Biomedical subjects

T Harrach

Publications and source records attributed to T Harrach.

4 recordsLinked to original sources

PubMed1998

Isolation and characterization of two forms of an acidic bromelain stem proteinase.

Two forms of an acidic bromelain proteinase isolated from crude bromelain, an extract from pineapple stem, were found by a two-step FPLC purification procedure. The basic main components were removed by cation exchange chromatography and the breakthrough fraction was further resolved by anion exchange chromatography into 15 protein fractions, only two of which, called SBA/a and SBA/b, were proteolytically active. These components were characterized by electrospray mass spectroscopy (ESMS), isoelectric focusing, N-terminal amino acid sequence analysis, monosaccharide analysis, and enzymatic parameters. The molecular masses of SBA/a and SBA/b were determined by ESMS to be 23,550 and 23,560, respectively. The isoelectric points (pI) of the two bands of SBA/a were 4.8 and 4.9; SBA/b focused as a single band at pI = 4.8. Partial N-terminal amino acid sequences (11 residues) were identical to SBA/a and SBA/b and identical with those of stem bromelain, the basic main proteinase of the pineapple stem, and fruit bromelain, the acidic main proteinase of the pineapple fruit. Both components are highly glycosylated; hydrolysis of SBA/a yielded about twofold more monosaccharide per protein than SBA/b. The comparison of the catalytic properties of SBA/a with those of SBA/b revealed no relevant differences in the hydrolysis of three peptidyl-NH-Mec substrates and in the inhibition profiles using chicken cystatin and E-64, indicating that these components can be considered as two forms of a single enzyme. Both forms are scarcely inhibited by chicken cystatin and slowly inactivated by E-64, hence are nontypical cysteine proteinases of the papain superfamily.

Amino Acid Sequence↗

PubMed1997

Hair concentrations and self-reported abuse history of 20 amphetamine and ecstasy users.

Hair samples of 20 volunteers of the techno-music scene, who more or less regularly consumed ecstasy tablets and speed and anonymously reported their abuse history, were analyzed in one to seven 3 cm segments for amphetamine (A), methamphetamine (MA), methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), methylenedioxyethamphetamine (MDE) and N-methyl-1-(1,3-benzodioxol-5-yl)-2-butylamine (MBDB) by digestion in 1 M NaOH, subsequent extraction with C18 Bond Elut columns, derivatization with pentafluoropropionyl anhydride and GC/MS-SIM measurements using deuterated standards of A, MA, MDA and MDMA. The concentrations were in the regions 0.1 to 4.8 ng/mg for A (17 samples), 0.05 to 0.89 ng/mg for MDA (16 samples), 0.1 to 8.3 ng/mg for MDMA (16 samples), 0.12 to 15 ng/mg for MDE (13 samples) and 0.21 to 1.3 ng/mg for MBDB (2 samples). MA was not detected. For comparison the frequency and the concentration of these drugs in 124 different ecstasy tablets were determined by HPLC. The drug concentration in the hair segments were compared with the volunteers' reports. Despite the enormous interindividual differences qualitatively an increase of the total concentration of MDA, MDMA and MDE in the proximate 3 cm segments with increasing ecstasy abuse frequency during the last three month before sampling is recognized. In the individual comparison with the chronological consumer reports in most cases a longer interruption or a change of the abuse intensity is not clearly seen at the segment concentrations. As a reason the incorporation of the drugs from sweat into elder hair regions and the slow removal by washing are discussed.

Adolescent↗

PubMed1995

Isolation and partial characterization of basic proteinases from stem bromelain.

Crude bromelain extracts from pineapple stems (Ananas comosus) were fractionated by two-step FPLC-cation-exchange chromatography. At least eight basic proteolytically active components were detected. The two main components F4 and F5 together with the most active proteinase fraction F9 were characterized by SDS-PAGE, mass spectroscopy, multizonal cathodal electrophoresis, partial amino acid sequence, and monosaccharide composition analysis. F9 amounts to about 2% of the total protein and has a 15 times higher specific activity against the substrate L-pyroglutamyl-l-phenylanalyl-l-leucine-p-nitroanilide (PFLNA) than the main component F4. The molecular masses of F4, F5, and F9 were determined to 24,397, 24,472, and 23,427, respectively, by mass spectroscopy. Partial N-terminal amino acid sequence analysis (20 amino acids) revealed that F9 differs from the determined sequence of F4 and F5 by an exchange at position 10 (tyrosine-->serine) and position 20 (asparagine-->glycine). F4 and F5 contained fucose, N-acetylglucosamine, xylose, and mannose in ratio of 1.0:2.0:1.0:2.0, but only 50% of the proteins seem to be glycosylated, whereas F9 was found to be unglycosylated. Polyclonal antibodies (IgG) against F9 detected F4 and F5 with tenfold reduced reactivity. The pH optimum of F4 and F5 was between pH 4.0 and 4.5 and for F9 close to neutral pH. The kinetic parameters for PFLNA hydrolysis were similar for F4 (Km 2.30 mM, kcat 0.87 sec-1 and F5 (Km 2.42 mM, kcat 0.68 sec-1), and differed greatly from F9 (Km 0.40 mM, kcat 3.94 sec-1).

Amino Acid Sequence↗

PubMed1994

Bromelain protease F9 reduces the CD44 mediated adhesion of human peripheral blood lymphocytes to human umbilical vein endothelial cells.

The thiol protease bromelain has been shown to remove T-cell CD44 molecules from lymphocytes and to affect T-cell activation. We investigated the effect of a highly purified bromelain protease F9 (F9) on the adhesion of peripheral blood lymphocytes (PBL) to human umbilical vein endothelial cells (HUVEC). Preincubation of the lymphocytes with F9 reduced the adherence to about 20% of unstimulated and to about 30% of phorbol-dibutyrate (P(Bu)2) stimulated lymphocytes. Using flow cytometry, both crude bromelain and protease F9 reduced the expression of CD44, but not of LFA-1, on PBL. F9 was about 10 times more active than crude bromelain; at 2.5 micrograms/ml of F9 about 97% inhibition of CD44 expression was found. A mAb against CD44 was tested and found to block the F9-induced decrease in PBL-binding to HUVEC. The results indicate that F9 selectively decreases the CD44 mediated binding of PBL to HUVEC.

Antibodies, Monoclonal↗