on the end of this highly flexible connection. If such a structured region were bound to other parts of the enzyme complex for a major part of its time, its resonances might be broadened sufficiently to evade detection by 1H n.m.r. spectroscopy.

scholarly journals Plant mitochondrial pyruvate dehydrogenase complex: purification and identification of catalytic components in potato

1998 ◽  
Vol 334 (3) ◽  
pp. 571-576 ◽  
Author(s):  
A. Harvey MILLAR ◽  
Carina KNORPP ◽  
Christopher J. LEAVER ◽  
Steven A. HILL
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Specific Activity ◽  
Divalent Cations ◽  
Pyruvate Dehydrogenase Complex ◽  
Protein Sequences ◽  
Protein Band ◽  
Sds Page ◽  
Molecular Masses ◽  
Α Subunits ◽  
Dehydrogenase Complex

The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberosum cv. Romano) tuber mitochondria was purified 40-fold to a specific activity of 5.60 µmol/min per mg of protein. The activity of the complex depended on pyruvate, divalent cations, NAD+ and CoA and was competitively inhibited by both NADH and acetyl-CoA. SDS/PAGE revealed the complex consisted of seven polypeptide bands with apparent molecular masses of 78, 60, 58, 55, 43, 41 and 37 kDa. N-terminal sequencing revealed that the 78 kDa protein was dihydrolipoamide transacetylase (E2), the 58 kDa protein was dihydrolipoamide dehydrogenase (E3), the 43 and 41 kDa proteins were α subunits of pyruvate dehydrogenase, and the 37 kDa protein was the β subunit of pyruvate dehydrogenase. N-terminal sequencing of the 55 kDa protein band yielded two protein sequences: one was another E3; the other was similar to the sequence of E2 from plant and yeast sources but was distinctly different from the sequence of the 78 kDa protein. Incubation of the mPDC with [2-14C]pyruvate resulted in the acetylation of both the 78 and 55 kDa proteins.

scholarly journals Purificaton of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus and resolution of its four component polypeptides

1980 ◽  
Vol 189 (1) ◽  
pp. 161-172 ◽  
Author(s):  
C E Henderson ◽  
R N Perham
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Bacillus Stearothermophilus ◽  
Pyruvate Dehydrogenase Complex ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Yield ◽  
Icosahedral Symmetry ◽  
Enzyme Complex ◽  
Molecular Masses ◽  
Dehydrogenase Complex

1. The pyruvate dehydrogenase complex was purified from Bacillus stearothermophilus in high yield. The specific activity (about 40nkat/mg of protein) was substantially lower than that of the pyruvate dehydrogenase complex from Escherchia coli (about 570nkat/mg of protein) measured at 30 degrees C under the same conditions. 2. The relative molecular masses of the four types of polypeptide chain i the complex were estimated by means of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to be 57 000, 54 000, 42 000 and 36 000 respectively. These polypetide chains showed no evidence of seriously anomalous behavior during tests of electrophoretic mobility. 3. The enzyme complex was resolved into its constituent proteins by means of gelfiltration on Sepharose CL-6B in the presence of 2M-KI, followed by chromatography on hydroxyapatite in the presence of 8M-urea. These harsh conditions were necessary to cause suitable dissociation of the enzyme complex. 4. The amino-acid compositions of the four constituent proteins after resolution were determined and their chain ratios were measured for several preparations of the complex. Some variability was noted between preparations but all samples contained a significant molar excess of the chains thought to contribute the pyruvate decarboxylase (EC 1.2.4.1) activity. 5. From the relative molecular masses and chain ratios of the four constituent proteins, it was calculated that the empirical unit must be repeated at least 50 times to make up the assembled complex. This conclusion is fully consistent with the demonstration by means of electron microscopy of apparent icosahedral symmetry for the Bacillus stearothermophilus complex, implying a 60-fold repeat. The structure stands in sharp contrast with the octahedral symmetry (24-fold repeat) of the Escherichia coli enzyme.

Regulation of pyruvate dehydrogenase complex in ischemic rat heart

1984 ◽  
Vol 246 (6) ◽  
pp. H858-H864 ◽  
Author(s):  
T. B. Patel ◽  
M. S. Olson
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Pyruvate Dehydrogenase Complex ◽  
Total Activity ◽  
Control Flow ◽  
Enzyme Complex ◽  
Multienzyme Complex ◽  
Flow Rates ◽  
Perfused Rat Heart ◽  
Dehydrogenase Complex

The effect of flow-induced ischemia on the rate of pyruvate decarboxylation and the activation state of the pyruvate dehydrogenase multienzyme complex was investigated in the isolated, perfused rat heart. Pyruvate dehydrogenase activity in the heart decreased significantly during flow-induced ischemia and was a function of changes in the activation state (i.e., active/total activity) of the enzyme complex. In the absence of pyruvate, the activation state of pyruvate dehydrogenase decreased from nearly 100% active at the normal flow rate (10 ml/min) to 20% active as the flow was reduced to 0.5 ml/min. At high pyruvate levels (5 mM), the activation state increased from nearly 70% active at control flow rates to 100% active during ischemia. At an intermediate pyruvate concentration (0.5 mM), the enzyme complex was maintained at a relatively low activation state (30–35% active) throughout the range of flow rates tested. Ischemia caused elevated perfusate lactate concentrations only when the flow rates were less than 5.0 ml/min. The activation state of the pyruvate dehydrogenase complex in hearts perfused with glucose was also decreased during ischemia.

scholarly journals Inhibition of pyruvate dehydrogenase complex by moniliformin

1986 ◽  
Vol 233 (3) ◽  
pp. 719-723 ◽  
Author(s):  
P S Gathercole ◽  
P G Thiel ◽  
J H S Hofmeyr
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Inhibitory Action ◽  
Pyruvate Dehydrogenase Complex ◽  
Time Dependent ◽  
Enzyme Complex ◽  
First Order ◽  
Saturation Kinetics ◽  
Thiamin Pyrophosphate ◽  
Inhibition Reaction ◽  
Dehydrogenase Complex

The mechanism for the inhibition of pyruvate dehydrogenase complex from bovine heart by moniliformin was investigated. Thiamin pyrophosphate proved to be necessary for the inhibitory action of moniliformin. The inhibition reaction was shown to be time-dependent and to follow first-order and saturation kinetics. Pyruvate protected the pyruvate dehydrogenase complex against moniliformin inactivation. Extensive dialysis of the moniliformin-inactivated complex only partially reversed inactivation. Moniliformin seems to act by inhibition of the pyruvate dehydrogenase component of the enzyme complex and not by acting on the dihydrolipoamide transacetylase or dehydrogenase components, as shown by monitoring the effect of moniliformin on each component individually. On the basis of these results, a suicide inactivator mechanism for moniliformin on pyruvate dehydrogenase is proposed.

Sign in / Sign up

Export Citation Format

Share Document