Fluorometric measurement of alkaline phosphatase and aminopeptidase activities in the order of 10−14 mole

Greenberg, Leonard J.

doi:10.1016/0006-291x(62)90029-3

Cited by 152 publications

(33 citation statements)

References 8 publications

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“…AlaAP and CysAP activities were measured fluorometrically using as substrates the amino acyl-β-naphthylamides (aaNNap) AlaNNap and CysNNap, according to the modified method of Greenberg [22]described elsewhere [23]: 25 µl of plasma was incubated during 30 min at 25°C with 1 ml of the substrate solution: 2.14 mg/100 ml AlaNNap or 5.63 mg/100 ml CysNNap, 10 mg/ 100 ml bovine serum albumin (BSA), and 10 mg/100 ml dithiothreitol (DTT) in 50 m M of phosphate buffer, pH 7.4, for AlaAP and 50 m M HCl-Tris buffer, pH 6, for CysAP.…”

Section: Methodsmentioning

confidence: 99%

Plasma Aminopeptidase Activities in Rats after Left and Right Intrastriatal Administration of 6-Hydroxydopamine

Banegas

Prieto²,

Vives³

et al. 2004

Neuroendocrinology

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Asymmetries in the neuroendocrine system extend from central structures to paired endocrine glands and their innervation. In addition to the well-known asymmetry in the function of brain dopamine, there are also asymmetries in the peripheral response to experimental hemi-parkinsonism, performed by means of lesions of the nigrostriatal system with 6-hydroxydopamine (6-OHDA) injections into the left or right hemisphere. Therefore, it is speculated that the neuroendocrine system would also be asymmetrically affected in experimental hemi-parkinsonism. Aminopeptidases (AP) play a major role in the control of peptide concentration at both central and peripheral levels in tissues and blood, thus reflecting the functional status of their endogenous substrates. Therefore, to evaluate the peripheral response of hemi-parkinsonism, we have performed a comprehensive study of plasma AP activities after lesions of the nigrostriatal system with 6-OHDA administered into either left or right striatum of adult male rats. Saline was injected into control groups. AlaAP, CysAP, AspAP and GluAP activities were determined in plasma, using specific arylamides as substrates. Plasma AlaAP activity increased 3-fold (p < 0.001) whereas AspAP activity decreased by 30% (p < 0.05) after lesion of the right hemisphere. In contrast, CysAP and GluAP activities increased significantly after lesion of the left hemisphere by 200 and 50%, respectively (p < 0.05). The main discovery of the present results demonstrates that experimental hemi-parkinsonism affects differentially the plasma AP activities depending on the hemisphere in which the lesion is performed. This suggests that the circulating hormones, susceptible to be hydrolyzed by these enzymatic activities, are also modified.

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Section: Methodsmentioning

confidence: 99%

Plasma Aminopeptidase Activities in Rats after Left and Right Intrastriatal Administration of 6-Hydroxydopamine

Banegas

Prieto²,

Vives³

et al. 2004

Neuroendocrinology

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“…To ensure that the activity of the TRH-degrading ectoenzyme is specifically measured by this test, the incubation mixture was supplemented with 2-iodoacetamide (2 mM) or L-5-oxoprolyl-diazomethylketone (1 pM) [26] to inactivate any residual activities of the soluble pyroglutamate aminopeptidase that may still be present when crude enzyme preparations are used. However, despite the extremely sensitive detection method [19], the activity of the TRH-degrading ectoenzyme could only be determined accurately in samples containing relatively high enzyme concentrations. This fact is easily explained by the kinetic data.…”

Section: Substrate Specificity and Kinetical Analysismentioning

confidence: 99%

“…Preparations containing high enzymic activities were diluted appropriately with buffer A containing 2% bovine serum albumin to minimize the adsorption to glass. After the initial steps of purification, the enzymic activity could also be measured by the fluorometric detection method [19] using ~-5-oxoprolyl-~-naphthylamide as substrate (0.1 mM in buffer A containing 2 m M 2-iodoacetamide or 1 pM L-5-oxoprolyldiazomethane). The liberation of /?-naphthylamine was determined by use of a Perkin-Elmer LS-3B fluorescence spectrometer (excitation at 340 nm; emission at 410 nm).…”

Section: Enzyme Assaymentioning

confidence: 99%

Purification and Characterization of the Thyrotropin‐releasing‐hormone‐degrading Ectoenzyme

