Isolation of Acid-Resistant Urinary Trypsin Inhibitors by High Performance Liquid Chromatography and Their Characterization by N-Terminal Amino-Acid Sequence Determination

Hochstrasser, K; Reisinger, P. W. M.; Albrecht, Gerd J.; Wächter, Elmar; Schönberger, Öyvind L.

doi:10.1515/bchm2.1984.365.2.1123

Cited by 23 publications

(15 citation statements)

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“…Hochstrasser et al have recently reported the partial loss of carbohydrate by purifying the urinary inhibitor HI-30 by HPLC, while using a solvent containing trifluoroacetic acid [4]. We have demonstrated that perchloric acid treatment did not dissociate uronic acid and antiprotease activity and did not modify either the mobility of UTI I in SDS-PAGE.…”

Section: Discussionmentioning

confidence: 55%

“…The amount of glycosaminoglycan removed by chondroitinase accounts for approximately 20% of the molecule, i.e. 6000 Da and the true M , of UTI I (protein + glycosaminoglycan) should therefore be approximately 32000, as described by Hochstrasser et al [4,11]. The overestimation to 44000 in SDS-PAGE may be an artefact, probably due to the presence of the glycosaminoglycan chain.…”

Section: Discussionmentioning

confidence: 93%

“…This inhibitor has an apparent molecular mass of 30 kDa, as estimated by gel chromatography [6], but 40 kDa in SDS-PAGE [4].…”

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confidence: 99%

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The major human urinary trypsin inhibitor is a proteoglycan

et al. 1986

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The major urinary trypsin inhibitor (MI 44000), isolated from human urine, contains 35% carbohydrate. In addition to N-acetylglucosamine and neutral sugars (primarily mannose and galactose), the carbohydrate moiety contains hexuronic acid and N-acetylgalactosamine and corresponds to a glycosaminoglycan. This carbohydrate chain is an integral component of the inhibitor: it does not dissociate from the inhibitor when using dissociative conditions such as sodium dodecyl sulfate, guanidinium chloride, or by increasing ionic strength or mixing with cetylpyridinium chloride.This glycosaminoglycan chain is sensitive to chondroitinase ABC or testicular hyaluronidase digestion and corresponds to slightly sulfated chondroitin 4-sulfate or 6-sulfate. After treatment by these enzymes, the urinary inhibitor has a lower molecular mass (MI 26000) but still inhibits trypsin.Several acid-resistant trypsin inhibitors with molecular masses, estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE), ranging from 40 kDa -45 kDa, have been isolated from human urine [l -41. All these inhibitors show an immunological cross-reactivity with intera-trypsin inhibitor.Hochstrasser et al.[5] first described a physiological urinary inhibitor having an apparent molecular mass of 34 kDa as calculated from its specific activity. This inhibitor (HI-30) has an apparent molecular mass of 30 kDa, as estimated by gel chromatography [6], but 40 kDa in SDS-PAGE [4].We previously described the isolation and characterization of a human urinary trypsin inhibitor (UTI) with the same aminoterminal sequence as 81. This sequence was recently found to be identical with that of inter-a-trypsin inhibitor [9, 101; those data tend to confirm the hypothesis that the different urinary trypsin inhibitors isolated so far are derived from inter-a-trypsin inhibitor.The proportion and nature of the carbohydrate reported for those inhibitors is variable, ranging from 10% to 50% [l,3, 71. According to Hochstrasser et al. [ll] the carbohydrate content of HI-30 accounts for 50% of the total weight and includes two carbohydrate chains: one is attached N-glycosidically via an asparaginyl residue, the other is 0-glycosidically linked to the polypeptide via a seryl residue in position 10. This last chain is made up of at least 48 Nacetylgalactosamine, 2 glucose, 18 galactose and 2 N-acetylneuraminic acid residues [6]. Such a composition is quite unusual for a glycoprotein.In this report we describe a slightly modified procedure for isolation of urinary trypsin inhibitor and we present evidence that it consists a proteoglycan. MATERIALS AND METHODSSephacryl S-200, DEAE-Sephacel and ConA-Sepharose were purchased from Pharmacia, DEAE-Trisacryl was from IBF. Acrylamide and N-N'-methylenebisacrylamide were from BDH, agarose (Indubiose A 37) from IBF. Bovine pancreatic trypsin (treated with Tos-Phe-CH2C1) and L-benzoylarginine p-nitroanilide were purchased from Merck, chondroitinase ABC (from Proteus vulgaris) and bovine testicular hyaluronidase were from ...

