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Biomedical subjects

A J Barrett

Publications and source records attributed to A J Barrett.

At least 271 records · Page 15Linked to original sources
PubMed1985

How should acute lymphoblastic leukaemia relapsing after bone-marrow transplantation be treated?

21 of 63 patients given bone-marrow transplantation (BMT) for acute lymphoblastic leukaemia (ALL) relapsed between day 75 and day 1126 after BMT. 19 patients were given leukaemic induction treatment with standard combination chemotherapy. 7 patients achieved a sustained remission, of whom 5 are alive in remission 88-1240 days after relapse; the 2 others died during a second BMT. 2 of the remaining 14 obtained partial remission but all 14 ultimately died with relapsed leukaemia. A favourable outcome for ALL relapsing after BMT was predicted by the pace of the disease: the 5 survivors had an interval between diagnosis and first relapse of more than 2 years and a disease-free period after BMT of 500 days or more. These results indicate that further conventional chemotherapy should be used rather than a second marrow transplant to prolong disease-free survival in patients with ALL relapsing after BMT.

Adolescent↗
PubMed1985

The proteolytic activities of chymopapain, papain, and papaya proteinase III.

The three proteinases present in papaya latex: papain (EC 3.4.22.2) chymopapain and papaya proteinase III (EC 3.4.22.6), were standardized by active-site titration, and compared in proteolytic activity against azocasein, serum albumin and cartilage proteoglycan. The activities were all of the same order, although there were differences in pH dependence. SDS-polyacrylamide gel electrophoresis of the early products of digestion of albumin and phosphorylase a showed very similar patterns for the three papaya proteinases. Kinetic parameters for hydrolysis of benzyloxycarbonyl-phenylalanyl-arginyl-7(4-methyl)coumarylamide were determined for the three enzymes. Values for kcat/Km varied only within a factor of 2, but the individual constants were much higher for papain than for chymopapain and papaya proteinase III. In contrast to the results obtained with the synthetic substrate, the kinetic parameters for the initial hydrolysis of succinyl-albumin were very similar for the three papaya proteinases. This was consistent with their similar proteolytic activities in other assays.

Albumins↗
PubMed1985

Human liver cathepsin L.

Cathepsin L was purified to apparent homogeneity from human liver obtained post mortem. It was necessary to treat the homogenate at pH 4.2 and 37 degrees C to release active enzyme. The purification procedure involved ion-exchange chromatography on carboxymethyl-Sephadex and the Mono S column of a Pharmacia fast-protein-liquid-chromatography system. The enzyme was found to consist of two polypeptide chains of Mr 25 000 and 5000. The larger chain was shown to contain the active-site cysteine residue. Human cathepsin L proved to be similar to the rat and rabbit enzymes in regard to kinetic constants for the substrate benzyloxycarbonylphenylalanylarginine 7-(4-methyl)coumarylamide and rates of inactivation by the active-site-directed reagents benzyloxycarbonylphenylalanylphenylalanyldiazomethane and benzyloxycarbonylphenylalanylalanyldiazomethane. Thus clear characteristics of cathepsin L are now emerging, and these should simplify the identification of the enzyme in other tissues and species.

Cathepsin B↗
PubMed1985

Distribution of cystatin C (gamma-trace), an inhibitor of lysosomal cysteine proteinases, in the anterior lobe of simian and human pituitary glands.

Cystatin C, a protein inhibitor of lysosomal cysteine proteinases, was demonstrated by immunohistochemical techniques to be present in most luteinizing hormone- (LH-)containing cells in simian and human adenohypophyses. Immunoreactivity of cystatin C was also found in simian adrenocorticotrophic hormone- (ACTH-)containing cells localized to an area corresponding to the pars intermedia but not in the ACTH-containing cells of the anterior pituitary lobe of monkey. No immunoreactivity of cystatin C was detected in the growth hormone- (GH-) and prolactin-containing cells of monkey and man.

Adrenocorticotropic Hormone↗
PubMed1984

Chymopapain. Chromatographic purification and immunological characterization.

