Protein iodination with solid state lactoperoxidase

David, Gary S.; Reisfeld, Ralph A.

doi:10.1021/bi00702a028

Cited by 433 publications

(116 citation statements)

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“…Lipoprotein lipase (EC 3.1.1.34) described [8]. 1251-labeled lipoprotein lipase was prepared by the lactoperoxidase method [9] and then repurified and separated from unreacted iodide by chromatography on heparin-Sepharose. The resulting material had the same specific enzyme activity as the unlabeled preparation, and retained its ability to bind to heparin-Sepharose.…”

Section: Methodsmentioning

confidence: 99%

Lipoprotein Lipase: Some Effects of Activator Proteins

Bengtsson

Olivecrona

1980

European Journal of Biochemistry

118

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This paper considers how apolipoprotein CII from human plasma lipoproteins and T1 and T2 proteins from egg yolk lipoproteins stimulate the activity of lipoprotein lipase. These activator proteins stabilized the enzyme much more effectively than a thousandfold higher concentration of albumin did, indicating a direct interaction with the enzyme. The effects of the activators were seen also at 1 M NaC1. Thus, forces other than electrostatic are implicated. Centrifugation experiments showed that '251-labeled lipase bound equally well to the emulsion droplets in the absence of activator protein as in its presence. This was true even under conditions when the activator caused a severalfold increase in the rate of hydrolysis. Thus, the activator makes enzyme at the interface more effective in hydrolysis. By optimizing the conditions it was possible to obtain almost as high rates of triglyceride hydrolysis in the absence as in the presence of activator. Thus, the main effect of the activator protein is probably not on a rate-limiting chemical step. Under most conditions, the rate of hydrolysis was much below optimal and activator increased it. This was always the case with phosphatidylcholine/triglyceride emulsions, where the activator enhanced hydrolysis of both lipids. Other experiments showed that the activator enhanced triglyceride hydrolysis in the absence of phospholipids and phospholipid hydrolysis in the absence of triglycerides. It is suggested that interaction with activator orientates the enzyme and/or the lipid substrate for effective hydrolysis at the surface of lipoproteins/model substrates.Lipoprotein lipase is a glycoprotein enzyme which hydrolyzes acylglycerols in plasma lipoproteins at the capillary endothelium (review in [l]). Activator proteins, present on the lipoproteins, enhance the enzyme activity [2]. The primary structure of the human activator, apolipoprotein CII, is known [3], and the activity has been localized to its C-terminal part [4,5]. How it promotes hydrolysis is however not known. The action of the lipase involves several steps [6], any of which could be the site of the activator effect; adsorption of the lipase to the lipid-water interface of the insoluble substrate, formation of an enzyme-substrate (Michaelis) complex at the interface, the chemical steps of catalysis, and finally removal of the products from the site of lipolysis. The following paper [7] considers the mechanism of the product inhibition. This paper considers the possible effects of activator proteins on the other three steps. MATERIALS AND METHODSLipoprotein lipase was purified from bovine skim milk by chromatography on heparin-Sepharose as Enzyme. Lipoprotein lipase (EC 3.1.1.34) described [8]. 1251-labeled lipoprotein lipase was prepared by the lactoperoxidase method [9] and then repurified and separated from unreacted iodide by chromatography on heparin-Sepharose. The resulting material had the same specific enzyme activity as the unlabeled preparation, and retained its ability to bind to heparin-Sepharose. Lipa...

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Section: Methodsmentioning

confidence: 99%

Lipoprotein Lipase: Some Effects of Activator Proteins

Bengtsson

Olivecrona

1980

European Journal of Biochemistry

118

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“…Highly purified CS (15), factor B (16), factor D (17), ,B1H (6), C3bINA (6), nP (18), and partially purified nephritic factor (19) 20 Ml of rabbit serum were removed from a stock reaction mixture and washed once in GVBE and then once in Mg-GVB. At the end of the kinetic experiment, cells were assayed for the presence of hemolytically active bound C3b as described above.…”

Section: Methodsmentioning

confidence: 99%

Initiation of the alternative pathway of complement: recognition of activators by bound C3b and assembly of the entire pathway from six isolated proteins.

Schreiber¹,

Pangburn²,

Lesavre³

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

147

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ABSTRACr An intact alternative pathway of complement activation was assembled from six isolated proteins present at their respective physiological concentrations (C3, 1200 ug/ml; factor B, 200 ttg/ml; factor D, 2 Aig/ml; 311H, 560 Wig/ml; C3b inactivator, 34 gg/ml; and native properdin, 20 jg/ml) Initiation of the pathway required the presence of five of these proteins not including properdin. The initial C3 convertase of the system was shown to be a fluid-phase rather than a surface-bound enzyme. The ability of the pathway to discriminate between activator and nonactivator was found to reside in the bound C3b molecule. When bound to the surface of an activator through its labile binding site, C3b interacts with surface structures of the activator through another site on the molecule.This interaction results in diminished jPlH binding to C3b and thereby allows the bound C3b molecule to escape control and participate in C3 convertase formation. Thus, initiation of the alternative pathway is a two-step process, the first being nonspecific and the second being discriminatory. (7,8) made the crucial observation that both C3b and the resultant CS convertase deposited on the surface of activators are relatively protected from destruction by the regulatory proteins. In this laboratory it was shown that protection of bound C3b is due to restriction of #1H binding to C3b. It was further shown that restriction of control could be generated by defined chemical rndifications of a nonactivator thereby converting it to an actimtor (9-11). These findings aid in explaining the known ability of activators to allow amplification of the pathway in the presence of the controlling proteins.

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“…Before binding studies, the cell preparations were exposed to 0.85% ammonium chloride for 5 min to lyse the contaminating erythrocytes, washed, and resuspended in the incubation medium (Hanks' solution, pH 7.4, containing 0.1% bovine serum albumin) (7) at various concentrations. lodinated CCF was obtained using the solid phase lactoperoxidase method (8) (5), and migrated the same distance (2.5-2.7 cm) from the cathode on polyacrylamidegel electrophoresis as did the unlabeled material (5).…”

Section: Methodsmentioning

confidence: 99%