R. Carrell - Academia.edu (original) (raw)

Papers by R. Carrell

The Journal of Clinical Endocrinology & Metabolism, 2010

Background: Only 5% of circulating cortisol is active and unbound to carrier proteins. Because co... more Background: Only 5% of circulating cortisol is active and unbound to carrier proteins. Because cortisol levels vary rapidly due to the pulsatile nature of cortisol secretion, the dynamics of cortisol binding are critical determinants of tissue levels of free cortisol and consequent hormonal signaling. The major glucocorticoid carrier protein is corticosteroid binding globulin (CBG), a member of the serpin family that undergoes conformational changes to bind and release hormones. This mechanism has been noted to be temperature responsive, and we have now investigated the effects of temperature on the binding of human CBG to both cortisol and progesterone. Methods: Recombinant human CBG was synthesized and used for binding studies with cortisol and progesterone between 34 and 43 C. Binding was monitored by recording the change in intrinsic protein fluorescence. Binding of the steroids to the other major carrier, serum albumin, was measured in a similar manner. Results: There was no effect of temperature on the interaction between human serum albumin and either cortisol or progesterone. The association of both cortisol and progesterone with CBG is more than three orders of magnitude greater than that with HSA, and this interaction was extremely responsive to changes in temperature. The affinity of both cortisol and progesterone for CBG drops approximately 16-fold as temperature increases from 35 to 42 C. Conclusions: This study clearly shows that even within the clinically relevant range of temperatures found in humans, CBG acts as a protein thermocouple that is exquisitely sensitive to temperature change and will release cortisol in response to fever or external sources of heat. This has major implications for our understanding of cortisol regulation in febrile patients.

Journal of Liquid Chromatography, 1981

The application of high performance liquid chromatography on reversed-phase columns for the trypt... more The application of high performance liquid chromatography on reversed-phase columns for the tryptic mapping of haemoglobin variants is reported. The effect of flow rate and gradient shape on resolution and column efficiencies has been examined using acetonitrile-water-orthophosphoric acid elution systems. With these conditions it is possible to recognise sequence changes for variant haemoglobins including HbC, HbS, HbE, HbJ Cambridge and

Advances in Experimental Medicine and Biology, 1997

The development of our knowledge of the serpins illustrates the advantages of considering a prote... more The development of our knowledge of the serpins illustrates the advantages of considering a protein superfamily as a whole. The serpins have all retained a common tertiary structure despite the individual evolution of diverse functions; for example, the homology of the plasma protease inhibitor α1-antitrypsin is closer to that of corticosteroid binding globulin than is the homology of the two heparin-binding plasma inhibitors — antithrombin and heparin cofactor II — one to another. This retention of a well conserved structure necessarily requires the retention of strong homologies in primary and secondary structures in all the members of the family, across functions as well as species. For this reason, from the beginning, the study of the serpins has been a collective process with our understanding of the function of each member being greatly strengthened by parallel studies of other serpins. This has been particularly true of the lessons learnt from the human dysfunctional variants; one by one they have provided clues as to the normal function in individual members but when considered together with structural studies, in terms of the family as a whole, they have opened our understanding to a degree that far surpasses the contribution of more conventional approaches.

FEBS Letters, 1988

Antithrombin Glasgow is a hereditary abnormal antithrombin that has lost thrombin inhibitory acti... more Antithrombin Glasgow is a hereditary abnormal antithrombin that has lost thrombin inhibitory activity. It was isolated from the plasma of a 41-year-old male with a history of thrombotic events. Antithrombin Glasgow was purified from plasma using heparin-Sepharose chromatography at pH 7.4 eluting with increasing concentrations of NaCl. The normal protein eluted with 0.9 mol/l NaCl and Glasgow with 1.05 mol/l NaCI. Electrophoresis in agarose at pH 8.6 showed the variant to migrate more anodally than normal. The C-terminal small fragment resulting from catalytic cleavage with elastase between P, and P4 of the reactive loop was isolated and sequenced. This showed the replacement of the arginine at residue 3 by a histidine. This is residue 393 in the intact molecule. The findings suggest that heparin, on binding, interacts indirectly with the reactive centre region of antithrombin.

