Lawrie Austin: the man and the scientist.
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Australia↗
Biomedical subjects
M T Hearn
Publications and source records attributed to M T Hearn.
Duchenne muscular dystrophy and dystrophin: sequence homology observations.
Duchenne muscular dystrophy (DMD) is a genetically transmitted disease characterized by progressive muscle weakness and usually leads to death. DMD results from the absence, deficiency or dysfunction of the protein dystrophin. Analysis of protein data bases, including homology alignments and domain recognition patterns, have located highly significant correlations between dystrophin and other calcium regulating proteins. In particular, a major portion of the dystrophin sequence has been found to contain repeating units of approximately 100 amino acid residues. These repeating units were found to exhibit significant homology to troponin I. Troponin I has been found to bind to the calcium binding proteins calmodulin and troponin C. The regions of highest homology were characterized by patterns of high localization of charged amino acids and thus could represent a possible calmodulin or troponin C surface accessible binding site. Since subcellular localization studies have indicated that dystrophin is associated with the triadic junction, these findings imply that dystrophin could be involved in controlling intracellular calcium homeostasis.
Amino Acid Sequence↗
Structure and function of the heparin-binding (fibroblast) growth factor family.
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Amino Acid Sequence↗
Rapid isolation of rat brain nuclei on percoll gradients.
A rapid isotonic method for fractionation of nuclei from rat brain is described. This procedure is based on the use of discontinuous colloidal silica gel (Percoll) gradients. We start from a 63,000-g purified nuclear pellet (fraction P3) isolated from gray matter and white matter separately. This is followed by fractionation of fraction P3 in an initial differential centrifugation step on five-step Percoll gradients producing six nuclear fractions designated 1, 2, 3 (gray matter) and 4, 5, 6 (white matter). Fractions 2, 4, and 5 obtained from this centrifugation are heterogeneous. These fractions are subfractionated further under isopycnic conditions using five-step Percoll gradients to yield subfractions 2b, 4b, and 5c. Three methods were used to characterize the nuclear types. First, light and electron microscopic examination was used to identify the nuclei in each preparation and to assess the purity of each preparation. Second, the activities of RNA polymerase I and II were monitored. Third, the protein/DNA ratios of the nuclear fractions were determined. Fraction 1 was enriched in neuronal nuclei; fractions 2b and 4b in astrocytic nuclei; and fractions 3, 5c, and 6 in nuclei of oligodendrocytes. RNA polymerase I and II activity was highest in fraction 1, which also displayed the highest protein/DNA ratio. Electron microscopy showed that the various classes of nuclei are congruent to 90% pure. Therefore, the procedure described here is suitable for obtaining highly purified neuronal and three types of glial nuclei from rat brain.
Animals↗
High-performance liquid chromatography of amino acids, peptides and proteins. C. Characterisation of coulombic interactive regions on hen lysozyme by high-performance liquid anion-exchange chromatography and computer graphic analysis.
The molecular characteristics of the dominant anion-exchange binding site of hen egg white lysozyme (HEWL) has been investigated using a combination of high-performance liquid chromatographic techniques and computer graphic analysis of the X-ray crystallographic structure. These studies have indicated that the site of highest electrostatic potential, in terms of the density of negatively charged amino acid side chains, is located around the catalytic cleft area. The four residues tentatively identified to be involved in the electrostatic binding domain were aspartic acid 48, 52, 101 and glutamic acid 35. The number of these charged groups correlated with the maximum value of the chromatographically determined retention parameter (Zc value). Variations in the range of experimental Zc values obtained under different elution conditions have been interpreted in terms of conformational flexibility of the structural domains of HEWL which result in the opening or closure of the catalytic cleft during the retention process.
Amino Acid Sequence↗
Evaluation of factors which affect column performance with immobilized monoclonal antibodies. Model studies with a lysozyme-antilysozyme system.
