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Biomedical subjects

A J Barrett

Publications and source records attributed to A J Barrett.

At least 253 records · Page 14Linked to original sources
PubMed1986

Cathepsin L inactivates alpha 1-proteinase inhibitor by cleavage in the reactive site region.

The lysosomal cysteine proteinases cathepsin L and cathepsin B were examined for their effect on the neutrophil elastase inhibitory activity of human alpha 1-proteinase inhibitor (alpha 1PI). Human cathepsin L catalytically inactivated human alpha 1PI by cleavage of the bonds Glu354-Ala355 and Met358-Ser359 (the serine proteinase inhibitory site). Cathepsin B did not inactivate alpha 1PI, even when equimolar amounts of enzyme were employed. Cathepsin L is the first human proteinase shown to catalytically inactivate alpha 1PI. These findings, in conjunction with other reports, suggest that alpha 1PI contains a proteolytically sensitive region encompassing residues 350-358. Taken together with the discovery of the elastinolytic activity of cathepsin L (Mason, R. W., Johnson, D. A., Barrett, A. J., and Chapman, H. A. (1986) Biochem. J. 233, 925-927), the present findings emphasize the possible importance of cathepsin L in the pathological proteolysis of elastin and diminish the role that can be attributed to cathepsin B in such processes.

Binding Sites↗
PubMed1986

Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue.

The dimethylmethylene blue assay for sulphated glycosaminoglycans has found wide acceptance as a quick and simple method of measuring the sulphated glycosaminoglycan content of tissues and fluids. The available assay methods have lacked specificity for sulphated glycosaminoglycans in the presence of other polyanions, however, and have not discriminated between the different sulphated glycosaminoglycans. We now describe a modified form of the dimethylmethylene blue assay that has improved specificity for sulphated glycosaminoglycans, and we show that in conjunction with specific polysaccharidases, the dimethylmethylene blue assay can be used to quantitate individual sulphated glycosaminoglycans.

Chondroitin Lyases↗
PubMed1986

Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids.

Six cysteine proteinase inhibitors were isolated from human urine by affinity chromatography on insolubilized carboxymethylpapain followed by ion-exchange chromatography and immunosorption. Physicochemical and immunochemical measurements identified one as cystatin A, one as cystatin B, one as cystatin C, one as cystatin S, and one as low molecular weight kininogen. The sixth inhibitor displayed immunochemical cross-reactivity with salivary cystatin S but had a different pI (6.85 versus 4.68) and a different (blocked) N-terminal amino acid. This inhibitor was tentatively designated cystatin SU. The isolated inhibitors accounted for nearly all of the cysteine proteinase inhibitory activity of the urinary pool used as starting material. The enzyme inhibitory properties of the inhibitors were investigated by measuring inhibition and rate constants for their interactions with papain and human cathepsin B. Antisera raised against the inhibitors were used in immunochemical determinations of their concentrations in several biological fluids. The combined enzyme kinetic and concentration data showed that several of the inhibitors have the capacity to play physiologically important roles as cysteine proteinase inhibitors in many biological fluids. Cystatin C had the highest molar concentration of the inhibitors in seminal plasma, cerebrospinal fluid, and milk; cystatin S in saliva and tears; and kininogen in blood plasma, synovial fluid, and amniotic fluid.

Body Fluids↗
PubMed1986

Purification of cathepsin B by a new form of affinity chromatography.

Human cathepsin B was purified by affinity chromatography on the semicarbazone of Gly-Phe-glycinal linked to Sepharose 4B, with elution by 2,2'-dipyridyl disulphide at pH 4.0. The product obtained in high yield by the single step from crude starting material was 80-100% active cathepsin B. The possibility that this new form of affinity chromatography may be of general usefulness in the purification of cysteine proteinases is discussed.

2,2'-Dipyridyl↗
PubMed1986

Human low-Mr kininogen contains three copies of a cystatin sequence that are divergent in structure and in inhibitory activity for cysteine proteinases.

We point out that human low-Mr kininogen contains three cystatin-like sequences, rather than two, as had previously been thought. The protein was purified by affinity chromatography on carboxymethyl-papain-Sepharose, and subjected to limited proteolysis by trypsin and chymotrypsin. Fragments were isolated, and three corresponding to the individual cystatin-like domains were identified. By comparison with the known amino acid sequence of the protein they were numbered 1 to 3 from the N-terminus. Domain 1 was not found to have any inhibitory activity for cysteine proteinases, which is consistent with the absence of residues that are highly conserved in inhibitors of the cystatin superfamily, and have previously been suggested to be essential for activity. Domain 2 was a good inhibitor of chicken calpain, and also papain and cathepsin L. Domain 3 showed negligible inhibition of calpain, but inhibited papain and cathepsin L strongly. The probable arrangement of disulphide bonds in the heavy chain of low-Mr kininogen is deduced from the homology with the cystatins and other evidence contained in the present paper.

Amino Acid Sequence↗
PubMed1986

Elastinolytic activity of human cathepsin L.

The hydrolysis of a tritiated elastin substrate by the human cysteine proteinases cathepsins B and L has been studied. Cathepsin L was found to be at least 100-fold more active on this substrate than cathepsin B. The specific activity of cathepsin L at pH 5.5 for hydrolysis of elastin was about the same as that of pig pancreatic elastase at its optimum pH of 8.8.

