Affinity precipitation of enzymes

Larsson, Per‐Olof; Mosbach, Klaus

doi:10.1016/0014-5793(79)80211-2

Cited by 91 publications

(15 citation statements)

References 7 publications

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“…The progressive atrophy of type II fibers may therefore partly explain the reduced muscle creatine in the elderly. Moreover, the reduction of creatine in the muscle of the elderly is in line with previous evidence that documents an increased oxidative process in aged skeletal muscles, for example, with decreased dependence on glycolysis [58] and a decrease of lactate dehydrogenase [59]. Smith et al (1998) first reported an increase in muscle PCr (30%) in middle-aged adults (58 years) as a result of short-term intake of high doses of creatine (0.3 g/kg/day for 5 days) [52].…”

Section: 2supporting

confidence: 90%

Novel Insights on Nutrient Management of Sarcopenia in the Elderly

Rondanelli¹,

Faliva²,

Monteferrario³

et al. 2016

Clinical Nutrition and Aging

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Sarcopenia is defined as a syndrome characterized by progressive and generalized loss of muscle mass and strength. The more rationale approach to delay the progression of sarcopenia is based on the combination of proper nutrition, possibly associated with the use of dietary supplements and a regular exercise program. We performed a narrative literature review to evaluate the till-now evidence regarding (1) the metabolic and nutritional correlates of sarcopenia; (2) the optimum diet therapy for the treatment of these abnormalities. This review included 67 eligible studies. In addition to the well recognized link between adequate intake of proteins/amino acids and sarcopenia, the recent literature underlines that in sarcopenic elderly subjects there is an unbalance in vitamin D synthesis and in omega-6/omega-3 PUFA ratio. Given the detrimental effect of these metabolic abnormalities, a change in the lifestyle must be the cornerstone in the treatment of sarcopenia. The optimum diet therapy for the sarcopenia treatment must aim at achieving specific metabolic goals, which must be reached through accession of the elderly to specific personalized dietary program aimed at achieving and/or maintaining muscle mass; increasing their intake of fish (4 times/week) or taking omega-3 PUFA supplements; taking vitamin D supplementation, if there are low serum levels.

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Section: 2supporting

confidence: 90%

Novel Insights on Nutrient Management of Sarcopenia in the Elderly

Rondanelli¹,

Faliva²,

Monteferrario³

et al. 2016

Clinical Nutrition and Aging

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“…Bifunctional NAD analogues, bis-NAD, have been described as useful reagents for affecting affinity precipitation of enzymes (10). In this report we describe the use of such bis-NAD analogues to obtain an immobilized two-enzyme system in which the two different active sites are facing one another.…”

Section: )mentioning

confidence: 99%

“…Beef heart lactate dehydrogenase (520 units/mg of protein), pig heart lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3; 136 units/mg of protein), NAD, NADH, pyruvate, and oxalate were purchased from Sigma. Benzyl alcohol, acetaldehyde, and silica plates for TLC were from Merck (Darmstadt, Federal Republic of Germany), tresyl chloride was from Fluka (Buchs, Switzerland), Sepharose and DEAE-Sephacel were from Pharmacia (Uppsala, Sweden), and bis-NAD II (10) N6-[(6-aminohexyl)carbamoylmethyl]-NAD (13) can be obtained from Sigma. bis-NAD I was synthesized according to the procedure for bis-NAD II (10) but with hydrazine instead of adipic acid dihydrazide.…”

Section: Methodsmentioning

confidence: 99%

Site-to-site directed immobilization of enzymes with bis-NAD analogues.

Månsson¹,

Siegbahn²,

Mosbach³

1983

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

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Lactate dehydrogenase (L-lactate:NAD' oxidoreductase, EC 1.1.1.27) and alcohol dehydrogenase (alcohol: NAD' oxidoreductase, EC 1.1.1.1) have been crosslinked with glutaraldehyde on agarose beads. The crosslinking was performed while the two enzymes were spatially arranged with their active sites facing one another with the aid of a bis-NAD analogue. Subsequently the bis-NAD analogue was allowed to diffuse out. By using a third enzyme, lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3), which was also coupled to the same beads and which competes with lactate dehydrogenase for the NADH produced by alcohol dehydrogenase, the effect of site-tosite directed immobilization was studied. It was found that much more NADH than was theoretically expected (50% instead of 19% of produced NADH) was oxidized by lactate dehydrogenase, which indicates that the NADH was preferentially channeled to lactate dehydrogenase due to the juxtapositioned active sites of the two enzymes.Several coimmobilized multistep enzyme systems have been described in the literature (1-6). For example, an immobilized system composed of the sequence malate dehydrogenase/citrate synthase showed a consistantly higher overall steady-state rate (6). More recently, a four-enzyme sequence (7) and even the enzymes of a complete metabolic cycle, the urea cycle, have been coimmobilized to supports (8). In the latter case, the immobilized cyclic enzyme system again was more efficient than the corresponding soluble system. These effects have been partly attributed to the close proximity of the enzymes and partly to the diffusional restrictions imposed by the Nernst unstirred layer around the enzymes (2, 9).

