B Grego

Ludwig Institute for Cancer Research Australia, Melbourne, Victoria, Australia

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Publications (47)146.18 Total impact

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    ABSTRACT: 1. A 78 kDa protein (p78) has been partially purified from washed membranes isolated from the corpus of porcine gastric mucosa. The purification was monitored by covalent cross-linking of iodinated [Nle15]-gastrin. 2. A single N-terminal sequence extending for 33 amino acids was obtained from the p78 preparation. Partial sequences totalling 192 amino acids were also obtained from 14 tryptic and 3 Staphylococcal V8 peptides. 3. 10 peptides plus the N-terminal sequence were derived from a previously unsequenced protein which was distantly related to the product of the E. coli fadB gene (Baldwin G. S. (1993) Comp. Biochem. Physiol. 104B, 55-61). The remaining 7 peptides were derived from the beta-subunit of the gastric H+/K(+)-ATPase. 4. The gastrin-binding activity remained in association with p78, and could be separated from the beta-subunit of the gastric H+/K(+)-ATPase, during chromatography on tomato lectin-Sepharose. 5. We conclude that p78 binds gastrin, and is a novel member of the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase family of enzymes.
    International Journal of Biochemistry 05/1994; 26(4):529-38.
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    ABSTRACT: 1. An iron-binding glycoprotein has been purified to homogeneity from porcine gastric mucosa. 2. The molecular weight (80,000), amino acid composition, carbohydrate content, N-terminal amino acid sequence, tryptic map, stoichiometry of iron binding (2 mol/mol), visible absorption spectrum of the ferric complex and chromatographic behaviour of the gastric protein are all strikingly similar to the corresponding properties of porcine serum transferrin. 3. The quantity of the gastric protein (1.3 mg/g wet weight) present in the gastric mucosa suggests that it is not serum transferrin (plasma concentration 1.8 mg/ml) contaminating the tissue. 4. A role for transferrin in the uptake of dietary iron by the gastrointestinal tract is proposed.
    Comparative biochemistry and physiology. B, Comparative biochemistry 02/1990; 95(2):261-8. · 2.07 Impact Factor
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    ABSTRACT: The progesterone receptor form B has been isolated to apparent homogeneity from large scale preparations of laying hen oviduct cytosol. The quantities obtained were sufficient to monitor the separation of tryptic peptides on HPLC columns. Using a multi-dimensional microbore HPLC peptide purification protocol, several peptides were isolated in homogeneous form and sequenced up to 34 steps at the sub-40 pmol level using a gas phase sequenator. One of the peptides showed a striking homology with sequences of the putative steroid binding domain of the human glucocorticoid receptor; this region is also conserved in the human and chick estrogen receptor.
    Molecular and Cellular Endocrinology 09/1987; 52(3):177-84. · 4.04 Impact Factor
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    ABSTRACT: A high-performance liquid chromatographic procedure for recovering subnanomole amounts of protein from SDS/polyacrylamide gel electroeluates in a form suitable for gas-phase sequence analysis has been developed. By a judicious choice of reversed-phase column packing, proteins can be retained at high concentrations of n-propanol (90-100%) where sodium dodecylsulfate and acrylamide gel-related contaminants are washed through the column. Retained proteins can be recovered from the column in high yield (greater than 90%) by the simultaneous adding of an ion-pairing reagent into the mobile phase and elution with a gradient of decreasing n-propanol concentration (i.e. an 'inverse or negative gradient'). Furthermore, by using a steep gradient (e.g. 50%/min) at a low flow rate (20-200 microliters/min) the proteins can be recovered in less than 100 microliters and can be used for gas-phase sequence analysis without further manipulation. This procedure is independent of sodium dodecylsulfate concentration (up to 1.2% w/v) in sample loading volumes of up to 1.5 ml. Microbore columns (2.1 mm internal diameter) have been employed for recovering small amounts of protein (1-100 micrograms from electroeluates of protein-containing gel spots while conventional columns (4.6 mm internal diameter) were used for isolating larger amounts of protein (greater than 500 micrograms) from electroeluates of preparative gel bands. The general utility of this inverse-gradient high-performance liquid chromatography procedure has been demonstrated by its successful application in recovering a wide variety of proteins from sodium dodecylsulfate gel electroeluates in a form suitable for N-terminal sequence analysis in the 10-500 pmol range.
    European Journal of Biochemistry 06/1987; 165(1):21-9. · 3.58 Impact Factor
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    ABSTRACT: The first six N-terminal amino acid residues of the 85-90K non-estrogen binding component of the calf uterine, molybdate-stabilized estradiol receptor have been determined by Edman degradation. After affinity chromatography of the stabilized receptor oligomer, the 85-90K unit was purified to homogeneity by preparative gel electrophoresis using electroelution for protein recovery. Inverse-gradient high performance liquid chromatography provided the 85-90K protein suitable for amino-terminal sequence analysis.