Methods are described for the automated evaluation of affinity columns by frontal boundary analysis. These methods were used to evaluate the performance of immunoaffinity columns based on antilysozyme monoclonal antibody-lysozyme immunoaffinity system. This model system enabled the effects of (i) matrix activation and (ii) the density of immobilized antibody on the change in specific activity of immobilized antibody to be quantitatively assessed. Experimental data were accumulated with carbonyldiimidazole-activated Fractogel HW65F and Trisacryl GF2000 resins and cyanogen bromide-activated Sepharose 4B. An increase in the molar ratio between the concentration of the active groups on the activated matrix and the concentration of immobilized antibody ligands did not result in significant change in the specific activity of the immobilized antibody in the immunochromatographic system. However, increased antibody density with the Fractogel HW65F resin resulted in an increase in the apparent heterogeneity of antibody binding sites for lysozyme and a significant decrease in the specific activity of the immobilized antibody. Furthermore, data from size-exclusion studies with these immunoaffinity matrices demonstrated that at high antibody densities, the accessibility of the immobilized antibody was further decreased due to steric resistance as the antigen size increased.
Animals↗
High-performance liquid chromatography of amino acids, peptides and proteins. XCVIII. The influence of different displacer salts on the bandwidth properties of proteins separated by gradient elution anion-exchange chromatography.
The influence of eight different displacer salts on the bandwidth properties of four globular proteins separated by a high-performance anion-exchange chromatography has been investigated. Proteins were eluted under gradient conditions with a range of alkali metal halide salts, in which the anion and cation were varied in the series F-, Cl- and Br- and Li+, Na+ and K+, respectively. The experimentally observed bandwidths (sigma v,exp) were found to deviate significantly from peak widths (sigma v,calc) predicted on the basis of plate theory for small molecules. For data accumulated under conditions of varied gradient time and constant flow-rate the solute bandwidth ratios (sigma v,exp/sigma v,calc) increased in the order Br- less than Cl- less than F- at low values of the gradient steepness parameter, b, or increasing column residence times. In addition, systematic changes in the cation influenced the bandwidth ratios (sigma v,exp/sigma v,calc) in the order K+ less than Na+ less than Li+. Significant deviations between predicted and observed bandwidth values were also observed under elution conditions of constant gradient time and varied flow-rate. The results of the present study further demonstrate the complex nature of the interaction between protein solutes and coulombic chromatographic surfaces.
Anions↗
High-performance liquid chromatography of amino acids, peptides and proteins. XCIX. Comparative study of the equilibrium refolding of bovine, porcine and human growth hormone by size-exclusion chromatography.
The equilibrium refolding of bovine, porcine and human growth hormone and ovine prolactin in guanidine hydrochloride has been investigated using high-performance size-exclusion chromatography (HPSEC). It was found that bovine and porcine growth hormones exhibited very similar refolding behaviour. However, the renaturation of human growth hormone followed a different pathway. In particular, the folding transition of human growth hormone occurred at 4.7 M guanidine hydrochloride compared to 3.8 and 3.5 M for the bovine and porcine molecules, respectively, and 3.5 M for ovine prolactin. The refolding mechanism of an internally clipped fragment derived from partial tryptic digestion, exhibited similar folding properties to the corresponding intact molecule. The internally clipped analogue existed as a relatively larger molecule under fully denaturing conditions. Reduction followed by carboxymethylation resulted in growth hormone molecules with significantly reduced stability and altered folding properties. The results have been correlated with differences in structure to further demonstrate the utility of HPSEC in the study of protein folding and stability.
Alkylation↗
High-performance liquid chromatography of amino acids, peptides and proteins. CIII. Mass transfer resistances in ion-exchange and dye-affinity chromatography of proteins.
Adsorption equilibria and rate kinetics have been investigated for the binding of several proteins, with different molecular geometries, to several ion-exchange and dye-affinity chromatographic resins with varying pore size and protein accessibilities. The pore geometry was shown to play a significant role in the protein capacity and loadability of both the ion-exchange and dye-affinity resins. For example the Fractogel HW75-Cibacron Blue F3GA affinity sorbent had the greatest capacity for the small protein, lysozyme, compared to the other Fractogel HW-Cibacron Blue F3GA sorbents, and similarly, the ion-exchange resins, such as DEAE-Fractogel 65, bound more human serum albumin (HSA), as opposed to the larger protein, ferritin. The apparent diffusion of protein from the bulk phase to the ligands/ionic sites was calculated to be considerably restricted when the pore to protein size ratio was small, as is the case of DEAE Fractogel 65/ferritin system, and the dye-affinity Fractogel HW55/HSA system. In these circumstances, pore diffusivity was calculated to be up to 100-fold smaller than bulk diffusivity.
