Determination of α2-macroglobulin as trypsin-protein esterase

Ganrot, P. O.

doi:10.1016/0009-8981(66)90037-4

Cited by 362 publications

(100 citation statements)

References 13 publications

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“…functional approach to the determination of cti-proteinase inhibitor and o^-macroglobulin which is suitable for routine clinical chemical analyses (12)(13)(14)(15). The analytic criteria of the amidolytic assay are comparable to those of methods measuring the antigenic levels of the proteinase inhibitors.…”

Section: Discussionmentioning

confidence: 99%

“…Both proteins wcre determined by a chromogenic subslrate assay (Boehringer Mannheim), the principles of which have bccn described in detail previously (12)(13)(14)(15)18).…”

Section: Funclional Delermination Of U ι-Proteinase Inhibitor Andmentioning

confidence: 99%

“…More recently, chromogenic Substrates have been introduced which offer the possibility of determining the functional, i.e. the proteinase inhibitor activity, of the two proteins (12)(13)(14)(15). Although the amidolytic assay with the chromogenic Substrates has been evaluated and compared with immunological methods in sera of healthy persons (13^15), pathologic specimens have not been studied extensively so far.…”

Section: Introductionmentioning

confidence: 99%

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Amidolytic and Immuno-Nephelometric Determination of α1-Proteinase Inhibitor and α2-Macroglobulin in Serum with Calculation of Specific Inhibitor Activities in Health and Disease

Gressner¹,

Peltzer²

1984

Clinical Chemistry and Laboratory Medicine

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Summary:In sera of healthy persons (n = 50) and patients with a variety of diseases (n = 197) the two major proteinase inhibitors, αι-proteinase inhibitor (oti-antitrypsin) and ai-macroglobulin, were measured by two methods: a chromogenic (amidolytic) Substrate assay to assess the functional activities, and a laser nephelometric method to determine the immunoreactive concentrations of the respective proteins. The specific proteinase inhibitor activities defined s the number of inhibitor units per g inhibitor protein were calculated.The precision and accuracy of both assays were found to be similar, showing a satisfactory correlation of results for the sera of healthy persons (r ?= 0.916 for cta-macroglobulin and 0.988 for cti-proteinase inhibitor). In diseased individuals the correlation was lower than in normal persons (0.862 for a2-macroglobulin and 0.907 for αϊ-proteinase inhibitor). A poor correlation was obtained in patients with liver diseases (r = 0.586 for αι-proteinase inhibitor and 0.852 for ai-macroglobulin).Reference ranges were established for functional and immunological concentrations and for specific inhibitor activities, respectively. Normal values followed a Gaussian distribution.In patients with various diseases including those with acute phase response, the specific inhibitor activities of ctrproteinase inhibitor re reduced significantly; this is because inhibitor activity shows a smaller relative increase than immunoreactivity. Among the various diseases, no significant differences were noted. The specific inhibitor activity of az-macroglobulin changed significantly only in patients with carcinoma, liver diseases and trauma. Follow up of some patients shows also intraindividual Variation of specific proteinase inhibitor activities. Amidolytische und immun-nephelometrische Bestimmung von a/-Proteinase-Inhibitor und

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Section: Discussionmentioning

confidence: 99%

“…Both proteins wcre determined by a chromogenic subslrate assay (Boehringer Mannheim), the principles of which have bccn described in detail previously (12)(13)(14)(15)18).…”

Section: Funclional Delermination Of U ι-Proteinase Inhibitor Andmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Amidolytic and Immuno-Nephelometric Determination of α1-Proteinase Inhibitor and α2-Macroglobulin in Serum with Calculation of Specific Inhibitor Activities in Health and Disease

Gressner¹,

Peltzer²

1984

Clinical Chemistry and Laboratory Medicine

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“…After 2 minutes, any uncomplexed trypsin was inhibited by addition of 30 pg of soybean trypsin inhibitor (0.5 mg/ml in H2O; Sigma), which does not inhibit azM-complexed trypsin molecules (14). Complex formation was confirmed by SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

Plasma from rheumatoid arthritis patients does not contain abnormally high levels of α₂‐macroglobulin–proteinase complexes