    Biochemical and Biophysical Research Communications 03/1987; 143(1):218-24. · 2.28 Impact Factor
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    ABSTRACT: A 75-kDa glycoprotein (P75) has been purified to homogeneity from washed membranes isolated from the corpus of porcine gastric mucosa. The purification procedure employed chromatography on concanavalin A-Sepharose and DEAE-Sepharose, and preparative polyacrylamide gel electrophoresis. Reversed-phase microbore high-performance liquid chromatography was employed to fractionate and purify a number of tryptic peptides generated from approximately 1100 pmol purified P75. The use of reversed-phase microbore (2.1 mm internal diameter) columns facilitated the purification of subnanomole amounts of polypeptides in small volumes (40-60 microliter) suitable for loading onto the gas-phase sequencer without further concentration. N-Terminal amino acid sequence analyses were performed on the intact polypeptide and on 13 tryptic peptides and one Staphylococcus protease V8 peptide, yielding 170 unique assignments; this corresponds to approximately 26% of the molecule. A comparison of this amino acid sequence information with the cDNA-deduced primary structure of a 70-kDa heat-shock-related protein (P72), which is expressed in normal rat liver reveals that these protein sequences are almost identical, differing in only 1 of the 170 positions positively assigned thus far. The probable correspondence of P72 with the 78-kDa glucose-regulated protein (GRP78) isolated from hamster fibroblasts has been reported.
    Protein sequences & data analysis 02/1987; 1(1):7-12.
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    ABSTRACT: The identification, isolation and aminoterminal sequencing of two S-genotype-associated proteins from style extracts of Lycopersicon peruvianum Mill. is reported. There is a high level of homology between these two sequences and with the amino-terminal sequences of other S-allele-associated glycoproteins isolated from Nicotiana alata Link et Otto. These sequences were obtained by a new high-sensitivity method of selected twodimensional gel analysis followed by electroelution and purification of proteins by inverse-gradient high-performance liquid chromatography before sequencing.
    Planta 10/1986; 169(2):184-191. · 3.38 Impact Factor
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    ABSTRACT: The complete amino acid sequence of the plastocyanin from the green alga Enteromorpha prolifera has been determined by Edman degradation of the intact molecule and fragments produced by enzymatic cleavage of the polypeptide chain with chymotrypsin, Staphylococcus aureus protease, proline-specific endopeptidase, Lys-C endopeptidase and trypsin. The molecule consists of 98 amino acid residues with a calculated relative molecular mass of 10103. The amino acid sequence of E. prolifera plastocyanin shows a high degree of homology with those plastocyanins from other algae and higher plants. In particular, the four residues which are copper ligands in other plastocyanins and in the bacterial electron transport protein azurin (two histidines, one cysteine and one methionine) are conserved. Five out of the six acidic amino acid side-chains which create an 'acidic patch' on the surface of plastocyanin from Populus nigra var. italica [Colman, P. M. et al. (1978) Nature (Lond.) 272, 319-324] are conserved in the amino acid sequence of E. prolifera plastocyanin.
    European Journal of Biochemistry 07/1986; 157(3):497-506. · 3.58 Impact Factor
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    ABSTRACT: The complete amino acid sequence of the plastocyanin from the green alga Enteromorpha prolifera has been determined by Edman degradation of the intact molecule and fragments produced by enzymatic cleavage of the polypeptide chain with chymotrypsin, Staphylococcus aureus protease, proline-specific endopeptidase, Lys-C endopeptidase and trypsin. The molecule consists of 98 amino acid residues with a calculated relative molecular mass of 10103. The amino acid sequence of E. prolifera plastocyanin shows a high degree of homology with those plastocyanins from other algae and higher plants. In particular, the four residues which are copper ligands in other plastocyanins and in the bacterial electron transport protein azurin (two histidines, one cysteine and one methionine) are conserved. Five out of the six acidic amino acid side-chains which create an ‘acidic patch’ on the surface of plastocyanin from Populus nigra var. italica [Colman, P. M. et al. (1978) Nature (Lond.) 272, 319–324] are conserved in the amino acid sequence of E. prolifera plastocyanin.
    European Journal of Biochemistry. 05/1986; 157(3):497 - 506.
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    ABSTRACT: Self-incompatibility in flowering plants is controlled by the S gene. A complementary DNA clone encoding a style protein of Nicotiana alata which segregates with the S2 allele has now been sequenced. The S-allele-associated style components in different genotypes of N. alata and in Lycopersicon peruvianum, another self-incompatible species in the family Solanaceae, are homologous.
