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Biomedical subjects

A J Barrett

Publications and source records attributed to A J Barrett.

At least 307 records · Page 17Linked to original sources
PubMed1982

Evolution of alpha 2-macroglobulin. The demonstration in a variety of vertebrate species of a protein resembling human alpha 2-macroglobulin.

Plasma or serum samples from a large number of vertebrate species were screened for the presence of a papain-binding protein resembling human alph a 2-macroglobulin (alpha 2M). The screening method depended on the unique property of alpha 2M of binding proteinases in such a way that the enzyme retains partial activity against low-molecular-weight substrates. A papain-binding protein was detected in serum from members of all the major vertebrate taxa. In mammals, birds, reptiles and amphibians the protein had an Mr similar to that of human alpha 2M (725 000), but in fish, including dipnoans, actinopterygians, elasmobranchs and cyclostomes, the papain-binding protein was of Mr about 360 000. Of the invertebrate species tested, all of which were arthropods, two were negative, but the horseshoe crab, an arachnid, did possess a papain-binding protein, although this was heterogeneous in electrophoresis and differed from alpha 2M in resisting inactivation by methylamine. From the results, and a detailed study of the properties of the fish papain-binding protein described in an accompanying paper [Starkey, Fletcher & Barrett (1982) Biochem. J. 205, 97-104], it seems that alpha 2M first appeared in an ancestor of all modern vertebrates as a protein of Mr 360 000 and that the larger macroglobulin (Mr 725 000) first appeared in an ancestor of the tetrapods.

Animals↗
PubMed1982

Evolution of alpha 2-macroglobulin. The purification and characterization of a protein homologous with human alpha 2-macroglobulin from plaice (Pleuronectes platessa L.) plasma.

A papain-binding protein (PB-protein) was purified to homogeneity from the plasma of plaice (Pleuronectes platessa L.). PB-protein inhibited the activity of trypsin and pancreatic elastase (serine proteinases), thermolysin (a metalloproteinase) and papain (a cysteine proteinase). Presaturation of PB-protein with trypsin prevented the subsequent inhibition of thermolysin, and vice versa. Only catalytically active endopeptidases were bound by PB-protein. The catalytic activity of trypsin bound by PB-protein was inhibited by 95% against an insoluble protein substrate, but only by 38% against a low-molecular-weight synthetic substrate. The remaining activity of the bound trypsin was partially protected against further inhibition by soya-bean trypsin inhibitor. Trypsin bound by PB-protein showed a decrease of 67% in its reactivity with antibodies. The inhibitory activity of PB-protein was inactivated at pH 8.0 by methylamine (0.2M) or dithiothreitol (1 mM). The inhibition of proteinases by plaice PB-protein shows the distinctive characteristics of inhibition by human alpha 2-macroglobulin, and it is concluded that the plaice protein is a homologue of the human macroglobulin.

Animals↗
PubMed1982

Evolution of alpha 2-macroglobulin. The structure of a protein homologous with human alpha 2-macroglobulin from plaice (Pleuronectes platessa L.) plasma.

The plaice (Pleuronectes platessa L.) papain-binding protein previously demonstrated to be homologous with human alpha(2)-macroglobulin, and designated plaice alpha(2)-macroglobulin homologue or alphaMh, was shown to be a glycoprotein of s(20,w) 11.86S. In polyacrylamide-gel pore-limit electrophoresis under non-denaturing conditions plaice alphaMh migrated to the same position as half-molecules of human alpha(2)-macroglobulin, and treatment with methylamine or a proteinase caused no change in its electrophoretic properties. Either denaturation in urea (4m) or mild reduction by dithiothreitol (1mm) partially dissociated plaice alphaMh into half-molecules. Denaturation with reduction further dissociated the protein into quarter-subunits. In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions plaice alphaMh dissociated into subunits of M(r) 105000 (I) and 90000 (II). Approximately equal amounts of each subunit were formed, and peptide ;mapping' showed subunits I and II to be distinct polypeptide chains. Under alkaline denaturing conditions, a proportion of the I chains of alphaMh were cleaved into fragments of M(r) about 60000 and 40000. This cleavage was favoured by reducing conditions and prevented by prior inactivation of the alphaMh with methylamine. [(14)C]Methylamine allowed to react with alphaMh became covalently linked to subunit I. These properties suggested the existence of an autolytic site on subunit I analogous to the autolytic site of human alpha(2)-macroglobulin. Reaction of alphaMh with a proteinase resulted in cleavage of a fragment of M(r) 10000-15000 from subunit I. A proportion of the proteinase molecules trapped by alphaMh became covalently linked to the inhibitor. A scheme is proposed for the evolution of human alpha(2)-macroglobulin and plaice alphaMh from a common ancestral protein, which may also have been an ancestor of complement components C3 and C4.