Chymopapain (EC 3.4.22.6) was purified from commercially available spray-dried latex of papaya (Carica papaya) fruit by (NH4)2SO4 fractionation and fast protein chromatography on the Mono S cation-exchange column. Multiple forms of chymopapain separated chromatographically were shown to be immunologically identical. A major form was isolated and found to be homogeneous by several criteria, and fully active, and its N-terminal amino acid was identified as tyrosine. Latex from fresh unripe papaya fruit contained predominantly one form of chymopapain, and it is concluded that chymopapain is a single enzyme distinct from the other cysteine proteinases of C. papaya latex.

Amino Acids↗
PubMed1984

Inhibition of cysteine proteinases and dipeptidyl peptidase I by egg-white cystatin.

The interactions between egg-white cystatin and the cysteine proteinases papain, human cathepsin B and bovine dipeptidyl peptidase I were studied. Cystatin was shown to be a competitive reversible inhibitor of cathepsin B (Ki 1.7 nM, k-1 about 2.3 X 10(-3) s-1). The inhibition of dipeptidyl peptidase I was shown to be reversible (Ki(app.) 0.22 nM, k-1 about 2.2 X 10(-3) s-1). Cystatin bound papain too tightly for Ki to be determined, but an upper limit of 5 pM was estimated. The association was a second-order process, with k+1 1.0 X 10(7) M-1 X s-1. Papain was shown to form equimolar complexes with cystatin. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of complexes formed between papain or cathepsin B and an excess of cystatin showed no peptide bond cleavage after incubation for 72 h. The reaction of the active-site thiol group of papain with 5,5'-dithiobis-(2-nitrobenzoic acid) at pH 8 and 2,2'-dithiobispyridine at pH 4 was blocked by complex-formation. Dipeptidyl peptidase I and papain were found to compete for binding to cystatin, contrary to a previous report. The two major isoelectric forms of cystatin were found to have similar specific inhibitory activities for papain, and similar affinities for papain, cathepsin B and dipeptidyl peptidase I. This, together with specific oxidation of the N-terminal serine residue with periodate, showed the N-terminal amino group of cystatin 1 to be unimportant for inhibition. General citraconylation of amino groups resulted in a large decrease in the affinity of cystatin for dipeptidyl peptidase I. It is concluded that the interaction of cystatin with cysteine proteinases has many characteristics similar to those of an inhibitor such as aprotinin with serine proteinases.

Binding Sites↗
PubMed1984

Human plasma alpha-cysteine proteinase inhibitor. Purification by affinity chromatography, characterization and isolation of an active fragment.

Human plasma alpha-cysteine proteinase inhibitor (alpha CPI) was purified by a two-stage method: affinity chromatography on S-carboxymethyl-papain-Sepharose, and high-resolution anion-exchange chromatography. The protein was obtained as a form of Mr about 64 000 and material of higher Mr (about 100 000). In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with reduction, both forms showed a major component of Mr 64 000. An antiserum was raised against alpha CPI, and 'rocket' immunoassays showed the mean concentration in sera from 19 individuals to be 35.9 mg/dl. Both low-Mr and high-Mr forms of alpha CPI were confirmed to be sialoglycoproteins by the decrease in electrophoretic mobility after treatment with neuraminidase. alpha CPI was shown immunologically to be distinct from antithrombin III and alpha 1-antichymotrypsin, two serine proteinase inhibitors from plasma with somewhat similar Mr values. alpha CPI was also distinct from cystatins A and B, the two intracellular low-Mr cysteine proteinase inhibitors from human liver. Complexes of alpha CPI with papain were detectable in immunoelectrophoresis, but dissociated to free enzyme and intact inhibitor in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The stoichiometry of binding of papain was close to 1:1 for both low-Mr and high-Mr forms. alpha CPI was found to be a tight-binding inhibitor of papain and human cathepsins H and L (Ki 34 pM, 1.1 nM and 62 pM respectively). By contrast, inhibition of cathepsin B was much weaker, Ki being about 35 microM. Dipeptidyl peptidase I also was weakly inhibited. Digestion of alpha CPI with bromelain gave rise to an inhibitory fragment of Mr about 22 000, which was isolated.

Blood Proteins↗
PubMed1984

The place of human gamma-trace (cystatin C) amongst the cysteine proteinase inhibitors.

