PubMed Health⌕ Search

Biomedical subjects

C W Akey

Publications and source records attributed to C W Akey.

23 records · Page 2Linked to original sources
PubMed1983

Electron microscopy of beef heart F1-ATPase crystals.

The structure of thin crystalline plates of beef heart F1-ATPase has been investigated by a combination of electron microscopy and computer-based image processing. Both negatively stained thin crystals and thin sections of embedded crystals were used in the analysis. Some inherent twinning was observed in the thin crystals and two distinct orthorhombic crystal forms present in the microcrystal population were characterized. The form I crystals are space group P2(1)2(1)2 with unit cell parameters of a = 164 A, b = 324 A, and c = 118 A. The form I crystals have 1 molecule of beef heart coupling factor-ATPase/asymmetric unit and averaged reconstructions of projections of the (001) and (100) planes allowed the deduction of the packing of single F1-ATPase complexes in the crystals. The form II crystals have unit cell parameters of a = 156 A, c = 162 A, beta = 90 degrees and are either space group P2(1)2(1)2 or P222(1). Furthermore, based on the results presented in this report, it is clear that the monoclinic crystalline inclusions which have been observed in human mitochondria are not directly related to the form I or form II crystals of the F1-ATPase.

Animals↗
PubMed1983

Equivalence of the projected structure of thin catalase crystals preserved for electron microscopy by negative stain, glucose or embedding in the presence of tannic acid.

Thin crystals of beef liver catalase have been examined by electron microscopy following various preservation procedures. In the first part of this investigation, micrographs of three principal projections were obtained from thin sections of micro-crystals embedded in the presence of tannic acid. Computer reconstructions confirmed the space group assignment of P2(1)2(1)2(1) and permitted the packing arrangement of the catalase tetramers to be deduced to a resolution of about 20 A. These results corroborate the packing model for this crystal form proposed by Unwin (1975) on the basis of molecular modeling of one projection. In the second part of this investigation, the projected structures of the thin crystals in various preserving media were compared. The negative contrasting of crystals embedded in the presence of tannic acid was confirmed by direct comparison with non-embedded, negatively stained thin platelet crystals. In addition, good agreement at 20 A resolution was observed between the structure of negatively stained crystals and the structure of crystal platelets preserved in glucose and examined by low-dose methods, while moderate agreement was established with the published data of Taylor (1978) for crystals embedded in thin ice films. Tannic acid alone was also found to serve as a suitable medium for preserving catalase crystals to a resolution of 3 X 7 A as judged by electron diffraction. Overall, we demonstrate that projections obtained from thin sections of catalase crystals embedded in the presence of tannic acid can provide a reliable, negatively contrasted representation of the protein structure to 20 A resolution. Examination of sectioned crystals could thus provide a useful adjunct to X-ray crystallographic studies of protein crystals and three-dimensional reconstruction of crystal thin sections should ultimately be possible.

Animals↗
PubMed1983

Electron microscopy and single molecule averaging of subunit-deficient F1-ATPases from Escherichia coli and spinach chloroplasts.

The morphology of F1-ATPases lacking one or more small subunits has been investigated by minimal-beam electron microscopy of close-packed monolayers of molecules. Computer-based rotational analyses of single molecules were performed on reconstituted 3-subunit F1-ATPase (-delta epsilon) from Escherichia coli and both 3-subunit (-delta epsilon) and 4-subunit (-delta) F1-ATPase from chloroplasts. Optical diffraction measurements of close-packed arrays revealed maximal dimensions of 122 +/- 4 A and 129 +/- 9 A for 3-subunit ECF1 and 4-subunit CF1, respectively. Molecules which displayed either hollow or solid hexagonal morphologies were observed in all preparations. Averaged reconstructions were obtained from molecules with hollow morphologies in 3-subunit preparations and demonstrated strong hexagonal symmetry in projection with a central, stain-filled cavity. The average reconstruction obtained from molecules with the solid morphology in 4-subunit CF1 preparations, was also strongly hexagonal with six peripheral units ringed about a central subunit. Differences between hollow and solid morphologies cannot be attributed solely to the presence or absence of the delta and epsilon subunits; therefore, the two image types may represent staining variants of a common structure. Overall, the reconstructions are consistent with an alpha 3 beta 3 gamma stoichiometry for the coupling factors from both E. coli and chloroplasts.

Chloroplasts↗
PubMed1977

Dissociation and reconstitution of human ferroxidase II.

The ferroxidase II protein from human serum is large and structurally complex. It possesses protein-bound lipid and copper components which are essential for the maintenance of its catalytic activity. Treatment of ferroxidase II with 8 M urea, 6 M guanidine hydrochloride, or 6 M guanidine hydrochloride and alkylation does not result in the dissociation of the enzyme into subunits. However, treatment with sodium dodecyl sulfate results in the dissociation of ferroxidase II into two nonidentical subunits, designated S-I and S-II. S-I contains little phospholipid, cholesterol, or copper and has a molecular weight of 3.8-3.9 X 10(5). In contrast, S-II contains bound phospholipid, cholesterol, and copper and has a molecular weight of 2.2-2.4 X 10(5). The lipid compositon of S-II is identical with the native enzyme. Sodium dodecyl sulfate-free S-I exhibits no ferroxidase activity. Immediately following removal of sodium dodecyl sulfate, S-II exhibits ferroxidase activity but S-II rapidly loses its activity in the absence of S-I. The separated subunits spontaneously reassociate upon removal of the sodium dodecyl sulfate to yield a fully active enzyme which chemically appears identical with native ferroxidase II. Furthermore, the reconstituted enzyme is stable. Both native and reconstituted ferroxidase II may be stored at 4 degrees C for 6 weeks without any loss in activity. This suggests that S-II, the copper and lipid-containing subunit, is the catalytic subunit and that S-I is essential for the stabilization of the enzymic activity of S-II. These results provide insight into the molecular structure and chemical composition of ferroxidase II and suggest that the complete native structure of ferroxidase II is required for the maintenance of i-s functional integrity.

Apoenzymes↗