Biochemical Journal, 2003

Murine serpin 2A is expressed at high levels in haemopoietic progenitors and down-regulated on di... more Murine serpin 2A is expressed at high levels in haemopoietic progenitors and down-regulated on differentiation. When it is constitutively expressed in the multipotent haemopoietic cell line, FDCP-Mix, it causes a delay in differentiation and increased clonogenic potential. The serpin is also dramatically up-regulated on T-cell activation. It has an unusual reactive site Cys-Cys sequence, a unique C-terminal extension and lacks a typical cleavable N-terminal signal sequence. In spite of these features, the protein is not a member of the ovalbumin—serpin family, but is instead most closely related to human antichymotrypsin. We have shown that the serpin is intracellular with prominent nuclear localization. Transverse urea gradient gels and CD studies show that the protein undergoes the stressed—relaxed conformational change typical of inhibitory serpins. However, we have not detected complex-forming activity with a set of proteases. Thermal denaturation studies also show that the prot...

Biochimica et Biophysica Acta (BBA) - Protein Structure, 1974

Thrombosis and Haemostasis, 1997

Human α-1-antitrypsin (AT) was reacted with hydroxylamine at pH 9.0 giving cleavage at an Asn-Gly... more Human α-1-antitrypsin (AT) was reacted with hydroxylamine at pH 9.0 giving cleavage at an Asn-Gly bond. A fragment of molecular weight 8,500 was released and this was isolated and sequenced. The fragment had the same carboxy terminal amino acid sequence as intact ...

XIth International Congress on Thrombosis and Haemostasis

Four families with AT-III variants of decreased heparin affinity have been compared with families... more Four families with AT-III variants of decreased heparin affinity have been compared with families with a quantitative deficiency of AT-III. The affinity variants had a lower incidence of thrombosis and, unlike thedefiency variants, had no increase in plasma FDF (defined by anti-D-neo ELISA assay). These results support a major physiological role for the progressive activity of AT-III.The characterisation of the affinity variants provides information onthe heparin-binding site. Variants were isolated from plasma of heterozygotes by NaCl elution gradient from a heparin-Sepharose column. Maps of tryptic and SA V8 peptides were compared on TLC and HPLC with analysis of aberrant peptides. Confirmation was by gas-phase automated sequencing.Two variants were unequivocally identified.1) Rouen-I (47Arg→His) wasfoundin a heterozygote with a history of a single cerebral occlusion.2) Rouen-II (47Arg→Ser) was in a forty years old heterozygote with an acute myocardial infarction but no other hist...

Journal of Biological …, 1985

Blood Coagulation & Fibrinolysis, 1995

Three unrelated families have been identified with mutations involving asparagine 187. Two of the... more Three unrelated families have been identified with mutations involving asparagine 187. Two of these families are asymptomatic and were identified during the screening of random blood donors, whilst the third has a history of recurrent thromboembolic disease. In two families the mutation (6460 AAC-->GAC) results in an asparagine to aspartate substitution and is associated with normal immunological levels of antithrombin but a reduction in functional activity. In the third family the mutation (6462 AAC-->AAA) results in an asparagine to lysine substitution at residue 187 and is associated with a parallel reduction in both immunological and functional antithrombin levels. Asparagine 187 is located in the middle of the F helix of antithrombin and forms the major link between the F helix and strand 3 of the A sheet. The F helix is seen to overlie the A sheet of the molecule and moves with strands 2 and 3 of this sheet as they open to allow entry of the reactive site loop to form strand 4. Substitutions of asparagine 187 are, therefore, likely to disrupt this sliding movement leading to a loss of inhibitory activity.

