Use of physiological substrates for zymograms of disaccharidases after separation in immobilized pH gradients

Sinha, Pranav; Seidel, Mike; Righetti, Pier Giorgio; Bause-Niedrig, Ingrid; Köttgen, E.

doi:10.1016/0165-022x(89)90004-3

Cited by 3 publications

(4 citation statements)

References 18 publications

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“…2-5), referring to values at the pH optimum, described by the suppliers. This staining procedure is obviously highly sensitive when compared with 5-1 0 mU of alkaline phosphatase [15] and 5-50 mU of disaccharidases by the glucose dehydrogenase reaction [4].…”

Section: Discussionmentioning

confidence: 99%

“…Visualization using hexokinase (HK)/glucose-6-phosphate dehydrogenase (G6PDH) as intermediary enzymes, instead of glucose dehydrogenase, was not successful due to the additional reaction of the intermediary enzymes and also because Correspondence: Dr. Hidehiko Mukasa. Department of Chemistry, National Defense Medical College, 2-Namiki 3-Chome, Tokorozawa, Saitama, 359 Japan Abbreviations: F6P, fructose-6-phosphate; BFase, B-fructofuranosidase; FTase, fructosyltransferase; GlP, glucose-1-phosphate; G6P, glucose-6phosphate; G6PDH, glucose-6-phosphate dehydrogenase; aG, p-nitrophenyl-a-D-glucoside; f3G, p-nitrophenyl-f3-o-glucoside; aGase, a-D-glucosidase; PGase, P-D-glucosidase; HK, hexokinase; IEF, isoelectric focusing; aM, p-nitrophenyl-a-D-mannoside; M6P, mannose-6phosphate; BM, p-nitrophenyl-P-D-mannoside; aMase, a-D-mannosidase; BMase, B-D-mannosidase; NBT, Nitroblue Tetrazolium; PGI, phosphoglucose isomerase; PGL, phosphogluconolactone; PGM, phosphoglucomutase; PI, isoelectric point; PMI, phosphomannose isomerase; PMS, phenazine methosulfate; PMSH2, reduced form of phenazine methosulfate; SPase, sucrose phosphorylase of the high solubility in water of the two intermediates, namely glucose and glucose-6-phosphate (G6P) [4].…”

Section: Introductionmentioning

confidence: 99%

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Glucose, fructose, mannose and/or glucose‐1‐phosphatereleasing activity stains for glycosidases and glycosyltransferases in gels after isoelectric focusing

1994

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beta-Fructofuranosidase, alpha-glucosidase, beta-glucosidase, alpha-mannosidase, beta-mannosidase, sucrose phosphorylase, glucosyltransferase and fructosyltransferase were separated by isoelectric focusing and sensitively detected to be slightly diffuse and insoluble spots in thin-layer gels, supported by a glass plate, by release of monosugars or a sugar phosphate, followed by conversion to glucose-6-phosphate (G6P) and then by reduction of NADP+ to NADPH, terminated by the formation of reduced Nitroblue Tetrazolium (NBT). Approximately 1-10 mU of enzyme was focused and the gel, after washing with a buffer, was partially dried and directly stained by uniformly spreading on the gel surface a staining medium containing sucrose or nitrophenyl glycosides as substrates, intermediary enzymes such as hexokinase, mutase and/or isomerase, NADP+, ATP, Mg+, phenazine methosulfate (PMS) and NBT. Specific staining procedures for each of these activities, on sucrose or on the glycosides as substrates, and staining procedures for multiple activities are described, with the conditions necessary for optimal development.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Glucose, fructose, mannose and/or glucose‐1‐phosphatereleasing activity stains for glycosidases and glycosyltransferases in gels after isoelectric focusing

1994

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“…Colorimetric (20). In this reaction, shown in Reaction 1, tetrazolium is reduced to a strongly colored formazan dye ( max Ϸ 500 nm):…”

Section: Resultsmentioning

confidence: 99%

Enzyme-Based Sensor Arrays for Rapid Characterization of Complex Disaccharide Solutions