Adsorption↗
High-performance liquid chromatography of amino acids, peptides and proteins. XCVII. The influence of the gradient elution mode and displacer salt type on the retention properties of closely related protein variants separated by high-performance anion-exchange chromatography.
The influence of different elution modes, gradient times and flow-rates on the relative retention of closely related variants of carbonic anhydrase and ovalbumin has been investigated using high-performance ion-exchange chromatography. Three isoform species of carbonic anhydrase and four isoforms related to ovalbumin eluted by anion-exchange chromatography were characterised by isoelectric focusing and sodium dodecylsulphate-polyacrylamide electrophoresis. Gradient retention data were collected using several different alkali metal halides as the displacer salt, in order to systematically evaluate the effect on selectivity of different anions and cations in the series F-, Cl- and Br-, and Li+, Na+ and K+. While the selectivity between the different ovalbumin isoform species remained essentially constant with each displacer salt, solute Zc-values [J. Chromatogr., 458 (1988) 27] varied with the type of salt. In contrast, non-parallel retention plots were obtained for the carbonic anhydrase isoforms with the Zc values different for each isoform. Furthermore, significant differences in chromatographic behaviour for these proteins were observed between experiments carried out under gradient elution conditions with either varied gradient time and constant flow-rates or fixed gradient time and varied flow-rates. These results are discussed in terms of the influence of column residence time and protein-salt interactions of the solute's interactive ionotope and the concomitant effects these structural perturbations may have on chromatographic behaviour.
Amino Acids↗
High-performance liquid chromatography of amino acids, peptides and proteins. XCV. Thermodynamic and kinetic investigations on rigid and soft affinity gels with varying particle and pore sizes: comparison of thermodynamic parameters and the adsorption behaviour of proteins evaluated from bath and frontal analysis experiments.
The thermodynamic constants, associated with the interaction of three proteins with triazine dye affinity sorbents, have been derived from bath and frontal analysis experiments. In cases where mass-transfer restrictions are very high, calculation of the thermodynamic constants directly from frontal analysis experiments could not be achieved. In such cases, a portion of the adsorbate was always present in the effluent, a situation which has its effect as the split peak phenomenon. With Fractogel-based triazine dye affinity sorbents none of the test proteins applied in frontal analysis were adsorbed. A similar behaviour was observed for a Cellufine sorbent during the adsorption of human serum albumin and the Blue Sepharose CL6B sorbent during the adsorption of alcohol dehydrogenase, which displayed much slower apparent adsorption kinetics than observed in the bath experiments. These phenomena were shown to be associated with changes in the gel structure, caused in part by the column packing procedure. Silica-based sorbents performed better in the adsorption of lysozyme in the column mode than soft-gel affinity sorbents, as was evident in the higher capacities and steeper breakthrough curves. At high protein concentrations (feedstock concentration greater than 0.2 mg/ml) breakthrough curves obtained with small- and large-particle-size sorbents, but of constant pore size, were found to be identical. This finding demonstrates that the use of small-particle-size sorbents (e.g. particle diameter, dp less than or equal to 5 microns) for the preparative isolation of proteins may not be justified when operating in the overload mode. With other higher-molecular-weight proteins and the silica-based sorbent systems examined, the small-particle-size sorbents (dp = 5 microns) displayed less symmetrical shapes of their breakthrough curves than the larger-particle-size and soft-gel sorbents. This behaviour was further exacerbated when non-porous glass or silica-based sorbents were utilized. These non-porous affinity sorbents displayed nearly rectangular breakthrough shapes at the onset of the adsorption process, but comparatively slow adsorption kinetics became evident as saturation was approached. This phenomenon has been attributed to surface rearrangement and/or reorientation of the adsorbed proteins, particularly with sorbents of high ligand densities.
Amino Acids↗
The effect of hysterectomy and bilateral oophorectomy in women with severe premenstrual syndrome.
The etiology of premenstrual syndrome is unknown, although this syndrome is linked to the menstrual cycle. Fourteen women with severe, debilitating premenstrual syndrome volunteered for a study of therapy by hysterectomy, oophorectomy, and continuous estrogen replacement. All had completed their families and had failed to benefit from previous medical treatment. The diagnosis and severity of premenstrual syndrome were assessed by means of prospective charting and psychological evaluation. All patients had clearly cyclic symptoms and psychological scores consistent with a major disruption of their lives before surgery. Six months after surgery, premenstrual syndrome symptom charting revealed complete disappearance of a cyclic pattern with scores equivalent to those of a normal population. Psychological measures 6 months after operation showed dramatic improvement in mood, general affect, well-being, life satisfaction, and overall quality of life. Surgical therapy, involving oophorectomy, hysterectomy, and continuous estrogen replacement, is effective in relieving the symptoms of premenstrual syndrome and is indicated for a small, selected group of women.
