Studies of the Electron Transfer System

Fleischer, Sidney; Brierley, Gerald P.; Klouwen, H.M.; Slautterback, David B.

doi:10.1016/s0021-9258(18)50157-5

Cited by 336 publications

(15 citation statements)

References 41 publications

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“…Removal of this environment inactivates the enzyme and a suitable environment can then be provided by the addition of appropriate phospholipids. These data satisfy the criteria for phospholipid-dependence (Fleischer et al, 1962) and confirm our earlier conclusions (Graham & Wood, 1969;Attwood et al, 1971).…”

Section: U4supporting

confidence: 91%

“…We have now examined the phospholipid-dependence of UDP-glucuronyltransferase more closely, using guinea-pig microsomal fractions prepared with 154mm-KCI where this modulation mechanism predominates. The main criteria required to demonstrate the phospholipid-dependence of a membranebound enzyme (Fleischer et al, 1962) are that removal of phospholipid from the membrane should be correlated with loss of enzyme activity and that activity should be restored to lipid-depleted fractions by addition of the appropriate phospholipids. Although previous work strongly suggests that UDP-glucuronyltransferase is phospholipid-dependent, there are serious objections to the validity of some of the conclusions.…”

mentioning

confidence: 99%

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The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Phospholipid depletion and re-activation of guinea-pig liver microsomal enzyme

Graham¹,

Pechey²,

Toogood³

et al. 1977

Biochemical Journal

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More than 80% of the phospholipid component of guinea-pig liver microsomal membranes (prepared with 154mM-KCl) was removed by treatment with phospholipase A followed by extraction of the lysophosphatides and fatty acids produced with albumin. Delipidation strongly inactivated the highly active UDP-glucuronyltransferase of these preparations and activity was restored by mixtures of phosphatidylcholine and lysophosphatidylchlone. However, small quantities of lysophosphatides were still associated with the delipidated fractions after extraction with albumin and might have influenced the inactivation and re-activation observed. To eliminate these uncertainties, microsomal proteins and phospholipids were separated by gel filtration on Sephadex G-150 in the presence of cholate. This technique also strongly inactivated the enzyme but did not generate membrane-active phospholipid degradation products. High transferase activity was again restored to the delipidated protein by choline glycerophosphatides. These results confirm the view that the fully active form of microsomal UDP-glucuronyltransferase is phospholipid-dependent.

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Section: U4supporting

confidence: 91%

mentioning

confidence: 99%

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Phospholipid depletion and re-activation of guinea-pig liver microsomal enzyme

Graham¹,

Pechey²,

Toogood³

et al. 1977

Biochemical Journal

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“…It has, for instance, been suggested that cholesterol regulates permeability through its effect on the fluidity of the hydrocarbon chain layer (43), and in model systems sphingomyelin was found a necessary component for the simulation of action potentials (183) . That some lipids may have specific functions, such as cardiolipin in mitochondria or the linolenic acid-containing galactolipids of chloroplasts, is suggested mainly by their consistent occurrence in these organelles and by reactivation experiments on lipid-depleted structures (18,88) . Whether they participate directly in these functions or through their effects on protein is not known, and their participation may be quite unrelated to their role in forming a bilayer .…”

Section: Discussionmentioning

confidence: 99%

Current Models for the Structure of Biological Membranes

Stoeckenius

Engelman

1969

The Journal of Cell Biology

246

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“…Heavy beef heart mitochondria (HBHM) were prepared by the method of Hatefi and Lester (21); submitochondrial electron transport particles (ETP~) by the method of Smith and Hansen (22), and liver microsomes by the method of MacLennan et al (23). Mitochondrial phospholipid was prepared by the method of Fleischer et al (24).…”

Section: Control Particlesmentioning

confidence: 99%

“…Protein was determined either by the biuret method of Gornall et al (31) or by the method of Lowry et al (32). Lipid phosphorus was determined after the extraction of particles in chloroform:methanol by the method of Fleischer et al (24).…”

Section: Enzymic Assaysmentioning

confidence: 99%

The Isolation of Retinal Outer Segment Fragments

McConnell

1965

The Journal of Cell Biology

109

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Bovine retinal outer segment fragments were isolated by density gradient centrifugation in a high centrifugal field. Assays of the final preparation for enzymes of the mitochondrial respiratory chain indicated mitochondrial contamination not in excess of 1 per cent. Glucose-6-phosphatase and TPNH-cytochrome c reductase activities, presumably diagnostic for microsomes, were also absent. Electron micrographs did not disclose the presence of significant numbers of particles other than fragments of the outer segment discs. The red flagments were characterized by an ascorbate-oxidizing system and a high lipid content.Electron microscopic observations by numerous investigators (1-6) have established fundamental similarities between the ultrastructure of the outer segment of the retinal receptor and that of other membranous structures such as the mitochondrion and chloroplast. Wald (7-9), MacNichol (10, 11), and others have presented unequivocal evidence that the outer segment houses the carotenoid visual pigments required for vision in bright or dim light. Futterman (12-14) has undertaken to elaborate the mechanism which governs carotenoid maintenance and metabolism. Nonetheless, investigation of the device by which the energy of light absorbed by the carotenoid is ultimately transduced into an electrical impulse has been very meagre. The starting assumption of our studies has been that this device must have certain enzymic principles in common with energy-conserving mechanisms found in organelles possessing similar membranous ultrastructure.While all investigators may not share this assumption, all must agree that the serious study of the enzymology of this unique organelle demands its physical isolation from contaminating particles. It now appears inescapable that such isolation has not been achieved in the past. The standard sucrose suspensions of outer segments, from which extracts of the visual pigments are classically derived (8, 9, 15-18), are almost certainly contaminated by mitochondria. Our own observation of the light sensitivity of the ATPase found in digitonin extracts of rhodopsin (19) must in due course be reexamined on this account. The purpose of the present communication is to describe the methodology we have used to isolate the outer segments, and to present the criteria used in evaluation of the completeness of the separation.Many investigators have used one or more modifications of the method of Saito (20), which entails breaking off large numbers of outer segments, or their distal fragments, by shaking the retina in dilute sucrose and then filtering the suspension through gauze. Despite claims made for this method, we have found that the outer segment does not invariably break free of the mitochondrialaden inner segment. This appendage, complete with connecting cilium and enclosed mitochondria, can be seen with the electron microscope in suspensions of outer segments prepared in this way. The inner segments are not removed by gauze filtration, and are not easily removed by sedimentation. ...

show abstract

Studies of the Electron Transfer System

Cited by 336 publications

References 41 publications

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Phospholipid depletion and re-activation of guinea-pig liver microsomal enzyme

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Phospholipid depletion and re-activation of guinea-pig liver microsomal enzyme

Current Models for the Structure of Biological Membranes

The Isolation of Retinal Outer Segment Fragments

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