Curey

Salazar

Oliveira

et al. 2002

Analytical Biochemistry

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“…This promising approach does not allow digest conditions to be optimized independently of spatial (pI) resolution, however, because the small cleavage products diffuse rapidly. [30] To limit diffusion, we propose scoring focused, unfixed, unstained IPG gels with a sharp template, knife, or razor blade, creating ca. 2-mm gel sections, yet leaving the polyester backing to which the slices are bound, intact.…”

Section: Bottom-up Proteomicsmentioning

confidence: 99%

Top-down, bottom-up, and side-to-side proteomics with virtual 2-D gels

Loo

Hayes

Yang

et al. 2005

International Journal of Mass Spectrometry

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Intact protein masses can be measured directly from immobilized pH gradient (IPG) isoelectric focusing (IEF) gels loaded with mammalian and prokaryotic samples, as demonstrated here with murine macrophage and Methanosarcina acetivorans cell lysates. Mass accuracy and resolution is improved by employing instruments which decouple the desorption event from mass measurement; e.g., quadrupole time-of-flight instruments. MALDI in-source dissociation (ISD) is discussed as a means to pursue top-down sequencing for protein identification. Methods have been developed to enzymatically digest all proteins in an IEF gel simultaneously, leaving the polyacrylamide gel attached to its polyester support. By retaining all gel pieces and their placement relative to one another, sample handling and tracking are minimized, and comparison to 2-D gel images is facilitated. MALDI-MS and MS/MS can then be performed directly from dried, matrix-treated IPG strips following whole gel trypsin digestion, bottom-up methodology.Side-to-side proteomics, highlighting the link between virtual and classical 2-D gel electrophoresis, is introduced to describe a method whereby intact masses are measured from one side (the IEF gel), while proteins are identified based on analyses performed from the other side (the SDS-PAGE gel). Keywords: archaea, proteomics, virtual 2-D gel electrophoresis, immobilized pH gradient gels, Methanosarcina acetivoransAbbreviations: 2-D PAGE, two-dimensional polyacrylamide gel electrophoresis; CHAPS, 3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate; DTT, dithiothreitol; HDL, high density lipoprotein; ICAT, Isotope coded affinity tagging; IEF, isoelectric focusing; IPG, immobilized pH gradient; ISD, in-source dissociation; MudPIT, multidimensional protein identification technology; PSD, post-source decay; QqTOF, quadrupole time-of-flight; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid; TOF, time-of-flight 3 IntroductionIntact mass measurements, in concert with protein identifications, can alert one to 1) coor post-translationally modified proteins, 2) alternative splice variants, 3) RNA editing events, or Without the link to protein identity, an intact mass has limited value. Hence, tying the intact masses recovered from virtual 2-D gels to unambiguous protein identities and further to descriptors is important. We consider 3 ways to forge this link; two are well-described in popular parlance as: "top-down" and "bottom-up" [9,10]. We call the third, "side-to-side", because it highlights the link to classical 2-D gel electrophoresis: namely that isoelectric focusing constitutes one side/dimension/axis/gel in classical 2-D analysis, while SDS-PAGE constitutes the second side/dimension/axis/gel. In side-to-side analysis, one measures the intact mass from one side (the IEF gel), while securing the protein identification from an analysis performed on the other side (the SDS-PAGE gel).Top-down proteomics strategies provide both accurate intact mass and sequence da...

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Use of physiological substrates for zymograms of disaccharidases after separation in immobilized pH gradients

Cited by 3 publications

References 18 publications

Glucose, fructose, mannose and/or glucose‐1‐phosphatereleasing activity stains for glycosidases and glycosyltransferases in gels after isoelectric focusing

Glucose, fructose, mannose and/or glucose‐1‐phosphatereleasing activity stains for glycosidases and glycosyltransferases in gels after isoelectric focusing

Enzyme-Based Sensor Arrays for Rapid Characterization of Complex Disaccharide Solutions

Top-down, bottom-up, and side-to-side proteomics with virtual 2-D gels

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