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Section: Discussionmentioning

confidence: 55%

Section: Discussionmentioning

confidence: 93%

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The major human urinary trypsin inhibitor is a proteoglycan

et al. 1986

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“…The Ͼ50-kDa bands we detected here most likely correspond to heavy chain components, P␣I or ITI. The 21-kDa BK we found corresponds to either the ϳ25-kDa BK form identified by Suzuki et al (43) as deglycosylated BK or a 20-kDa fragment of BK, also known variously as urinary trypsin inhibitor 2 (UTI-2) (15,22) or uristatin (37). LMM bands (Ͻ20 kDa) are products of proteolytic degradation.…”

Section: Discussionmentioning

confidence: 58%

Urine protein markers distinguish stone-forming from non-stone-forming relatives of calcium stone formers

Bergsland

Kelly²,

Coe³

et al. 2006

American Journal of Physiology-Renal Physiology

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We have investigated urine protein inhibitors of calcium oxalate crystallization to determine whether variations in these proteins are associated with kidney stone disease and whether protein measurements improve the identification of stone formers compared with conventional risk factors (RF). Using Western blotting, we studied variations in the electrophoretic mobility patterns and relative abundances of crystallization-inhibitory proteins in urine from 50 stone-forming (SF) and 50 non-stone-forming (NS) first-degree relatives of calcium SF patients, matched by gender and age. Standard urine chemistry stone risk measurements were also made. Multivariate discriminant analysis was used to test the association of these proteins with nephrolithiasis. Differences in form and abundance of several urine proteins including inter-␣-trypsin inhibitor (ITI), prothrombin fragment 1 (PF1), CD59, and calgranulin B (calB) were found to be associated with stone formation. By multivariate discriminant analysis, measurements of forms of PF1, ITI, and calB in men and ITI and CD59 in women, classified 84% of men and 76% of women correctly by stone status. In contrast, standard urine chemistry RF identified only 70% of men correctly and failed to distinguish female SF from NS. Thus a small subset of protein measurements distinguished SF from NS far better than conventional RF in a population of relatives of calcium SF,

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“…view of the suspicion that NC may also be a relative of ITI: recent findings have suggested its identity to HI-14 [ 113], a fragment of bikunin known to be present in human urine [134].…”

Section: Osteopontin ( Uropontin )mentioning

confidence: 99%

Glycosaminoglycans, proteins, and stone formation: adult themes and child’s play

Ryall

1996

Pediatr Nephrol

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The relative infrequency of renal stones in children is probably the main reason for the paucity of literature devoted to the study of urolithiasis in pediatric patients. Nonetheless, when pediatricians do address the issue, the contents of their papers reflect those prevalent in the adult literature; with one notable exception. Papers dealing with the potential role of urinary macromolecules in pediatric stone disease are very scarce indeed; to my knowledge, only four have been published in the English literature in the last 15 years. One of these is to be found in this issue and, like the remaining three, it compares the urinary excretion of glycosaminoglycans in healthy children and those with stones. This article briefly reviews the history of the association of urinary macromolecules, particularly glycosaminoglycans and proteins, with calcium oxalate urolithiasis, and discusses in more detail the published experimental evidence for their fulfilling a determinant role in stone formation.

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Isolation of Acid-Resistant Urinary Trypsin Inhibitors by High Performance Liquid Chromatography and Their Characterization by N-Terminal Amino-Acid Sequence Determination

Cited by 23 publications

References 10 publications

The major human urinary trypsin inhibitor is a proteoglycan

The major human urinary trypsin inhibitor is a proteoglycan

Urine protein markers distinguish stone-forming from non-stone-forming relatives of calcium stone formers

Glycosaminoglycans, proteins, and stone formation: adult themes and child’s play

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