Amino Acid Sequence↗
PubMed1982

Identification of plasma kallikrein as an activator of latent collagenase in rheumatoid synovial fluid.

Rheumatoid synovial fluid contains an activator of latent collagenase from culture medium of pig synovium. The activator was purified by gel chromatography on Ultrogel AcA 44 and affinity chromatography on soybean trypsin inhibitor coupled to Sepharose 4B. The purified material was homogeneous on SDS-polyacrylamide gel electrophoresis with Mr 88 000. The activator had limited proteolytic activity against azo-casein, but showed amidase activity on Pro-Phe-Arg-NMec, Z-Phe-Arg-NMec, D-Val-Leu-Arg-NPhNO2 and D-Pro-Phe-Arg-NPhNO2, with an optimum at pH 8.0. Activity was completely inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor, leupeptin and Pro-Phe-Arg-CH2Cl, whereas lima bean trypsin inhibitor, Tos-Lys-CH2Cl, a specific inhibitor of factor XIIa from maize, EDTA and iodoacetate were not inhibitory. These properties of the activator suggested that it might be plasma kallikrein (EC 3.4.21.34), and the possibility was further examined. The activator was treated with [3H]diisopropyl fluorophosphate, and run in SDS-polyacrylamide gel electrophoresis with reduction; a radioautograph of the gel showed a pair of [3H]diisopropyl phosphoryl-labelled bands (Mr 36 000 and 34 000) identical to those obtained with authentic plasma kallikrein. Double immunodiffusion with monospecific antiserum against human plasma kallikrein confirmed the identification. This is the first demonstration of collagenase-activating activity of plasma kallikrein, and raises the possibility that activation of prokallikrein in the inflamed joint space may contribute to the disease process not only by the production of bradykinin, but also by activating latent collagenase.

Animals↗
PubMed1982

Evidence that extracellular cathepsin D is not responsible for the resorption of cartilage matrix in culture.

Cathepsin D, the major lysosomal aspartic proteinase, is responsible for the autolysis of cartilage at slightly acidic pH, and it has been suspected of making a significant contribution to the breakdown of the living tissue, such as in stimulated by retinol. Our finding, however, has been that neither inhibitory antibodies against cathepsin D, nor chemical inhibition with pepstatin, significantly decreases the rate of degradation of proteoglycan in the organ culture system. Most of the other proteinase inhibitors tested were similarly ineffective, although the EDTA and 1,10-phenanthroline inhibited the resorption by a cytotoxic effect. We conclude that although cartilage matrix degradation has clear characteristics of proteolytic process, the identity of the enzyme(s) responsible remains obscure.

Animals↗
PubMed1982

Action of rat liver cathepsin L on collagen and other substrates.

1. It has been found that cathepsin L is very susceptible to loss of activity through autolysis. When this is prevented by purification and storage of the enzyme as its mercury derivative, preparations are obtained with higher specific activity than previously. 2. Active-site titration shows, however, that even the new purification method does not give preparations in which the enzyme is 100% active. 3. Benzyloxycarbonylphenylalanylarginine 7-(4-methyl)coumarylamide has been discovered to be a very sensitive substrate for cathepsin L. Like all other known substrates for cathepsin L, however, it is also cleaved by cathepsin B. 4. Cathepsin L degrades insoluble collagen at pH 3.5 over 5-fold faster than at pH 6.0. The specific activity at pH 3.5 is 5-10-fold higher than that of cathepsin B (rat or human) or bovine spleen cathepsin N ('collagenolytic cathepsin'). 5. Qualitatively, the action of cathepsin L on collagen is similar to that of cathepsins B and N, i.e. selective cleavage of terminal peptides leads to conversion of beta- and higher components mainly to alpha-chains.