Bauer¹

1994

European Journal of Biochemistry

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The membrane-bound enzyme which catalyzes the degradation of thyrotropin-releasing hormone (TRH; Glp-His-Pro-NH,) could be released from membranes of rat and pig brain by treatment with trypsin under very mild incubation conditions. The solubilized enzyme was purified 200 000-fold, with an overall yield of 20%, by conventional chromatographic methods.The enzyme preparation appeared to be electrophoretically homogenous since SDSPAGE analysis revealed a single band with a molecular mass of 116000 Da. By gel-filtration chromatography, a molecular mass of 230000 Da was estimated, suggesting that the enzyme consists of two identical subunits.The enzyme could be identified as a glycoprotein by lectin-binding analysis and by the reduction of the molecular mass to 97000 Da upon treatment of the denatured enzyme with endoglycosidase-F/N-glycosidase F. In its native form, however, the enzyme was only partially deglycosylated and retained full enzymic activity.In addition to TRH, the enzyme also hydrolyzed ~-5-oxoprolyl-~-naphthylamide, and thus a convenient fluorimetric assay could be established to determine high enzyme activities. The hydrolysis of both substrates was found to obey Michaelis-Menten kinetics, but considerable differences in the respective K , and V,,, values were noticed. TRH (thyrotropin releasing hormone) not only acts as a hypothalamic hypophysiotrophic hormone (for review, see [ 1 and 21) but also elicits a variety of effects in the central nervous system [3 -51 indicating that in extrahypothalamic brain areas this peptide may possibly act as a neuromodulator or neurotransmitter. Such functions imply the existence of a highly efficient inactivation system to clear the target site for transmission of the next signal. There is considerable evidence that the inactivation of neuronally released TRH is catalyzed by a particular enzyme (for review, see [6]) that, [7 -91 similar to the TRH-degrading serum enzyme [lo], exhibits a high degree of substrate specificity. This peptidase is found in synaptosomal [7] and adenohypophyseal [ l l ] membrane fractions; in brain cultures it is only found on the surface of neuronal cells and not on glial cells [12-141. In the pituitary it is localized preferentially on lactotrophs [13] ; recent studies demonstrated that the activity of the adenohypophyseal enzyme is regulated by estradiol [14] and is stringently controlled by thyroid hormones [ 16 -181, suggesting that this enzyme itself might serve regulatory functions. To further our understanding of the biological function of this enzyme, we were interested in purifying it to electrophoretic homogeneity as a basis to develop new tools for further studies.Correspondence to K. Bauer, Max-Planck-Institut fur experimentelle Endokrinologie, Feodor-Lynen-Str. 7, D-30625 Hannover, GermanyAbbreviations. P-NA, 8-naphthylamide; TRH, thyrotropin-releasing hormone ; TRF, thyrotropin-releasing factor.Enzyme. Phospholipidase type I11 (EC 3.1.4.3). MATERIALS AND METHODS MaterialsTrypsin, chymotrypsin, proteinase K and endoglycosida...

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“…A Hitachi-Perkin-Elmer 2A spectrofluorometer was utilized for the assays which were carried out at 25\ s=deg\C with an excitation wavelength of 330 m\g=m\ and an emission wavelength of 405 m\g=m\ (Greenberg, 1962). Assay solutions consisted of (a) 3\m=.\6ml of 0-05 M-tris-HCl, 0-05 M-CaCl2, pH 8, (b) 0-05 ml of 50 µ -dissolved in dimethylformamide, and (c) 0-05 ml to 0-1 ml of enzyme solution.…”

mentioning

confidence: 99%

Biochemical Detection and Activation of an Inactive Form of a Trypsin-Like Enzyme in Rabbit Testes

Meizel¹

1972

Reproduction

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Fluorometric measurement of alkaline phosphatase and aminopeptidase activities in the order of 10−14 mole

Cited by 152 publications

References 8 publications

Plasma Aminopeptidase Activities in Rats after Left and Right Intrastriatal Administration of 6-Hydroxydopamine

Plasma Aminopeptidase Activities in Rats after Left and Right Intrastriatal Administration of 6-Hydroxydopamine

Purification and Characterization of the Thyrotropin‐releasing‐hormone‐degrading Ectoenzyme

Biochemical Detection and Activation of an Inactive Form of a Trypsin-Like Enzyme in Rabbit Testes

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