Evaluation of elastase and alpha 1-proteinase inhibitor-elastase uptake by polymorphonuclear leukocytes and evidence of an elastase-specific receptor.

Neither resting nor stimulated isolated human polymorphonuclear leukocytes did bind or ingest preformed complexes of alpha 1-proteinase inhibitor and unlabeled/125I-labeled human leukocyte elastase. In contrast, granulocytes bound unlabeled/125I-labeled elastase and the extent of binding was reduced in the presence of respiratory burst stimulators, such as 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, E. coli endotoxin, and N-formyl-L-methionyl-L-leucyl-L-phenylalanine. In association/dissociation and competition inhibition experiments it was demonstrated that granulocyte-elastase binding was specific and saturable. From Scatchard and non-linear regression analysis there was evidence of a two-class receptor model with independent binding sites. Calculated by the non-linear regression method assuming a two-class receptor model the characteristics of the high affinity/low capacity binding site were K1 = 216 +/- 129 X 10(6) l X mol-1 (means +/- s; n = 3) and R1 = 1.38 +/- 0.95 nmol X l-1 corresponding to 0.083 X 10(6) receptors per cell, whereas the low affinity/high capacity binding site had the characteristics K2 = 0.50 +/- 0.09 X 10(6) l X mol-1 and R2 = 237 +/- 103 nmol X l-1 corresponding to 14.3 +/- 6.2 X 10(6) receptors per cell.

[Effect of total hormone concentration on the determination of amount of binding in the hormone/receptor interaction between erythrocytes and insulin]. / Zum Einfluss der totalen Hormonkonzentration auf die Ermittlung von Bindungskenngrössen bei der Hormon/Receptor-Wechselwirkung zwischen Erythrocyten und Insulin.

The interaction of erythrocytes and [125I]insulin/insulin were studied up to a total insulin concentration of 409 mumol/l. Assuming a single class receptor model the evaluation of receptor affinity Ka and concentration R0 may be performed either by non-linear regression analyses with iteration procedures of R0, Ka and U (nonspecific binding), or by a linear regression analysis of the initial part of the Scatchard plot. Nonlinear fitting of data to a two class receptor model gives results that are reliable only for the high affinity receptor site. The non-definable step of R0 determination leads to uncertainties in results determined by the negative cooperativity model. From the results of this investigation and from considerations of signal modulation by receptor occupancy, some recommendations have been formulated for the evaluation of binding parameters; these should contribute to an improvement in the comparability of studies on the interactions of erythrocytes and insulin.

Insulin binding to erythrocytes from pregnant, postpartum, follicular and luteal states.

Specific binding of [125I] insulin to isolate erythrocytes from four groups of women was investigated: (A) pregnant subjects between weeks 38 and 40 of pregnancy (n = 18), (B) postpartum subjects within 6 days after delivery (n = 20), (C) normal women during the follicular phase of the menstrual cycle (n = 12) and (D) normal women during the luteal phase of the menstrual cycle (N = 11). Specific [125I] insulin binding (fraction), fasting plasma glucose concentrations (mmol/l) and the corresponding insulin concentrations (mU/l) were 0.074 +/- 0.012 / 4.00 +/- 0.58 / 29.4 +/- 21.4 for group A, 0.065 +/- 0.016 / 4.40 +/- 0.75 / 41.5 +/- 26.2 for group B, 0.052 +/- 0.008 / 4.58 +/- 0.62 / 6.7 +/- 4.0 for group C and 0.054 +/- 0.011 / 4.49 +/- 0.63 / 8.3 +/- 5.9 for group D. By using a modified Scatchard analysis, statistically significant differences were observed between the receptor affinities of the groups A and D, B and D, A and C. The receptor affinities and concentrations were not significantly different between the follicular and the luteal phases. From the data, no inverse correlation between the plasma insulin concentration and receptor binding was seen, i.e. the phenomenon of downregulation of insulin receptor concentration with hyperinsulinaemia seemed not to apply to erythrocytes.