Animals↗
PubMed1982

L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L.

1. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) at a concentration of 0.5 mM had no effect on the serine proteinases plasma kallikrein and leucocyte elastase or the metalloproteinases thermolysin and clostridial collagenase. In contrast, 10 muM-E-64 rapidly inactivated the cysteine proteinases cathepsins B, H and L and papain (t0.5 = 0.1-17.3s). The streptococcal cysteine proteinase reacted much more slowly, and there was no irreversible inactivation of clostripain. The cysteine-dependent exopeptidase dipeptidyl peptidase I was very slowly inactivated by E-64. 2. the active-site-directed nature of the interaction of cathepsin B and papain with E-64 was established by protection of the enzyme in the presence of the reversible competitive inhibitor leupeptin and by the stereospecificity for inhibition by the L as opposed to the D compound. 3. It was shown that the rapid stoichiometric reaction of the cysteine proteinases related to papain can be used to determine the operational molarity of solutions of the enzymes and thus to calibrate rate assays. 4. The apparent second-order rate constants for the inactivation of human cathepsins B and H and rat cathepsin L by a series of structural analogues of E-64 are reported, and compared with those for some other active-site-directed inhibitors of cysteine proteinases. 5. L-trans-Epoxysuccinyl-leucylamido(3-methyl)butane (Ep-475) was found to inhibit cathepsins B and L more rapidly than E-64. 6. Fumaryl-leucylamido(3-methyl)butane (Dc-11) was 100-fold less reactive than the corresponding epoxide, but was nevertheless about as effective as iodoacetate.

Binding Sites↗
PubMed1982

Bone marrow transplantation for acute lymphoblastic leukaemia.

Twenty patients have undergone bone marrow transplantation for acute lymphoblastic leukaemia (ALL). Eighteen patients were in complete remission and two had less than 10% leukaemic blasts in the marrow aspirate in the week prior to transplantation. Eighteen patients were grafted from HLA/MLC compatible siblings. One identical twin and one fully compatible parent were also used as donors. Two patients died of graft-versus-host disease and one of radiation-induced pneumonitis. Four have subsequently relapsed. Thirteen patients remain well and in remission from 202 to 1126 d post transplantation. These results show that morbidity and mortality from the bone marrow transplantation procedure is low. However, the major obstacle to permanent success in marrow transplantation of patients with ALL is recurrence of the disease (35% actuarial disease free survival at 1126 d).

Adolescent↗
PubMed1982

Haematopoietic stimulators in the serum of patients with severe aplastic anaemia.

Recent studies on sera from patients with aplastic anaemia show it to contain both stimulators and inhibitors of granulopoiesis. We have examined the serum of patients with aplastic anaemia in a short-term liquid culture system of human bone marrow which permits CFU-C proliferation. Increases in CFU-GM numbers over control values were observed with aplasia sera. All sera produced increased megakaryopoiesis, but no alteration in growth of the fibroblasts and macrophages of the adherent stromal layer. With one exception the sera had no significant direct colony stimulating activity when assayed in agar culture, but increased colony numbers were observed when the sera was incorporated into a sub-optimal feeder layer of peripheral blood leucocytes. When aplastic serum was added to adherent cell-depleted liquid cultures, the CFU-C stimulating property was lost, but increased megakaryopoiesis still occurred. These results indicate the presence of a direct-acting thrombopoietin and an indirect-acting stimulator of CFU-C proliferation in the serum of patients with aplastic anaemia, which may be related to the pathophysiology of the condition.

Adolescent↗
PubMed1982

Plasma arginine esterase in cystic fibrosis: kinetics of activation, identification as plasma kallikrein, reaction with alpha 2-macroglobulin and comparison with levels in normal plasma.

