Claude Lapresle

Fixation of penicilloyl groups to albumin and appearance of anti-penicilloyl antibodies in penicillin-treated patients.

Penicilloyl groups, which have been connected to penicillin allergy, are derived from penicillin by cleavage of the beta lactam ring and bind covalently to proteins. Fixation of penicilloyl groups was studied in seven patients given large amounts of penicillin. Penicilloyl groups were found essentially on the albumin molecule at sites not accessible to anti-penicilloyl antibodies, except after pronase digestion. The amount of penicilloyl groups was proportional to the cumulated doses of penicillin. The decline of penicilloyl groups with time after treatment interruption was exponential. The half-life of penicilloylated albumin was lower than or equal to that of normal albumin. The presence of anti-penicilloyl antibodies was demonstrated in 19 out of 34 penicillin-treated patients (including the seven mentioned above). The relative scarcity of penicillin allergy as compared with the frequent occurrence of anti-penicilloyl antibodies may be partly related to unavailable sites of penicilloyl groups within the albumin molecule.

The binding of penicillin to albumin molecules in bisalbuminemia induced by penicillin therapy

Abstract Bisalbuminemia in two penicillin-treated patients was detected by electrophoresis in polyacrylamide Agarose gel. The normal and fast albumins were isolated by chromatography on DEAE-Sephadex. Penicilloyl groups were measured by radioimmunoassay and found mostly on fast albumin. The amount of penicilloyl groups detected was markedly increased by enzymatic degradation of the albumin samples. Under these conditions, at least one penicilloyl group was found per mol fast albumin, which can explain its greater mobility.

Study of the antigenic structure of human serum albumin with monoclonal antibodies

Analysis of the antigenic structure of human serum albumin was undertaken using monoclonal antibodies. Nineteen antibodies were prepared and their specificities were studied using fragments which encompass the whole sequence of the albumin molecule. These antibodies recognized 13 different epitopes which are different from the one previously identified with two other monoclonal antibodies [Doyen et al., Immun. Lett. 3, 365-370 (1981)]. Among those 13 different epitopes, six were overlapping. Four epitopes were located on the N-terminal half of the albumin molecule. One of these required integrity of methionine 87 and the other three were overlapping and located around methionine 123. Eight epitopes were located on the C-terminal half of the albumin. Two of them were within the sequence, 330-422 and 299-496 respectively; the other six appeared to be topographic determinants which were altered or lost in the albumin fragments. A last epitope could not be located on any region of albumin. Four monoclonal antibodies directed against a given portion of the albumin molecule reacted slightly with another part of albumin, thus confirming the existence of an intramolecular cross-reactivity between the different domains of human albumin.

Evidence for the presence of large amounts of cathepsin E in rat spleen

Although cathepsin E is present in trace amounts in spleen from several species, it was found in large amounts in rat spleen. This observation can be correlated with the fact that spleen in the rat is an important organ in haemopoeisis.

Inhibition of cathepsin E by diazoacetyl-norleucine methyl ester

In a previous study [ 1 ] we described the inhibition of cathepsin D by diazoacetyl-norleuci~e methyl ester (DANME) * in the presence of copper ions. A similar inhibition has been observed before with pepsin [2, 3], in which an aspartic acid residue of the active site is blocked by the above reagent. This indicates a similar mechanism of catalytical action of the two enzymes. Cathepsin E is an intracellular proteinase of rabbit bone marrow [4] with an optimum activity in the acidic pH-range, resembling cathepsin D in its specificity as assayed with the oxidized B-chain of insulin as substrate. The present study was undertaken to establish whether cathepsin E could be inhibited by the same reagent as cathepsin D and to find similar features of the inhibition mechanisms.

Location of penicilloyl groups on CNBr fragments of the albumin from penicillin‐treated patients

Two fixation sites for penicilloyl groups on human albumin were demonstrated. Using CNBr cleavage the first site was located between methionine 123 and methionine 297 and the second one between methionine 297 and the C‐terminal residue. In both cases, penicilloyl groups were unmasked by pronase degradation or disulfide bond reduction.

Identification of two fixation sites for penicilloyl groups on the albumin molecule from penicillin-treated patients

Fixation of penicilloyl groups to albumin in penicillin‐treated patients was shown to involve histidine residues 146 and 338.

Enzyme immunoassay using monoclonal antibodies to study conformational changes of human serum albumin

A method for studying conformational changes induced in the human albumin molecule, either in its purified form or in serum, is described. Plates were coated with albumin or human serum at varying pHs and were reacted with peroxidase-labeled anti-albumin monoclonal antibodies of different specificities. The data showed that albumin molecules were coated in conformations induced as a result of pH changes, allowing us to demonstrate that pH modifications involved the N-terminal portion of the albumin molecule whether in its purified form or in serum. This method should be applicable to the study of conformational modifications in other proteins as well.

Specificity of two anti-human albumin monoclonal antibodies.

We have studied the reaction of two monoclonal anti-human albumin antibodies with various fragments of albumin comprising the entire molecule. Using solid phase assay, inhibition of enzyme immunoassay and of passive hemagglutination, they were shown to be specific for a fragment F1, of 6000 dalton, located near the C terminus of human albumin. In addition, these antibodies reacted weakly with fragment D which corresponds to the N terminal half of the molecule.

Etude de la specificite sur la chaine b de l'insuline des cathepsines d et e de lapin

Abstract The action of rabbit cathepsins D and E on the B chain of beef insulin has been studied. Cathepsin D hydrolyses the bonds Leu 11 -Val 12 , Glu 13 -Ala 14 , Ala 14 -Leu 15 , Leu 15 -Tyr 16 , Tyr 16 -Leu 17 , Leu 17 -Val 18 , Phe 24 -Phe 25 , Phe 25 -Tyr 26 , Tyr 26 -Thr 27 . Furthermore, it hydrolyses one of the bonds His 5 -Leu 6 or Leu 6 -Cys 7 , or both. Cathepsin E hydrolyses the bonds Leu 11 -Val 12 , Glu 13 -Val 14 , Leu 15 -Tyr 16 , Tyr 16 -Leu 17 , Phe 24 -Phe 25 , Phe 25 -Tyr 26 .