Release of [hydroxyproline3]-kinins by tissue kallikreins of pig, rat and man.

To determine the susceptibility of kininogens containing the recently described [Hyp3]-bradykinin moiety to cleavage by tissue kallikreins, we have studied the release of [Hyp3]-kinins from heat inactivated human plasma by purified tissue kallikreins. Kallikreins from man and pig were employed and compared with purified rat urinary kallikrein which is known to have a different cleavage specificity. Kinins released were separated by a modified reversed phase HPLC method and quantitated by bioassay and radioimmunoassay. Human urinary kallikrein and hog tissue kallikreins released 85-90% of the total kinins as Lys-bradykinin and 10-15% as [Hyp3]-Lys-bradykinin. In contrast, rat urinary kallikrein released 77% as bradykinin, 22% as [Hyp3]-bradykinin and negligible amounts of [Hyp3]-Lys-bradykinin from the identical substrate source indicating that rat tissue kallikreins prefer the Lys-Arg-bond within both unhydroxylated and hydroxylated kininogens. Therefore, hydroxylation of human kininogens apparently does not affect their ability to serve as substrates for tissue kallikreins with different cleavage specificities.

Correlation of two different assays for urinary kallikrein in normotensive and hypertensive subjects.

Possible differences in structure-function relationship of urinary kallikrein between normotensive and hypertensive individuals were analysed using two different assay systems which detect two distinct entities of the enzyme. A monospecific goat anti-human urinary kallikrein antibody was characterized by inhibition studies with the purified active enzyme and by trypsin activation of endogenous urinary prokallikrein. Analysis of the data revealed that the antibody is directed against active kallikrein by recognizing an epitope which is different from the catalytic site of the enzyme but which is being exposed together with the active site during trypsin activation of the proenzyme. A direct radioimmunoassay for urinary kallikrein was developed and correlated with the kinin generating activity of the enzyme by assessing endogenous active and trypsin activated kallikrein in the urine of normotensive and hypertensive subjects. Significant positive correlations were found between the two assays for both active and total kallikrein in normotensive and hypertensive subjects and the slopes of the respective regression lines were identical. These data do not provide evidence for a defective enzyme, a defective activation of the proenzyme or for the presence of an inhibitor of urinary kallikrein in essential hypertension.

Identification of [hydroxyproline3]-lysyl-bradykinin released from human kininogens by human urinary kallikrein.

The types of kinins released from purified native, single chain human high and low molecular mass kininogens (HMMKs and LMMKs, respectively) by purified human urinary kallikrein were separated by reverse-phase HPLC and quantitated by the rat uterus bioassay. [Hyp3]-lysyl-bradykinin, a recently discovered kinin, represented up to 58% of the biological activity released from 4 individual HMMK preparations purified from 4 different healthy volunteers. In contrast, the majority of the biological activity released from LMMKs purified from pooled plasma was identified as Lys-bradykinin and [Hyp3]-lysyl-bradykinin represented only 6.4 +/- 3.8%. These findings indicate posttranslation hydroxylation of human kininogens and suggest a preference of HMMKs for this modification.