Adult↗
Psychological assessment and follow-up after in vitro fertilization: assessing the impact of failure.
The present study was conducted to assess the immediate psychological impact of failed in vitro fertilization (IVF). Emotional status and marital functioning were also examined pre-IVF, and both demographic information and psychological test scores were evaluated as predictors of reaction to treatment failure. After a failed first cycle, both males and females showed significant increases in anxiety and depressive symptoms. Although group means were not clinically elevated and most participants were coping adequately, the prevalence of both mild and moderate depression increased substantially, particularly among women. In addition, women without children were a subgroup particularly vulnerable to the stress of failure. Predisposition towards anxiety, pre-IVF depressive symptoms, and fertility history were the most important predictors of emotional response. Treatment implications of these findings were discussed.
Adult↗
Protein phosphorylation in intact superior cervical ganglion during regeneration.
The incorporation of radioactive phosphate into proteins of both normal and regenerating superior cervical ganglion nerve of the rat is reported. Incorporation studies carried out by in vitro and in vivo methods are compared. In the in vitro method, excised intact ganglia or their homogenates were incubated in the presence of inorganic phosphate or ATP, respectively, under various conditions. Proteins were analyzed by gel electrophoresis followed by autoradiography, in which quantitative but not qualitative differences between regenerating and control cases were apparent. In the in vivo procedure, inorganic phosphate was injected into the living animal 4 h before removal of ganglia. At least fivefold more proteins became labeled in vivo than in vitro, whereas no similarity in the pattern of labeling between the two methods was observed. For example, the most heavily labeled protein in the in vivo method, tentatively identified as microtubule-associated protein-2, was not detected on autoradiograms of proteins labeled by the in vitro method. In this latter method, an 85-kDa species and growth-associated protein-43 were always labeled, and the extent of their phosphorylation was enhanced by the additional presence of phosphatidylserine and Ca2+, a result indicating that these labeled species are substrates of protein kinase C. The in vitro conditions also led to the labeling of proteins identified as alpha- and beta-tubulin. Comparison of the methods suggests that removal of the ganglion interferes with the function of protein phosphorylation systems and that this effect involves elements of the cytoskeleton.
Adenosine Triphosphate↗
Physicochemical and biological characterization of recombinant human inhibin A.
Recombinant human inhibin A was isolated from recombinant mammalian cell line culture media. Two forms of inhibin were identified with Mr of 34 and 31 Kd composed of subunits (alpha, beta) of 24 and 15 Kd and 21 and 15 Kd respectively. Both forms are bioactive in an inhibin in vitro bioassay and immunoactive with potencies comparable to or higher than purified bovine inhibin. Amino acid analyses and NH2-terminal sequences of each of the subunits are consistent with those predicted from their cDNA structures. The inhibin alpha- but not beta-subunit is glycosylated based on its binding to the lectins concanavalin A and wheat germ lectin. The difference in molecular weight of 31 and 34 Kd inhibin is attributed to variation in glycosylation of the alpha-subunit. The 31+34 Kd inhibin is heterogeneous on isoelectric focusing gels consisting of four isoforms in the pH range 6.2-7.6. Inhibition also exhibits in vivo biological activity by suppressing serum FSH but not LH in castrate male rats. These physicochemical and biological characteristics of recombinant human inhibin are similar to those described for native inhibin isolated from a variety of other species.
Amino Acid Sequence↗
Proteins as biological effectors.