Davies

Coughlan

Barrett

1987

Arthritis & Rheumatism

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We measured the levels of az-macroglobulin (azM)-proteinase complexes in the plasma of 18 patients with classic rheumatoid arthritis, 11 age-matched patients with noninflammatory back pain and osteoarthritis, and 8 healthy volunteers. In contrast with previous reports, we found no evidence of a2M-proteinase complexes in the plasma samples from individuals in any of the groups. In our assays, all activity that might have been the result of the presence of such complexes in the plasma samples proved instead to be an artifact attributable to contamination of the anti-a2M antibody immobilized on the AffiGel solid phase with a trypsin-like proteinase. When the contaminating activity was eliminated by pretreatment of the antibody with 1 mM diisopropyl fluorophosphate, no degradation of substrate was detected with any of the plasma samples. However, the ability of the solid-phase assay to detect and quantitate a2M-proteinase complexes when they are present was confirmed in control experiments with plasma samples to which preformed a2M-trypsin complexes had been added, or in which a2M-kallikrein complexes had been generated by activation of Hageman factor (coagulation factor XII). We therefore conclude that neither normal plasma nor that from rheumatoid arthritis patients contains measurable amounts of aZMproteinase complexes.It has been reported by Teodorescu and coworkers (1-6) that patients with classic seropositive rheumatoid arthritis (RA) consistently have abnormally high plasma concentrations of complexes of a2-macroglobulin ( a2M) with a trypsin-like proteinase. Such concentrations were not found in healthy individuals or patients with nonrheumatoid inflammatory arthritides. If confirmed, this observation could form the basis of a valuable laboratory test to distinguish patients with RA from those with other inflammatory arthritides. The report was surprising, however, in that complexes between proteinases and azM are known to be cleared very rapidly from the circulation, and so have not previously been found to accumulate to appreciable concentrations in the plasma (7,8).Teodorescu et a1 (6) used a modification of the solid-phase immunosorbent assay which was initially developed by Harpel and Hayes (9) for measuring a2M-proteinase complexes. In this assay, AffiGel agarose beads coated with monospecific antibodies against a2M are used to adsorb the complexes from the plasma. The enzymic activity of the adsorbed complexes is then measured as the hydrolysis of the low M , synthetic substrate, benzyloxycarbonyl-lvalylglycyl-L-arginine p-nitroanilide (Z-Val-Gly-ArgNEIPhNOZ), also known as Chromozym-Try . The activity detected in the arthritis plasma samples (5,6) was not fully characterized and was described as trypsinlike because of its action on Chromozym-Try, abroadspectrum trypsin substrate.In the present study, we attempted to repro-

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“…The a 2 M fraction was determined quantitatively by trypsin-protein esterase (TPE) activity, according to the method of Ganrot. 16 To 50 fi\ of each fractionated sample was added 0.9 ml of 0.1 M Tris HC1 buffer, pH 8.2, containing 0.02 M CaCl 2 and 100 /il of trypsin solution (1 mg/ml). After a thorough mixing, 100 /xg of soybean trypsin inhibitor solution (1 mg/ml), 0.85 ml of the Tris buffer, and 2.0 ml of benzoyl-arginine /?-nitroanilide solution were added.…”

Section: Purification Of A-macroglobuluimentioning

confidence: 99%

Formation of angiotensin II by tonin-inhibitor complex.

et al. 1988

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SUMMARY Enzymatic activity of tonin-o^-macroglobulin complex was studied in vitro and in vivo, using an immunoimmobilization technique. Tonin-ai-macroglobulin complex, which was immunologically immobilized by anti-a r macroglobulin antibody covalently coupled to agarose gels, could quantitatively hydrolyze angiotensin I and synthetic tridecapeptide renin substrate to form angiotensin II. However, the solid-phase antibody-bound tonin-a,-macroglobulin complex could not hydrolyze the plasma protein renin substrate. Phenylmethylsulfonyl fluoride, a serine protease inhibitor, inhibited both free tonin and the solid-phase antibody-bound tonin-a|-macroglobulin complex. The hydrolytic activity of the solid-phase antibody-bound tonin-«,-macroglobulin complex against angiotensin I was not inhibited by soybean trypsin inhibitor (molecular weight, 23,000), a potent inhibitor of free tonin. Taken together, these results suggest that tonin bound to c^-macroglobulin keeps the active site intact and that inhibition of the enzyme activity is due to a steric hindrance. When 500 /xl of tonin was administered intravenously to rats, the immunoimmobilization method was used to show that the tonin-a r -macroglobulin complex in the plasma formed angiotensin II. Thus, the tonin-a r macroglobulin complex in the plasma may be linked to some forms of hypertension through angiotensin II formation. (Hypertension 11: 63-70, 1988) KEY WORDS • tonin immunoimmobilization angiotensin I • angiotensin II • a r -macroglobulin

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Determination of α2-macroglobulin as trypsin-protein esterase

Cited by 362 publications

References 13 publications

Amidolytic and Immuno-Nephelometric Determination of α1-Proteinase Inhibitor and α2-Macroglobulin in Serum with Calculation of Specific Inhibitor Activities in Health and Disease

Amidolytic and Immuno-Nephelometric Determination of α1-Proteinase Inhibitor and α2-Macroglobulin in Serum with Calculation of Specific Inhibitor Activities in Health and Disease

Plasma from rheumatoid arthritis patients does not contain abnormally high levels of α₂‐macroglobulin–proteinase complexes

Formation of angiotensin II by tonin-inhibitor complex.

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Determination of α2-macroglobulin as trypsin-protein esterase

Cited by 362 publications

References 13 publications

Amidolytic and Immuno-Nephelometric Determination of α1-Proteinase Inhibitor and α2-Macroglobulin in Serum with Calculation of Specific Inhibitor Activities in Health and Disease

Amidolytic and Immuno-Nephelometric Determination of α1-Proteinase Inhibitor and α2-Macroglobulin in Serum with Calculation of Specific Inhibitor Activities in Health and Disease

Plasma from rheumatoid arthritis patients does not contain abnormally high levels of α2‐macroglobulin–proteinase complexes

Formation of angiotensin II by tonin-inhibitor complex.

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Plasma from rheumatoid arthritis patients does not contain abnormally high levels of α₂‐macroglobulin–proteinase complexes