Crystallization and Properties of Rabbit Skeletal Muscle Phosphofructokinase

Parmeggiani, Albamaria; Luft, John H.; Love, David; Krebs, Edwin G.

doi:10.1016/s0021-9258(18)99694-8

Cited by 225 publications

(54 citation statements)

References 41 publications

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“…2A) showed a homogeneous spread of individual particles that were interpreted to be PFK tetramers composed of pairs of dimer units separated by a narrow space. Higher polymers than tetramers were not observed in these images, which were similar to previously published EM patterns of rabbit muscle (1,28,44,45), chicken and pig liver (27,46), and human erythrocytes and muscle (1,47) PFKs. Reference free alignment, classification, and averaging of individual particles of human PFK-M showed an elongated molecule with apparent dimensions of 14.5 nm for the long axis and 9 nm for the short axis.…”

Section: Em Analysis Of Human Muscle and D Discodeum Pfksupporting

confidence: 90%

Electron microscopy analysis of mammalian phosphofructokinase reveals an unusual 3‐dimensional structure with significant implications for enzyme function

et al. 2010

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Phosphofructokinase is a sophisticated allosteric enzyme that is fundamental for the control of glycolysis. The structure of the bacterial enzyme is well characterized. However, little is known about the structural organization of the more complex enzyme from mammals. We have obtained the structure of human muscle phosphofructokinase in the presence of fructose 6‐phosphate at a resolution of 1.8 nm by electron microscopy (EM). Particles of the tetrameric enzyme corresponded to an elongated molecule (14.5 ×9nm) arranged into 2 dimeric subdomains. Image analysis and 3‐dimensional reconstruction showed the presence of a prominent channel in one of the dimers but not in the opposite one, revealing that they are in greatly different conformations. Fitting of bacterial structures into the EM model suggested disruption of the fructose 6‐phosphate catalytic and the fructose 2,6‐bisphophate allosteric sites in the cavity‐containing dimer. Therefore, the reported structure might have major implications for the function of mammalian phosphofructoki‐nase.—Arechaga, I., Martínez‐Costa, O. H., Ferreras, C., Carrascosa, J. L., Aragon, J. J. Electron microscopy analysis of mammalian phosphofructokinase reveals an unusual 3‐dimensional structure with significant implications for enzyme function. FASEB J. 24, 4960–4968 (2010). http://www.fasebj.org

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Section: Em Analysis Of Human Muscle and D Discodeum Pfksupporting

confidence: 90%

Electron microscopy analysis of mammalian phosphofructokinase reveals an unusual 3‐dimensional structure with significant implications for enzyme function

et al. 2010

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show abstract

“…Four AMP or fructose 6-phosphate binding units would make up a particle of this size. At low protein concentration and in the presence of ATP, Parmeggiani et al (1966) have observed a sedimentation coefficient of 8.4 S which would be consistent with a molecular weight of about 200,000.…”

Section: Discussionmentioning

confidence: 53%

“…One of these binding sites is identical with the AMP binding site and at least one must represent the catalytic site. Two other effects of ATP have been reported by Parmeggiani et al (1966). ATP appears to be obligatory for the crystallization of skeletal muscle P-fructokinase and is actually bound in the crystal lattice; and studies with the ultracentrifuge have indicated that ATP leads to a dissociation of the enzyme into a less highly aggregated form.…”

Section: Discussionmentioning

confidence: 90%

“…The enzyme solution was dialyzed in the cold for several hours against the buffer and, to remove bound ATP, was passed through a 20 X 4 mm column containing a mixture of one part of acid-washed charcoal and one part of coarse powdered cellulose. A 280:260 m/x ratio of 1.68 or greater indicated that ATP had been removed from the enzyme (Parmeggiani et al, 1966). The protein concentration was determined at 279 µ by using the extinction coefficient of 10.2 for a 1 % solution (Parmeggiani et al, 1966).…”

Section: Methodsmentioning

confidence: 99%

“…A 280:260 m/x ratio of 1.68 or greater indicated that ATP had been removed from the enzyme (Parmeggiani et al, 1966). The protein concentration was determined at 279 µ by using the extinction coefficient of 10.2 for a 1 % solution (Parmeggiani et al, 1966). Preparations were used for binding studies within 30 min of the time they were freed of ATP.…”

Section: Methodsmentioning

confidence: 99%

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