Simplified techniques for elution of proteins from polyacrylamide gel, staining, destaining, and isoelectric focusing

Bodhe, A.M.; Deshpande, Vasanti; Lakshmikantham, B.C.; Vartak, H.G.

doi:10.1016/0003-2697(82)90633-9

Cited by 13 publications

(2 citation statements)

References 20 publications

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“…198 (LKB Produkter, Stockholm, Sweden), replacing the Ultrodex with 40 strips of polyurethane foam (7 x 0.5 x 0.25 cm; total bed volume approximately 60 ml) (Bodhe et al 1982) placed on gel bond paper (24 x 9 cm). Preparative isoelectric focusing was carried out in an LKB Multiphor apparatus as described in LKB application note no.…”

Section: Methodsmentioning

confidence: 99%

Characterization of a major xylanase purified from Lentinula edodes cultures grown on a commercial solid lignocellulosic substrate

Mishra

Forrester

Kelley

et al. 1990

Appl Microbiol Biotechnol

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The white-rot basidiomycete Lentinula (syn.Lentinus) edodes (Berk.) Pegler is the dominant edible mushroom cultivated on wood. The major xylanase detected in cultures grown on a commercial oak wood medium was extracted, purified, and characterized. The enzyme was a non-debranching endo-fl-D-xylanase (l,4fl-D-xylan xylanohydrolase; E.C.3.2.1.8) highly specific for xylans, with a molecular weight of 41000 (on sodium dodecyl sulfate gels) and an isoelectric point of 3.6. With aspen glucuronoxylan as substrate, the enzyme showed optimal activity at pH 4.5-5.0 and 60 ° C, with a K,~ of 0.66 mg/ml and specific activity of 310 units/rag protein at 40 ° C. It was capable of hydrolyzing (forming reducing sugars from) 40%-50% of the hydrolyzable linkages in either glucuronoxylan or arabinoxylan. The enzyme produced xylose and major identifiable products in the xylobiose or xylotriose (and presumably larger) size range including xylobiose and xylooligosaccharides, but neither glucuronic acid nor arabinose. Products were also produced from arabinoxylan that appeared to be arabinoxylobiose and arabinoxylotriose.

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Section: Methodsmentioning

confidence: 99%

Characterization of a major xylanase purified from Lentinula edodes cultures grown on a commercial solid lignocellulosic substrate

Mishra

Forrester

Kelley

et al. 1990

Appl Microbiol Biotechnol

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“…Electrophoresis was carried out for 28 h at 100 V and 40 mA. After electrophoresis, the two protease bands were located on the gel, which were then cut, and the corresponding enzymes were eluted in KOH/borate, pH 7.5, by the method described by Bodhe et al [13]. The eluates, after dialysis, were concentrated by lyophilisation.…”

Section: Enzyme Purificationmentioning

confidence: 99%

Evidence for controlled autoproteolysis of alkaline protease

Phadatare¹,

Srinivasan²,

Deshpande³

1992

European Journal of Biochemistry

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The alkaline serine protease of Conidiobolus coronatus was shown to be involved in its conidial discharge [Phadatare, S., Srinivasan, M. C., Deshpande, M. (1989) Arch. Microhiul. 153,. To understand the regulation of conidial discharge, the mechanism of control of protease activity was investigated, which revealed the presence of two electrophoretically separable intracellular proteases (protease I and protease 11). The formation of smaller and less-active protease I1 coincided with the decrease in conidial discharge. In ordcr to trace the origin of protease 11, the corresponding purified extracellular enzymes were compared with respect to their biochemical, physicochemical and immunological properties. The biochemical properties, such as optimum pH and temperature, stability, sensitivity to metal ions and substrate specificity were closely similar for both proteases. Amino acid analysis revealed that protease I1 is completely similar to protease I, though protease I contains an additional portion which is not contained in protease 11. Western-blot ELISA, immunotitration and detcrmination of antigenic valencies also revealed the structural similarity between the two proteases. Purified protease I showed partial degradation to protease I1 in vitro, the process being sensitive to phenylmethylsulfonyl fluoride, indicating its proteolytic nature. These results suggest that the formation of a less-active protease by autoproteolysis represents a novel means of physiological regulation of protease activity, which in turn regulates the conidial discharge in C. coronatus.Thc release of conidia, a means of asexual reproduction in fungi, serves the purpose of wide dispersal of the organism. Little investigated fungi, Entomopkthora and Conidioholus, members of the order Entomophthorales, show a peculiar phenomenon of forcibly discharging mature conidia from a conidiophorc. The steps involvcd in this process were described by Sawyer [l], but its biochemical basis was not known.Many physiological functions have been attributed to serine proteases [2. 31. Using three independent approaches, we havc previously shown [4], that the serine protease of Conidiobolus coronutus is responsible for and regulates the triggering of the violent discharge of its conidia. The causal relationship betwecn the protease and the conidial discharge was demonstrated by the following experimental evidence : (a) a ininimum protease level of 1 U . mg-' is an absolute requirement for the initiation of conidial discharge; (b) a mutant identical with the parent strain in nutritional requirements, antibiotic sensitivity, colony morphology including conidia germination, rate of growth and extent of conidia formation, but with reduccd conidial discharge, showed decreased protease activity; (c) inhibition of the protease by phenylmethylsulfonyl fluoride (PheMeS0,F) affected the conidial discharge; (d) induction of the protease by casein resulted in the early onset or conidial discharge. As the extent of conidial discharge depends on the level of protease, a...

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