… et Biophysica Acta (BBA …, 1984

Journal of Biological Chemistry, Apr 10, 1985

Using the glyceraldehyde-3-phosphate dehydrogenase promoter, nonglycosylated human alpha 1-protei... more Using the glyceraldehyde-3-phosphate dehydrogenase promoter, nonglycosylated human alpha 1-proteinase inhibitor, representing 10% of the soluble cell protein, has been synthesized in yeast. Two forms of this protein were isolated with one being analogous to the human plasma protein and the other having the amino acid valine replacing methionine at position 358 (the P1 position). Both proteins were more sensitive to heat inactivation than the plasma form, and both had shorter half-lives in rabbits. These differences were presumably due to the absence of carbohydrate. Each protein could bind neutrophil elastase at a rate only slightly slower than that of human plasma alpha 1-proteinase inhibitor. However, the valine variant was stable to oxidation, while the P1 methionine-containing protein was readily inactivated. The specificity of alpha 1-proteinase inhibitor (methionine) was identical to that of the plasma form; however, the valine form could only effectively bind to neutrophil or...

Pathology, 1977

An investigation of the cord blood from full term twin infants revealed an additional haemoglobin... more An investigation of the cord blood from full term twin infants revealed an additional haemoglobin F component due to an abnormal alpha chain. The father of the twins, whose blood picture was normal, was shown to have normal alpha and beta polypeptide chains together with variant alpha and beta polypeptide chains. Electrophoresis showed that he had four major haemoglobin components. Separation of the haemoglobin fractions by column chromatography, globin preparation, chain separation, tryptic and chymotryptic digestion and peptide map preparation led to the identification of haemaglobin A, haemoglobin Strumica (alpha2 112His leads to Arg beta2), haemoglobin D Beograd (alpha2beta2 121 Glu leads to Val) and a hybrid haemoglobin Strumica D/Beograd (alpha2 112His leads to Argbeta2 121Glu leads to Val).

FACTORS XII AND XI

A number of the key inhibitors of coagulation and fibrinolysis have recently been shown to be mem... more A number of the key inhibitors of coagulation and fibrinolysis have recently been shown to be members of the same superfamily of serine protease inhibitors, the serpins. The archetypes of the group are alpha-l-antitrypsin and antithrombin and it includes antiplasmin, C1-inhibitor, heparin cofactor II and the newly recognised inhibitors of plasminogen activators and activated Protein C. Alignment of their structures shows that they have the same skeletal three-dimensional conformation and, by inference, the same general function mechanisms.The serpins have a reactive centre, primarily dependent on a single amino acid, exteriorly placed on a stressed peptide loop. This functions by offering the cognate protease a high-affinity substrate that resists complete cleavage to form a tight 1:1 complex of inhibitor and protease that is subsequently removed from the circulation. The loop is vulnerable to cleavage with resulting loss of inhibitory activity. This irreversible switch is utilised:...

Biochimica et Biophysica Acta (BBA) - Protein Structure, 1977

ABSTRACT

Trends in Biochemical Sciences, 1986

Blood, Jan 15, 1999

Antithrombin is shown to undergo a slow spontaneous conversion to its inactive latent conformatio... more Antithrombin is shown to undergo a slow spontaneous conversion to its inactive latent conformation with readily discernible amounts present in plasma on incubation at 37 degrees C for 72 hours. More rapid conversion occurs on incubation of isolated antithrombin at 41 degrees C or 50 degrees C, but the appearance on electrophoresis of free latent antithrombin is preceded by the formation, in reciprocal proportions, of a new slow band. This slow component is shown to be a heterodimer of active and latent antithrombin. It can be isolated as a single stable band either by incubation of antithrombin or by mixing equimolar proportions of active and latent antithrombin under the same conditions that give overnight crystallization of the active/latent antithrombin heterodimer. Similarly, equimolar addition of latent antithrombin to plasma results electrophoretically in a quantitative shift to the slower heterodimer mobility. Clinically, the presence of latent antithrombin is potentially del...