Cathepsin L↗
PubMed1986

The biochemistry of the action of chymopapain in relief of sciatica.

A study has been made of the mechanism of action of intradiscal injections of preparations of chymopapain in the treatment of sciatica. Such preparations were found to contain at least four distinct proteins, but enzymatically active chymopapain was the component mainly responsible for releasing glycosaminoglycan from cartilaginous tissue. Previous suggestions that an electrostatic interaction between chymopapain and glycosaminoglycan is important to the action of injected enzyme were not supported by the finding that both positively and negatively charged forms of chymopapain efficiently released glycosaminoglycan from cartilaginous tissue. In contrast, cysteine alone did not cause release of glycosaminoglycan. Chymopapain was found to be inhibited efficiently by the protein inhibitors, cystatin C and low molecular weight kininogen in vitro, and the possible relevance of this finding to the efficacy and safety of chemonucleolysis is discussed.

Cartilage↗
PubMed1986

Prediction and prevention of relapse of acute lymphoblastic leukaemia after bone marrow transplantation.

Results of bone marrow transplantation (BMT) in 63 adults and children with ALL transplanted in the 5-year period 1979-83 were analysed. Twenty-one patients (33%) relapsed, 25% of the group died in relapse and 19% died from complications of BMT. The actuarial disease-free survival at 6 years was 38%. Relapse after BMT could be predicted by standard prognostic diagnostic features such as age, sex, cell type and presenting blast cell count. Patients transplanted in first remission selected for their poor prognosis had a lower relapse risk than a similar group of poor prognosis patients transplanted in second or subsequent remission (P less than 0.05). Relapse following second and subsequent remission BMT was predicted by a score based on standard prognostic features or by the pace of the disease: patients with an interval of less than 2 years between diagnosis and first relapse having a 15% actuarial disease-free survival, compared with 81% for patients with an interval greater than 2 years (P less than 0.001). These results emphasize that ALL is a heterogeneous disease and establishes the importance of determining relapse risk when selecting BMT and other treatment schedules for ALL patients.

Acute Disease↗
PubMed1986

The cystatins: a diverse superfamily of cysteine peptidase inhibitors.

The cystatins comprise a homologous group of protein inhibitors of peptidases, of which the first to be studied in detail was chicken cystatin from egg-white. Three distinct protein families or "types" are recognized within the cystatin superfamily. The type 1 cystatins (also called "stefins") are the simplest in structure, being single chains of about 100 amino acids, with no disulphide bonds or carbohydrate. The Type 2 cystatins, which include the egg-white protein, have about 115 amino acids, and two disulphide loops, but still no carbohydrate. The Type 3 cystatins are the plasma kininogens in which each molecule contains three divergent copies of the typical cystatin sequence differing in activity as well as structure; these complex inhibitor molecules contain disulphide bonds and also carbohydrate groups. The cystatins inhibit most cysteine endopeptidases of the papain type, and also the exopeptidase dipeptidyl peptidase I. Each cystatin molecule has a single reactive site for all the peptidases it inhibits, but there are large differences in K(i) values for different combinations of cystatin and enzyme, and calpains are inhibited only by one of the segments of the kininogens. The cystatins have many important characteristics in common, but their differences in molecular structure imply different routes of biosynthesis, are associated with different in vivo distributions, and suggest a variety of physiological functions.

Animals↗
PubMed1985

Amino acid sequence of the intracellular cysteine proteinase inhibitor cystatin B from human liver.

The complete amino acid sequence of the cysteine proteinase inhibitor cystatin B (formerly named CPI-B) from human liver was determined. The 98-residue sequence (Mr = 11,175) was obtained by automated solid-phase Edman degradation of a large cyanogen bromide fragment and peptides generated by enzymatic cleavage. The protein starts with a blocked Met-Met sequence which is presumably N-acetylated. The sequence shows that human cystatin B is a member of the family of intracellular cystatins; it is 79% identical with cystatin beta from rat liver, but contains only a single cysteine. Human cystatin B is able to form a dimer stabilized by noncovalent forces.

Amino Acid Sequence↗
PubMed1985

Calpain inhibition by peptide epoxides.

A Ca2+-activated cysteine proteinase (calpain II) was purified from chicken gizzard smooth muscle by use of isoelectric precipitation, (NH4)2SO4 fractionation, chromatography on DEAE-Sepharose CL-6B, Reactive-Red 120-agarose and Mono Q. The apparent second-order rate constants for the inactivation of calpain by a series of structural analogues of L-3-carboxy-trans-2, 3-epoxypropionyl-leucylamido-(4-guanidino)butane (E-64) were determined. The fastest rate of inactivation was observed with L-3-carboxy-trans-2, 3-epoxypropionyl-leucylamido-(4-benzyloxy-carbonylamino)buta ne. It was possible to determine the active-site molarity of solutions of calpain by titration with E-64. When incubated with Ca2+, calpain underwent several steps of intermolecular limited proteolysis, via multiple pathways, followed by a slower loss of enzymic activity. The proteolytic steps preceding the loss of activity did not affect the rates of reaction of calpain with E-64 analogues.

Animals↗