Fluorimetric Assay of Enzymes

Roth, Marc

doi:10.1002/9780470110355.ch5

Cited by 31 publications

(1 citation statement)

References 340 publications

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“…The maximum activity in the culture supernatant was 15% of that identified in the cells of the same culture. Lipase activity and alkaline phosphatase activity was determined using the fluorogenic substrates 4-MU laurate and 4-MU phosphate, respectively [Roth, 1969]. The protein concentration was determined by the method of Bradford [1976] as supplied by Bio-Rad (Richmond, Calif., USA).…”

Section: Biochemical Techniquesmentioning

confidence: 99%

A <i>Brevibacillus choshinensis</i> System That Secretes Cytoplasmic Proteins

Horne¹,

Williams²,

Sutherland³

et al. 2004

Microb Physiol

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Brevibacillus choshinensis has previously been shown to be a useful strain for the secretion of heterologous proteins via the Sec secretory pathway. This pathway involves the secretion of proteins prior to folding, whereas the alternative TAT (twin-arginine translocation) pathway enables pre-folded proteins to be secreted. We have modified the signal peptide of the Brevibacillus expression vector pNCMO2 to accommodate a Sec avoidance signal as well as the twin arginines required for secretion via the TAT system. Use of this modified signal peptide with the phosphotriesterase OpdA enabled B. choshinensis transformants to express and secrete the enzyme in an active and substantially pure form. The system was also used successfully to secrete two cytoplasmic proteins, the phosphotriesterase HocA from Pseudomonas monteilii and the phenylcarbamate-degrading enzyme, PCD, from Arthrobacter oxydans. The inhibitors carbonyl cyanide m-chlorophenyl hydrazine and sodium azide were used to confirm that secretion was occurring via the TAT secretion pathway. The modified B. choshinensis system we have developed may have general utility in secreting a wide range of heterologous proteins in active and conveniently processed form.

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Section: Biochemical Techniquesmentioning

confidence: 99%

A <i>Brevibacillus choshinensis</i> System That Secretes Cytoplasmic Proteins

Horne¹,

Williams²,

Sutherland³

et al. 2004

Microb Physiol

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Determination of Glutamic and Aspartic Acids and Their Amides

1971

Methods of Biochemical Analysis

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Enzymatic activities during evolution of the cement gland in Xenopus tadpoles

Weets¹,

Picard²

1985

J. Exp. Zool.

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The activities of the hydrolases, a-L-fucosidase, N-acetyl-0-Dglucosaminidase (NAG), and zpacoumase appear to be involved in the regression of the cement gland in Xenopus laeuis. The involution of the gland is marked by an important rise of the total as well as the specific activities of a-L-fucosidase and zpacoumase. The specific activity of a-L-fucosidase and of zpacoumase increases about five-fold and eight-fold, respectively, after 12 days of development. Although total activity of NAG decreases during involution, its specific activity undergoes an increase of 93% after 12 days. In contrast, there is a dramatic loss in the total and specific activity of aldolase, used as a control enzyme.The behaviour of these four enzymes is strikingly different in the epidermal tissue adjacent to the gland. Their activity remains stable during the developmental period considered. These observations demonstrate that possible contamination of the explanted gland by surrounding tissue is not responsible for the observed increase in hydrolytic activity in the gland.It has been repeatedly demonstrated that lysosomal enzymes are involved in the tissue involution observed during vertebrate embryogenesis (Eeckhout, '69; Greenfield and Derby, '72; Hickey, '71; Patel, '70; Perriard, '71; Robinson, '70, '72; Scheib, '63, '77).The cement gland is an ectodermal larval organ located caudally to the mouth in most Anuran species. It secretes an abundant mucus that allows the tadpole to stick to immersed objects or the surface of the water where the oxygen tension is higher. The gland differentiates at the early neurula stage and reaches functional maturity at stage 28 (Nieuwkoop and Faber, '67). Shortly after its maturity, it involutes progressively and eventually disappears a t stage 50, about 12 days after its appearance. Regression of the gland proceeds at a precise rate and is independent of any hormonal influence (Weets and Picard, '79).Lyerla and collaborators demonstrated that the involution of the gland in Xenopus laeuis is characterized by an increased activity of lysosomal enzymes (Hsu and Lyerla, '77; Ling and Lyerla, '76). The gland in advanced regression (stage 45) had total and specific activities of acid phosphatase, P-glucuronidase, and N-acetyl-(3-D-glucosaminidase significantly greater than those observed in stage 35/36 glands. In contrast, the activity of the (3-galactosidase remained stable. The lysosomal location of these enzymes is well established in mammals. It has also been demonstrated in homogenates of total embryos and of stomachs of adult Xenopus that these enzymes have a maximal activity at an acid pH and are located in the cellular fraction sedimenting at 20,OOOg (Hsu and Lyerla, '77).The aim of the studies described in the present article was to extend our knowledge of the behaviour of different enzymes at several stages in the evolution of the gland. Cystein-proteinases and two enzymes belonging to the group of glucosidases, a-L-fucosidase and N-acetyl-(3-D-glucosaminidase, have been assayed. Develo...

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Fluorimetric Assay of Enzymes

Cited by 31 publications

References 340 publications

A <i>Brevibacillus choshinensis</i> System That Secretes Cytoplasmic Proteins

A <i>Brevibacillus choshinensis</i> System That Secretes Cytoplasmic Proteins

Determination of Glutamic and Aspartic Acids and Their Amides

Enzymatic activities during evolution of the cement gland in Xenopus tadpoles

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