Native gamma-trace, a small basic protein present in high concentration in cerebrospinal fluid, semen and neuroendocrine cells, but of unknown biological function, is shown to be a potent inhibitor of the cysteine proteinases papain, ficin, and human cathepsins B, H and L. It proves to be the tightest -binding protein inhibitor of cathepsin B so far discovered. The name cystatin C is proposed for gamma-trace to reflect the many similarities in activity and structure to chicken egg-white cystatin and mammalian cystatins A and B. The inhibition constants of cystatin C, taken together with its widespread distribution in human tissues and extracellular fluids, suggest that a physiological function could well be the regulation of cysteine proteinase activity.

Cathepsins↗
PubMed1984

Kinetics of tryptic hydrolysis of the arginine-valine bond in folded and unfolded ribonuclease T1.

We have used ribonuclease T1 and its chemically modified derivatives as substrates, and trypsin as proteinase, to investigate the kinetics of proteolysis of a specific peptide bond in the folded and unfolded conformations of a protein. Steady-state kinetic studies showed that Km = 0.27 mM and Kcat. = 2.45 s-1 for the tryptic hydrolysis of the Arg(77)-Val(78) peptide bond in unfolded ribonuclease T1. This Km is somewhat lower than, and the kcat. value similar to, values found for the tryptic hydrolysis of comparable bonds in small peptides. Our data for the initial velocity of hydrolysis of the Arg(77)-Val(78) bond in a solution of the folded protein indicate that the bond is at least 1700 times less rapidly hydrolysed in the folded than in the unfolded conformation of ribonuclease T1, and do not exclude the possibility that the bond is completely resistant to hydrolysis in the folded protein.

Arginine↗
PubMed1984

Cystatin-like cysteine proteinase inhibitors from human liver.

Cysteine proteinase inhibitor (CPI) forms from human liver were purified from the tissue homogenate by alkaline denaturation of cysteine proteinases with which they are complexed, acetone fractionation, affinity chromatography on S-carboxymethyl-papain-Sepharose and chromatofocusing. The multiple forms of CPI were shown immunologically to be forms of two proteins, referred to as CPI-A (comprising the forms of relatively acidic pI) and CPI-B (comprising the more basic forms). CPI-A and CPI-B are similar in their Mr of about 12400, considerable stability to pH2, pH11 and 80 degrees C, and tight-binding inhibition of papain, several related cysteine proteinases and dipeptidyl peptidase I. Ki values were determined for papain, human cathepsins B, H and L, and dipeptidyl peptidase I. The affinity of CPI-A for cathepsin B was about 10-fold greater than that of CPI-B, whereas CBI-B showed about 100-fold stronger inhibition of dipeptidyl peptidase I. For all the cysteine proteinases the liver inhibitors were somewhat less tight binding than cystatin. The resemblance of both CPI-A and CPI-B in several respects to egg-white cystatin is discussed. CPI-A seems to correspond to the epithelial inhibitor described previously, and CPI-B to the inhibitor from other cell types [Järvinen & Rinne (1982) Biochim. Biophys. Acta 708, 210-217].

Amino Acid Sequence↗
PubMed1984

Cystatin. Amino acid sequence and possible secondary structure.

The amino acid sequence of cystatin, the protein from chicken egg-white that is a tight-binding inhibitor of many cysteine proteinases, is reported. Cystatin is composed of 116 amino acid residues, and the Mr is calculated to be 13 143. No striking similarity to any other known sequence has been detected. The results of computer analysis of the sequence and c.d. spectrometry indicate that the secondary structure includes relatively little alpha-helix (about 20%) and that the remainder is mainly beta-structure.

Amino Acid Sequence↗
PubMed1984

Immunolocalization of human cystatins in neutrophils and lymphocytes.

The endogenous inhibitors of cysteine proteinases, cystatin A and cystatin B, have been localized in sections of human liver by indirect immunofluorescence microscopy. Cystatin A was localized in neutrophils and cystatin B was found in lymphocytes. Both types of cell were present in the blood vessels and within the sinusoids between the parenchymal cells. Cystatin A was also found in some of the parenchymal cells surrounding the blood vessels but, in contrast to the uniform fluorescence of the leucocytes, the staining in the parenchymal cells was granular and rather irregular.

Animals↗