α-Glucosidase and N-acetylglucosamine-6-sulphatase are the major mannose-6-phosphate glycoproteins in human urine

Most newly synthesized lysosomal enzymes contain a transient carbohydrate modification, mannose 6-phosphate (Man-6-P), which signals their vesicular transport from the Golgi to the lysosome via Man-6-P receptors (MPRs). We have examined Man-6-P glycoproteins in human urine by using a purified soluble fragment of the soluble cation-independent MPR (sCI-MPR) as a preparative and analytical affinity reagent. In a survey of urine samples from seven healthy subjects, the pattern of Man-6-P glycoproteins detected with iodinated sCI-MPR as a probe in a blotting assay was essentially identical in each, regardless of sex or age. Two bands of approx. 100 and 110 kDa were particularly prominent. Man-6-P glycoproteins in human urine were purified by affinity chromatography on immobilized sCI-MPR. Seven distinct bands revealed by SDS/PAGE and Coomassie Blue staining were subjected to N-terminal sequence analysis. The prominent 100 and 110 kDa Man-6-P glycoproteins were identified as N-acetylglucosamine-6-sulphatase and α-glucosidase respectively. This identification was confirmed by molecular mass determinations on the two major bands after deglycosylation. Sequence analysis revealed arylsulphatase A and several previously unidentified proteins as minor species. Man-6-P glycoproteins were also purified on an analytical scale to determine the proportion of a number of lysosomal enzyme activities represented by the mannose-6-phosphorylated forms. The lysosomal enzymes in urine containing the highest proportion of mannose-6-phosphorylated form were β-mannosidase (82%), hexosaminidase (27%) and α-glucosidase (24%). The profiles of Man-6-P glycoproteins detected by blotting in urine and plasma were not similar, suggesting that the urinary species are not derived from the bloodstream.

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The Reduced Aggregation Response Of Bernard-Soulier Platelets To Thrombin May Be Related To An Abnormal Glycoprotein V

10.1055/s-0038-1652009 ◽

1981 ◽

Author(s):

A T Nurden ◽

D Dupuis

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Human Platelets ◽

Surface Proteins ◽

Galactose Oxidase ◽

Blue Staining ◽

Lag Phase ◽

Coomassie Blue Staining ◽

Aggregation Response ◽

Coomassie Blue ◽

Sds Page

Both platelet membrane GP Ib and GP V have been proposed as receptors for the activation of human platelets by thrombin. Bernard-Soulier (B-S) platelets exhibit a reduced aggregation response to thrombin with a lag phase that precedes aggregation. When B-S platelets, whose surface proteins had been labelled with (125I), were analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by PAS-staining, Coomassie blue staining or autoradiography, the apparent absence of GP Ib and the normal presence of GP IIb, IIIa and IIIb was demonstrated. On the basis of such studies several authors have stated that “GP I” is the thrombin receptor. However, GP V is not located by the above procedure, requiring more sensitive analytical methods for its detection. To meet this requirement washed platelets isolated from 3 B-S patients have been treated sequentially with neuraminidase, galactose oxidase and sodium(3H,)-boro- hydride. The labelled platelets were analysed by SDS-PAGE using 7-12% gradient acrylamide gels and the (3H,)-labelled GP’s located by fluorography. In addition to the GP Ib defect the platelets of each B-S patient were lacking the band corresponding to GP V of normal platelets. In agreement with previous studies we observed that when (3H,)-labelled normal human platelets were incubated with thrombin GP V (Mr=82,000) was hydrolysed,and that this was accompanied by the appearance of a labelled glycopeptide (Mr=69,500) in the supernatant. When (3H)-labelled B-S platelets were treated with thrombin no labelled glycopeptide was located. GP V could therefore be either absetit from B-S platelets or have a modified carbohydrate composition rendering it insensitive to the analytical procedure used. Interpretations into the reduced aggregation response of B-S platelets to thrombin should be extended to include a possible GP V defect.

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Accelerated Coomassie Blue Staining and Destaining of SDS-PAGE Gels with Application of Heat

Methods in Molecular Biology - Protein Electrophoresis ◽

10.1007/978-1-61779-821-4_41 ◽

2012 ◽

pp. 471-479 ◽

Cited By ~ 6

Author(s):

Biji T. Kurien ◽

R. Hal Scofield

Keyword(s):

Blue Staining ◽

Coomassie Blue Staining ◽

Coomassie Blue ◽

Sds Page

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Studies on the Biochemistry of Urokinase

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651899 ◽

1977 ◽

Vol 38 (04) ◽

pp. 0801-0808 ◽

Cited By ~ 12

Author(s):

Eng Bee Ong ◽

Mercedes E. Soberano ◽

Alan J. Johnson ◽

Guenther Schoellmann

Keyword(s):

