The role of cell envelope phospholipid in the enzymatic synthesis of bacterial lipopolysaccharide: Binding of transferase enzymes to a lipopolysaccharide-lipid complex

Rothfield, Lawrence; Takeshita, Masazumi

doi:10.1016/0006-291x(65)90611-x

Cited by 25 publications

(16 citation statements)

References 9 publications

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“…Since the original observation by Fleischer, Klouwen, and Brierley (15) that phospholipids were required for mitochondrial electron transport, similar lipid-dependence has been demonstrated for several classes of enzymes, e.g., hydrolytic (glucose-6-phosphatase) (16), synthetic (the transferases required for bacterial cell envelope synthesis) (17), and now for the tissue factor system in blood coagulation. (12).…”

Section: Discussionmentioning

confidence: 83%

Characteristics and lipid requirements of coagulant proteins extracted from lung and brain: the specificity of the protein component of tissue factor

Nemerson¹

1969

J. Clin. Invest.

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A B S T R A C T Soluble proteins were prepared from bovine lung and brain which, when combined with phospholipids, had the biological characteristics of tissue factor. The proteins were solubilized from delipidated (heptane:butanol-extracted) acetone powders with deoxycholate, and precipitated by (NH4)2SO4 (30-60% saturation). These soluble proteins, which contained less than 1% phospholipid, were essentially inert in an assay for tissue factor. When they were recombined with phospholipids, however, activity increased by a factor of 500-1000. Phosphatidylethanolamine was the most active specific phospholipid, followed by phosphatidylcholine. Phosphatidylserine was inert. Mixed phospholipids were from two to four times more active than phosphatidylethanolamine. Maximum activity was obtained at phospholipid to protein ratios of 1.5: 1 (wt/wt), and when relipidation was performed in deoxycholate followed by dialysis against 0.05 M imidazole-0.375 M NaCl, pH 7.2.The proteins were characterized by gel filtration on Sephadex G-200; the activity eluted as a single peak with an apparent mol wt of 425,000. INTRODUCTIONWhen aqueous extracts derived from various tissues are added to plasma, the rate of coagulation after recalcification is markedly enhanced. It is now known that this is due to a specific interaction between the tissue extracts (tissue factor) and a plasma protein, factor VII (1, 2). Although the structure of tissue factor is not known, it has been clear for many years that it contains both a protein and lipid moiety (3). In a previous study on the lipid component of tissue factor, it was demonstrated that certain phospholipids were necessary for full biological activity to be achieved (4).Only some tissues contain significant amounts of tissue factor which strongly suggests that there are specific proteins within the active tissues that account for their coagulant activity. On the other hand, it has been stated that tissue factor is all, or almost all, lipid, (5, 6). Previously, we demonstrated that a lipid-poor protein obtained from brain tissue activated the extrinsic coagulant mechanism, i.e., it functioned as tissue factor (7).This reaction, however, occurred at a very slow rate, and it was therefore uncertain whether this protein represented a specific tissue factor protein, or a nonspecific protease.In The optimal conditions for the restitution of activity by phospholipids were determined for brain and lung proteins and were found to be essentially identical, as were their requirements for specific phospholipids. Preliminary characterization of the proteins was accomplished by gel filtration and ion exchange chromatography. The proteins had similar molecular weights (as determined by gel filtration), but behaved slightly differently on ion exchange chromatography. The chromatographic data suggest that within each organ there is but one active protein (or a family of closely related proteins), and that there may be molecular differences between the tissue factor proteins of lung and brain. METHODSPuri...

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Section: Discussionmentioning

confidence: 83%

Characteristics and lipid requirements of coagulant proteins extracted from lung and brain: the specificity of the protein component of tissue factor

Nemerson¹

1969

J. Clin. Invest.

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“…There are now numer ous accounts of delipidation of enzymes and other membrane proteins associated with loss of activity, and in some cases reactivation has taken place on addition of phospholipid (12)(13)(14)18). It is not yet known whether 7-GT activity is dependent on the presence of phospholipid, but the relatively rapid effect of DIPE extraction would argue more in fa vour of involvement of triglyceride or cho lesterol (8).…”

Section: Discussionmentioning

confidence: 99%

Influence of Detergents and Organic Solvent Extraction on Human Gamma-Glutamyltransferase Activity

Pr¹,

King²

1978

Enzyme

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Human serum and urinary γ-glutamyltransferase (γ-GT) have been found to react differently towards the detergents Triton X-100 and sodium lauryl sulphate (SDS). Serum γ-GT was virtually unaffected by Triton X-100 at a concentration of 5% whereas urinary γ-GT was 10-15% activated under similar conditions. There was a 100-fold difference in the response of serum and urinary γ-GT to SDS. The enzyme activity in urine was completely destroyed by 0.02% SDS, whereas it required 2.0% to destroy the serum enzyme. These latter differences, however, were found to result from the environments of the serum and urinary enzyme rather than to intrinsic factors within the molecule. Extraction of serum γ-GT from normal individuals with «-butanol resulted in little or no loss of activity from the aqueous phase, whereas more than 30% activity was lost from normal urines. Extraction using various mixtures of butanol and di-/sopropyl ether (DIPE) produced largely similar results, except that 25% DIPE: 75% butanol mixture produced a marked loss of activity from serum. It is suggested that γ-GT activity in human body fluids may be dependent on the presence of a lipid fraction.

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“…Tissue factor, in common with several mammalian and bacterial enzymes (29,30), exhibits a requirement for phospholipids. Although an appropriate polar group is essential for activity, the nonpolar portion of the molecule also contributes to the activity: catalytic hydrogenation of egg PC significantly reduced activity as did enzymatic removal of the ,-fatty acid.…”

Section: Methodsmentioning

confidence: 99%

The phospholipid requirement of tissue factor in blood coagulation

Nemerson¹

1968

J. Clin. Invest.

143

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The role of cell envelope phospholipid in the enzymatic synthesis of bacterial lipopolysaccharide: Binding of transferase enzymes to a lipopolysaccharide-lipid complex

Cited by 25 publications

References 9 publications

Characteristics and lipid requirements of coagulant proteins extracted from lung and brain: the specificity of the protein component of tissue factor

Characteristics and lipid requirements of coagulant proteins extracted from lung and brain: the specificity of the protein component of tissue factor

Influence of Detergents and Organic Solvent Extraction on Human Gamma-Glutamyltransferase Activity

The phospholipid requirement of tissue factor in blood coagulation

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