This chapter presented an overview of the role of proteins as biological effectors. From a simplistic point of view, based solely on comparison of the structural diversity of immunoglobulins, blood coagulation proteins, gonadotropins, growth factors, and antiproteins, it could be concluded that the functional mechanisms of these protein families bear little or no relationship. Despite this enormous divergency in structure-function relationship, there are in fact elements of commonality in their effector roles arising as a direct consequence of the ability of these classes of protein effectors to act as exquisite examples of the processes of biorecognition. All these case histories, and the numerous other familial case studies of protein effectors which could have been employed to illustrate the different functional roles of proteins, owe their biological properties to their primordial protein antecedents which have traversed the harsh wilderness of evolution in biorecognition phenomena and survived to elicit specific effector roles. Dictated by underlying physicochemical constraints, deceived at times by the lulling tones of the siren entropy, and constantly vulnerable to the vagaries of other more pervasive forms of biological networking and information transfer encoded in the genes of virus and invading microorganisms, protein biorecognition in higher life forms, and particularly in mammals, represents the finely tuned molecular avenues for the genome to transfer its information to the next generation. The examples summarized in this chapter illustrate the complex, and in disease states imperfect, functional potential of proteins to be manifested in the jigsaw of biorecognition and be realized in the network of nature's biological effectors. Proteins thus represent a diverse range of effector molecules whose properties are totally dependent on their conformational or topographic status. The three-dimensional structure defines active sites on the molecule through which intermolecular interaction and biorecognition phenomena can occur. The cipher for this surface topography is, of course, coded in the primary amino acid sequence. Much experimental work is being directed in these and other laboratories at elucidating the principles governing the folding of unique peptide sequences into three-dimensional structures. Further advances in the theoretical understanding of the thermodynamics of protein folding as observed by x-ray crystallography, nuclear magnetic resonance and other spectroscopic techniques will greatly aid this quest. In addition, more comprehensive computer-aided algorithms for structure simulation, improved models of protein conformational behavior, and greater insight into the molecular forces which control sequence nucleation will also be required.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acid Sequence↗
Chemical, physical, and biochemical concepts in isolation and purification of proteins.
In this chapter the physicochemical basis of protein retention in adsorption chromatography has been examined. It is evident from this treatment that significant developments are in process with regard to both the theory and practice of high-resolution chromatographic methods, particularly at the preparative level with adsorptive stationary phases. The recognition that most purification strategies must be based on multidimensional multistage procedures presents numerous challenges for the protein chemist, the chromatographic scientist, and the biochemical engineer. The urgency for these developments has been stressed by the potential of modern biotechnology to produce large quantities of new proteins that must be obtained in highly purified form. The capabilities that these advances present will prove catalytic in the materials sciences as new solid phases are examined with particular properties for large-scale utilization. Similarly, new initiatives in protein engineering will lead to greater utilization of the fusion handle approach for selective protein isolation and recovery. The realization of these capabilities will represent an enthusiastic and innovative method that will revolutionize the role of bioprocess development over the next decade.
Chromatography, Liquid↗
High-performance liquid chromatography of amino acids, peptides and proteins. XC. Investigations into the relationship between structure and reversed-phase high-performance liquid chromatography retention behaviour of peptides related to human growth hormone.
The gradient elution behaviour of eight synthetic peptides encompassing residues [6-13] of human growth hormone, i.e. Leu1-Ser-Arg-Leu-Phe-Asp-Asn-Ala8, has been investigated, by using an octadecylsilica, a butylsilica, and a polymeric fluorocarbon as stationary phases. Quantitative expressions, derived from the linear-solvent-strength theory and the general plate-height theory, were used to assess the influence of gradient time on the relative retention and bandwidths of these peptides. It was demonstrated that the chromatographic properties of the cyclised imide form involving Asp6 are consistent with the formation of a highly stabilised amphipathic helix, while the open-chain alpha- and beta-rearranged forms eluted as less rigid structures. The putative hydrophobic contact region consists of two leucine residues and one phenylalanine residue. From an analysis of the retention and bandwidth data obtained at pH 9, a surface-induced molecular reorientation of the beta-linked peptides was observed, in which the repulsion of the aspartyl carboxyl group from the hydrophobic stationary phase directs the C-terminal moiety away from the sorbent surface. Furthermore, the fluorocarbon sorbent exhibited characteristics favourable for use in preparative purification of these peptides. The present results demonstrate the sensitivity of reversed-phase high-performance liquid chromatography (RP-HPLC) to monitor small changes in the interactive behaviour of peptides with hydrocarbonaceous ligands and aquo-organic solvent combinations in reversed-phase systems. These observations further illustrate the general utility of HPLC for investigating the conformational behaviour of peptides at solid-liquid interfaces.
Amino Acids↗