Biomedical subjects

A D Rowan

Publications and source records attributed to A D Rowan.

43 records · Page 3Linked to original sources

PubMed1992

Investigation of structure function relationships in cathepsin B.

Previous suggestions from sequence alignment studies and examination of the recently determined X-ray crystal structures of cathepsin B point to roles for several specific residues in substrate binding and catalysis. The role of these groups is being examined by studying cathepsin B mutants produced using a yeast expression system. The substitutions Gly198Asp, Arg202Ala, His111Gln and Glu245Gln provide a mechanistic basis for the exopeptidase activity of cathepsin B and the ability of this cysteine proteinase to accept an arginine residue in the S2 subsite.

Amino Acid Sequence↗

PubMed1992

Antibodies to rat procathepsin B recognize the active mature enzyme.

Use of mature cathepsin B for immunization invariably yields antisera that react with the denatured protein but not with the native enzyme. This is thought to be due to spontaneous denaturation of the immunizing antigen on introduction into the animal. Recombinant rat procathepsin B has been expressed in yeast as a secreted product. A procathepsin B mutant (Cys29Ser), where autoprocessing is prevented, has been purified and used to raise a rabbit polyclonal antiserum. Both immunodiffusion analysis and an activity depletion assay demonstrated that this antibody recognized native mature cathepsin B. It appears that conformational epitopes existing on the active enzyme are lost on denaturation. The stability of the proenzyme however permits their presentation for antibody generation.

Animals↗

PubMed1990

Inhibition of cysteine proteinases by a protein inhibitor from potato.

The inhibitory specificity of a protein from potato tubers that inhibits cysteine proteinases (potato cysteine proteinase inhibitor, PCPI) has been compared with that of chicken egg-white cystatin. Most proteinases that are inhibited by cystatin were also inhibited by PCPI, but the potato inhibitor inhibited stem bromelain and fruit bromelain, which are not inhibited by cystatin, and for which no protein inhibitor of comparable potency has previously been described. In contrast, papaya proteinase IV was unaffected by PCPI as it is by the cystatins, and the exopeptidase, dipeptidyl peptidase I, is inhibited by cystatins, but was unaffected by PCPI. The differences in inhibitory specificity between these proteins may well reflect differences between superfamilies of cysteine proteinase inhibitors.

Cystatins↗

PubMed1990

The cysteine proteinases of the pineapple plant.

The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct.

Amino Acid Sequence↗

PubMed1990

Debridement of experimental full-thickness skin burns of rats with enzyme fractions derived from pineapple stem.

A limited in vivo study using 12 rats with full-thickness skin burns injuries was carried out. The animals were treated 24 h postburn with two newly discovered enzyme fractions derived from the stem of the pineapple (Ananas comosus). The results indicated that even debridement of the injury could be effected rapidly (within 4 h). Although the details of enzyme formulation and clinical application have yet to be established, these findings clearly suggest that two enzyme fractions from pineapple stem have potential as non-surgical debriding agents.

Animals↗

PubMed1989

Stem bromelain: amino acid sequence and implications for weak binding of cystatin.

The amino acid sequence of stem bromelain, the major cysteine proteinase from pineapple stem is described. It shows that the enzyme is a member of the papain superfamily of cysteine proteinases, but is not very closely related to any other known member of this group. The sequence shows mutation or deletion of several residues that have been conserved in cysteine proteinases examined previously, including Asn-175 (papain). We suggest that some of these changes have the effect of altering the active-site geometry of stem bromelain, and that this accounts for the resistance of the enzyme to inhibition by cystatins and E-64[L-3-carboxy-2,3-trans-epoxypropionylleucylamido(4-guanidino)b utane].

Amino Acid Sequence↗

PubMed1988

Ananain: a novel cysteine proteinase found in pineapple stem.

A previously unknown cysteine proteinase, named ananain, has been isolated from crude commercial pineapple stem bromelain. The purification procedure involved affinity chromatography on Sepharose-Gly-Phe-glycinaldehyde semicarbazone, and cation-exchange chromatography. The relative molecular mass of ananain was very similar to that of bromelain (25,000 and 26,000, respectively), but ananain differed greatly in specificity for hydrolysis of peptide and protein substrates. The new enzyme behaved as a typical cysteine proteinase in showing strong inhibition by chicken cystatin, whereas bromelain was scarcely affected. Ananain was also shown to be immunologically distinct from bromelain. The significance of the discovery of ananain for the interpretation of previous work on "bromelain" is pointed out.

Binding Sites↗