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“…Several efforts have been made for this purpose [l -31 but only the alcohol-dehydrogenase -NAD complex, reported by MAnsson et al [2], has been clearly demonstrated to have covalently bound NAD. As for the complex, recycling of the bound NAD by lactate or malate dehydrogenase [4] and kinetic properties of the alcohol dehydrogenase activity of the complex [5] have also been investigated.We have prepared poly(ethyleneglyco1)-bound NAD (PEG-NAD) as a macromolecular NAD derivative for enzyme reactors [6, 71. PEG-NAD has a unique structure in that one NAD molecule and one amino group are linked with Correspondence to I. Urabe,…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Covalent linking of poly(ethyleneglycol)‐bound NAD with Thermus thermophilus malate dehydrogenase

Eguchi

Iizuka

Kagotani

et al. 1986

European Journal of Biochemistry

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Poly(ethyleneglyco1)-bound NAD (PEG-NAD) was covalently linked to Thermus thermophilus malate dehydrogendse with a bifunctional reagent, 3,3'-(1,6-dioxo-1,6-hexanediyl)bis-2-thiazolidinethione. The covalently linked malate-dehydrogenase -PEG -NAD complex (MDH-PEG-NAD) was purified by DEAE-Sephadex column chromatography to remove unbound PEG-NAD, and fractionated by blue-Sepharose column chromatography into four preparations: MDH-PEG-NAD I, MDH-PEG-NAD 11, MDH-PEG-NAD 111 and MDH-PEG-NAD IV. The average numbers of NAD moieties covalently bound per subunit of MDH-PEG-NAD I, MDH-PEG-NAD 11, MDH-PEG-NAD I11 and MDH-PEG-NAD IV were 1.2, 1.2, 0.8 and 0.5, respectively, and the values were confirmed by sodium dodecyl sulfate/polydcrylamide gel electrophoresis. 60 -80% bound NAD moiety of these preparations of MDH-PEG-NAD was reduced by the enzyme moiety in the presence of Lmalate, and the specific activity of the enzyme moiety of the preparations was more than 80% that of the native enzyme.MDH-PEG-NAD I has the following properties. The K , value for exogeneous NAD is three times that of the native enzyme. The coenzyme activity of its NAD moiety is 20-40% that of native NAD for alcohol and lactate dehydrogenases. The complex catalyzes the oxidation of L-malate in the presence of the redox system of 5-ethylphenazinium ethyl sulfate and a tetrazolium salt with a rate constant of 0.11 s-l. The coenzyme moiety of the complex can also be recycled by coupled reactions of the active site of the same complex and alcohol dehydrogenase. These results indicate that MDH-PEG-NAD works as an NAD(H)-regeneration unit for coupled reactions.A covalently linked dehydrogenase-NAD complex is an attractive catalytic unit if the bound NAD can be used by other enzymes as well as by itself. Such a complex serves as an NAD(H)-regeneration unit in enzyme reactors and also provides many interesting questions about the effects of fixing a readily dissociable coenzyme in the vicinity of an enzyme. Several efforts have been made for this purpose [l -31 but only the alcohol-dehydrogenase -NAD complex, reported by MAnsson et al. [2], has been clearly demonstrated to have covalently bound NAD. As for the complex, recycling of the bound NAD by lactate or malate dehydrogenase [4] and kinetic properties of the alcohol dehydrogenase activity of the complex [5] have also been investigated.We have prepared poly(ethyleneglyco1)-bound NAD (PEG-NAD) as a macromolecular NAD derivative for enzyme reactors [6, 71. PEG-NAD has a unique structure in that one NAD molecule and one amino group are linked with Correspondence to I. Urabe,

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Affinity precipitation of enzymes

Cited by 91 publications

References 7 publications

Novel Insights on Nutrient Management of Sarcopenia in the Elderly

Novel Insights on Nutrient Management of Sarcopenia in the Elderly

Site-to-site directed immobilization of enzymes with bis-NAD analogues.

Covalent linking of poly(ethyleneglycol)‐bound NAD with Thermus thermophilus malate dehydrogenase

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