Affinity Chromatography ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Gel Filtration ◽

Blue Staining ◽

Single Chain ◽

Coomassie Blue Staining ◽

Coomassie Blue ◽

Sds Page ◽

Excess Substrate

SummaryDirect evidence for an active center histidine residue in urokinase (UK) was obtained with use of newly synthesized peptide chloroketones Ac-Gly-Lys-CH2C1 and Nle-Gly-Lys-CH2C1. Stoichiometric inactivation by DFP provided further evidence that UK is a serine protease. Essential histidine and serine residues were both located in the heavy chain of the 47,0 M. W.UK. The high M.W. form can be converted (catalytically) to the low M. W. form.9 partially purified human urinary UK preparations (5 with predominantly high M. W. UK), varying in purity and proportion of high and low M. W. forms, were found to be heterogeneous by a number of acrylamide electrophoretic procedures. 7 preparations had strikingly similar molar activities at excess substrate, except for the lower values found in 2 predominantly high M. W. UK preparations from the same supplier. 2 high M. W. UK preparations from another supplier showed a definite increase in activity when assayed at low plasminogen concentration, but this effect was abolished after gel filtration (Sephadex G-25), by further purification with affinity chromatography, or when assayed with excess plasminogen.The high and low M. W. forms of UK (47,000 and 33,400 M. W.), isolated and purified by Sepharose-EACA-agmatine affinity chromatography were shown to be homogeneous by Coomassie Blue staining after SDS-polyacrylamide gel electrophoresis (PAGE) and by 14C-DFP and 14C-NPGB incorporation before SDS-PAGE. Comparative properties of the high M. W. vs low M. W. forms were as follows: specific activity (104,000 IU/mg vs 226,000) ; 2 chains (33,100 and 18,600 M.W.) linked by disulfide(s) vs a single chain; pi 8.60 (major subform) and pi 8.90 (minor subform) vs pi 8.35, 8.60, 8.70 (major subform) and pi 8.05 (minor subform); and second order kinetics for DFP inactivation (400 vs 770 M−1 min−1). The molar activities were similar (9.6 × 109 and 10.2 × 109IUm/mole) for each form.

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Identification of timothy cultivars by SDS-PAGE analysis of seed storage proteins

Canadian Journal of Plant Science ◽

10.4141/cjps92-148 ◽

1992 ◽

Vol 72 (4) ◽

pp. 1215-1222 ◽

Cited By ~ 1

Author(s):

Q. Cai ◽

M. R. Bullen

Keyword(s):

Storage Proteins ◽

Seed Proteins ◽

Seed Storage ◽

Seed Storage Proteins ◽

Phleum Pratense ◽

Environmental Stability ◽

Blue Staining ◽

Coomassie Blue Staining ◽

Coomassie Blue ◽

Sds Page

SDS-PAGE analysis of seed proteins was carried out to identify the cultivars in the forage crop, timothy (Phleum pratense L.). Nineteen cultivars of timothy were examined. Among them five were from Europe and fourteen from North America. In total fifty protein bands were detected in mature seed extract by SDS-PAGE followed by Coomassie blue staining. Except for two pairs, all the cultivars were differentiated by SDS-PAGE analysis of seed storage proteins. In the electrophoretic profile, no protein bands were found to be specific either to European or to North American cultivars which is an indication of their genetic similarity. Twelve samples of cultivar Toro harvested from Alberta and Manitoba (Canada), Idaho and Minnesota (USA) were compared and no significant differences were found in their seed protein banding patterns, which suggests environmental stability of timothy seed proteins.Key words: SDS PAGE, timothy cultivar identification, seed storage proteins

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Purification and characterization of human brain prolyl endopeptidase

Biochemical Journal ◽

10.1042/bj2760237 ◽

1991 ◽

Vol 276 (1) ◽

pp. 237-244 ◽

Cited By ~ 44

Author(s):

S Kalwant ◽

A G Porter

Keyword(s):