Treatment of normal plasma with chloroform and ellagic acid yielded esterase activity against tosylarginine methyl ester, which reached a maximum within 2 h. After 2 h most or all of the activity as resistant to inhibition by soybean trypsin inhibitor (STI), but was still sensitive to the low molecular weight inhibitors di-isopropyl fluorophosphate, aprotinin and prolyl-phenylalanyl-arginyl chloromethane. The activity ran in gel chromatography with alpha 2 macroglobulin (ampha 2M), as if it were due to an alpha 2M proteinase complex. The generation of the arginine esterase activity by chloroform and ellagic acid was apparently dependent on the activation of factor XII, being blocked by Polybrene. In plasma pretreated with methylamine-HCl (an inactivator of alpha 2M), the arginine esterase was 95% sensitive to inhibition by STI. With regard to substrate specificity, inhibition characteristics, and gel chromatographic behaviour, it was indistinguishable from plasma kallikrein (EC 3.4.21.34, formerly 3.4.21.8). The chloroform and ellagic acid treatment of plasma resulted in a disappearance of prokallikrein simultaneous with the appearance of the arginine esterase. By these criteria, the arginine esterase activity was attributable entirely to plasma kallikrein either in its free form (methylamine-treated plasma) or bound to alpha 2M (buffer-treated plasma). Comparisons of STI-sensitive and STI-resistant arginine esterase activities of plasma samples from cystic fibrosis patients, obligate heterozyotes or other groups showed no significant differences in levels of activity, kinetics of activation or gel chromatographic behaviour. We conclude that cystic fibrosis is unrelated to any abnormality in plasma arginine esterase activity, contrary to some previous reports.

Carboxylic Ester Hydrolases↗
PubMed1982

A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures.

A rapid spectrophotometric procedure is described for the estimation of sulfated glycosaminoglycans in cartilage cultures. Papain digestion of tissue or culture medium provides glycosaminoglycans in solution for assay; an aliquot of the digest is mixed with the dye 1,9-dimethylmethylene blue. The assay is based on the metachromatic shift in absorption maximum which occurs when the dye is complexed with sulfated glycosaminoglycans. The reagent is stable, and the method is substantially free from interference, is sensitive to less than 1 microgram (4 micrograms/ml) of chondroitin sulfate, and provides a simple alternative to the traditional methods for glycosaminoglycan determinations.

Animals↗
PubMed1981

Reversal of clinical features of Hurler's disease and biochemical improvement after treatment by bone-marrow transplantation.

A one-year-old boy with type I H mucopolysaccharidosis (Hurler's disease) was given a bone-marrow transplant (BMT) from his mother in an attempt to replace the deficient enzyme, alpha-L-iduronidase (iduronidase). These is definite evidence of engraftment, the enzyme activity of the recipient's leucocytes reaching heterozygote levels within 37 days of the BMT. Graft-versus-host disease (GVHD) developed but was partially controlled by steroids. From 3-4 months after graft until the present (13 months after the graft) iduronidase activity has been present in the serum and the urine and there has been evidence of considerable degradation of glycosaminoglycans excreted in the urine. The hepatosplenomegaly has disappeared, corneal clouding has cleared, and deterioration in the child's development seems to have been arrested.

Aminoacridines↗
PubMed1981

The effects of leucocyte elastase on the mechanical properties of adult human articular cartilage in tension.

The effects of leucocyte elastase on the tensile properties of adult human articular cartilage were examined in detail in 99 specimens from hip, knee and ankle joints in the age range 16-83 years. The results showed that elastase reduced the tensile stiffness of cartilage, both at low stress and at fracture. The tensile strength of cartilage was also considerably reduced by the action of elastase. Biochemical analysis of the incubation media, and the specimens, revealed that 90%, or more, of the proteoglycan was released from the cartilage, whilst the release of collagen was negligible. Leucocyte elastase is known to degrade the non-helical terminal peptides of cartilage collagen molecules and thereby disrupt the main intermolecular cross-links in collagen fibrils. A previous study (Kempson, G.E., Tuke, M.A., Dingle, J.T., Barrett, A.J. and Horsfield, P.H. (1976) Biochim. Biophys. Acta 428, 741-760) showed the lack of effect of proteoglycan degradation alone on the tensile strength and stiffness of cartilage. The reduction in strength and stiffness recorded in the present study can, therefore, be attributed to the action of elastase on the collagen in cartilage and it emphasises the important of covalent intermolecular cross-links to the mechanical properties of collagen fibrils.

Adolescent↗