Human Brain ◽

Proteinase Inhibitors ◽

Polyclonal Antibodies ◽

Deae Cellulose ◽

Serine Proteinase ◽

Prolyl Endopeptidase ◽

Blue Staining ◽

Coomassie Blue Staining ◽

Coomassie Blue ◽

Sds Page

Prolyl endopeptidase (EC 3.4.21.26) was purified from human brain by a series of column-chromatographic steps using DEAE-cellulose DE-52, hydroxyapatite, phenyl-Sepharose, Sephacryl S-200 and f.p.l.c. (Mono Q). The enzyme was purified by a factor of 943 and was homogeneous in a SDS/polyacrylamide gel as judged by Coomassie Blue staining. The Mr estimated by SDS/PAGE is 79,600, and under native conditions on Sephacryl S-200 it is 85,600. Therefore the enzyme exists as a monomer. With benzyloxycarbonylglycylproline p-nitroanilide as substrate, the optimum pH of the enzyme is 6.8, and with the substrate concentration between 0.059 mM and 0.37 mM the Km is 9.0 x 10(-4) M. The pI of the enzyme is 4.75. The enzyme is classified as a serine proteinase, as it is strongly inhibited by di-isopropyl fluorophosphate. However, other serine proteinase inhibitors do not inhibit the enzyme significantly, suggesting that the active site of prolyl endopeptidase differs from that of classical serine proteinases such as trypsin. Polyclonal antibodies were raised against purified human brain prolyl endopeptidase in rabbits. Western-blot analysis, enzyme-inhibition assays, antibody binding and immunoprecipitation experiments indicated that the polyclonal antibodies are both specific and inhibitory to the enzyme activity.

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Analysis of Bufo arenarum oviductal secretion during the sexual cycle

Zygote ◽

10.1017/s0967199409005462 ◽

2009 ◽

Vol 17 (4) ◽

pp. 329-340 ◽

Cited By ~ 1

Author(s):

Claudia A. Crespo ◽

Inés Ramos ◽

Marcela F. Medina ◽

Silvia N. Fernández

Keyword(s):

Electrophoretic Pattern ◽

Sexual Cycle ◽

Blue Staining ◽

Coomassie Blue Staining ◽

Coomassie Blue ◽

Sds Page ◽

Bufo Arenarum ◽

Ovulatory Period

SummaryBufo arenarum oocytes are oviposited surrounded by jelly coats, one component of the extracellular matrix required for fertilization. The secretion, released to the oviductal lumen, was analysed by SDS-PAGE. The coomassie blue staining evidenced an electrophoretic pattern with molecules ranging between 300 and 19 kDa that showed variations in their secretion profiles during the sexual cycle. In the preovulatory period the densitometric analysis showed the presence of nine peaks with marked predominance of the 74 kDa molecule. Once ovulation has occurred, the jelly coats become arranged around the oocytes during their transit throughout the oviductal pars convoluta (PC), revealing the addition of three proteins only observed during this period, which suggests a differential secretion. Some of these proteins could not diffuse under any extraction treatment, indicating for them a structural or in situ function. Proteins of low molecular mass diffused totally while others showed a partial diffusing capacity. After ovulation a marked decrease in the relative amount of all the proteins released to the lumen, especially the 74 kDa protein, could be detected. During this period, unlike the other stages of the sexual cycle, a differential secretion pattern was observed along the PC. The histochemical analysis performed during the ovulatory period showed the presence of glycoconjugates including both acidic and neutral groups. The present results are in agreement with previous ultrastructural and histochemical studies that describe the role of Bufo arenarum jelly coats in fertilization.

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Profil Protein Trypanosoma evansi dari Daerah Geografis Berbeda di Indonesia Tahun 2012-2014 dengan Sodium Dodecil Sulphate Polyacrylamide Gel Electrophoresis (TRYPANOSOMA EVANSI PROTEIN PROFILE OF DIFFERENT GEOGRAPHICAL AREAS ORIGIN IN INDONESIA

jurnal veteriner ◽

10.19087/jveteriner.2017.18.4.526 ◽

2018 ◽

Vol 18 (4) ◽

pp. 526

Author(s):

Fitrine Ekawasti ◽

Ichwan Yuniarto ◽

Sulinawati Sulinawati ◽

Didik Tulus Subekti

Keyword(s):

Soluble Protein ◽

Serological Test ◽

Protein Profile ◽

Polyacrylamide Gel Electrophoresis ◽

Trypanosoma Evansi ◽

Blue Staining ◽

Protein Profiles ◽

Coomassie Blue Staining ◽

Coomassie Blue ◽

Sds Page

Surra outbreak in 2012 has led to more than 1,700 animals have died in the province of East Nusa Tenggara (NTT) Indonesia. Surra case sporadically continues throughout the year in various areas, especially Kalimantan, Banten as well as other areas. Some reports reveal differences in protein profiles among multiple isolates of T. evansi. Therefore the purpose of this research were to find out the protein profile of each isolate T. evansi in Indonesia and the possible biological differences among them. Eleven isolates originating from the province of East Nusa Tenggara, South Kalimantan and Central Kalimantan, Banten, Lampung and Bengkulu has been isolated and purified Using DEAE. Trypanosoma isolate were frezeethawing repeatedly to obtain soluble protein. Furthermore, soluble protein is treated with heating or without heating and then each was run on SDS PAGE with Coomassie Blue staining. The protein profiles of all isolates were compared each other. The results showed that eleven isolates of T. evansi in Indonesia has a very diverse protein profile. Then for the purposes of development of diagnostic kit can be used whole lysate cell (WCL) as stock antigen in serological test process.

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