    Nature 04/1986; 321:38-44. · 38.60 Impact Factor
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    ABSTRACT: A procedure for the purification of subnanomole levels of polypeptides has been developed. Reversed-phase high performance liquid chromatography on short (10 cm or less) microbore (1-2 mm internal diameter) columns has been used to fractionate and purify a number of tryptic peptides generated from approximately 600 pmol of purified murine plasma cell antigen PC-1, a major membrane glycoprotein on all cells secreting immunoglobulins. The use of reversed-phase microbore columns permits the recovery of subnanomole amounts of polypeptides from large volumes in high yield (greater than 90%) and in small eluent volumes (40-60 microL) which can be loaded directly onto the gas-phase sequencer without further concentration. This procedure avoids the severe sample loss which frequently occurs with other concentration procedures such as lyophilization and evaporation. The use of a photodiode-array detector for identifying tryptophan-containing peptides from on-the-fly, ultraviolet spectra is described. This procedure permits the selection of tryptophan-containing peptides from complex tryptic digests for use as candidate peptides for oligonucleotide probe construction. Automated Edman degradation was performed on seven tryptic peptides, yielding 110 unique assignments; this corresponds to approximately 11% of the molecule.
    International journal of peptide and protein research 03/1986; 27(2):201-7.
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    ABSTRACT: Epidermal growth factor (EGF) isolated from the submaxillary gland of the rat (rEGF) is missing the COOH-terminal five residues present in both mouse and human EGF. rEGF competes for the binding of 125I-labelled mEGF to human carcinoma cells with the same affinity as mEGF. rEGF and mEGF have identical mitogenic activities on mouse 3T3 fibroblasts, thus the C-terminal region of the sequence is not necessary for the in vitro activity of EGF. Using reversed-phase high-performance liquid chromatography, four molecular forms of EGF have been extracted from rat submaxillary glands. These forms represent rEGF, rEGF(2-48), rEGF(3-48) and rEGF(4-48); all forms appear to be equipotent in both the receptor binding and mitogenic assays. The isoelectric points of these rEGFs are in the range of pH 5.1 to 5.2. The primary structure of rEGF was determined from approximately 10 micrograms protein by sequence analysis of the intact molecule and fragments obtained from the reduced and alkylated protein by chemical cleavage with CNBr and enzymic cleavage with chymotrypsin and a proline-specific endopeptidase. Subnanomole amounts of generated peptides were purified to homogeneity by reversed-phase microbore high-performance liquid chromatography and analysed by automated Edman degradation in a gas-phase sequencer. There are 48 amino acid residues in the complete polypeptide chain which lacks alanine, phenylalanine, lysine and tryptophan. The amino acid sequence of rat epidermal growth factor is: Asn-Ser-Asn-Thr-Gly-Cys-Pro-Pro-Ser-Tyr-Asp-Gly-Tyr-Cys-Leu-Asn- Gly-Gly-Val-Cys-Met-Tyr-Val-Glu-Ser-Val-Asp-Arg-Tyr-Val-Cys-Asn-Cys -Val-Ile-Gly-Tyr-Ile-Gly-Glu-Arg-Cys-Gln-His-Arg-Asp-Leu-Arg. The calculated relative molecular mass from the sequence analysis is 5377.
    European Journal of Biochemistry 01/1986; 153(3):629-37. · 3.58 Impact Factor
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    ABSTRACT: The characteristic zero- and second-order derivative spectra of phenylalanine, tyrosine and trytophan are described and used to identify aromatic residues contained in sub-microgram amounts of polypeptides and proteins during their elution from reversed-phase short microbore columns under gradient conditions. Spectral data were acquired with a commercially available scanning diode array detector. The method allows the non-destructive identification of tryptophan residues in complex polypeptide mixtures such as tryptic maps and enables the selection and isolation of such peptides for amino acid sequence analysis. The sub-microgram level of sensitivity is due to the small peak volumes and cosequent elevated solute concentrations obtained on short (< 10 cm), microbore (2 mm I.D.) reversed-phase columns.
    Journal of Chromatography A - J CHROMATOGR A. 01/1986; 352:359-368.
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    ABSTRACT: We describe herein the use of reversed-phase high-performance liquid chromatography coupled with the novel application of short (10 cm or less) microbore columns (2 mm internal diameter) to fractionate and purify a number of tryptic peptides generated from approximately 200 pmol purified murine transferrin receptor. The use of reversed-phase microbore columns permits the recovery of submicrogram amounts of purified polypeptides in high yield (greater than 90%) in small eluent volumes (20-60 microliter). In this manner, purified polypeptides can be loaded directly onto the gas-phase sequencer without further manipulation. This procedure avoids sample loss, which frequently occurs with other forms of concentration (e.g. lyophilization, evaporation). The application of second-order-derivative ultraviolet spectroscopy, using a diode array detector, for the analysis of aromatic aminoacid-containing peptides in complex tryptic digests is described. N-terminal amino acid sequence analyses were performed on six tryptic peptides, yielding 105 unique assignments; this corresponds to approximately 14% of the molecule. A comparison of this amino acid sequence information with the primary structure of human transferrin receptor deduced from the mRNA sequence [Nature (Lond.) 311, 675-678 (1984); Cell 39, 267-274 (1984)] reveals, with the exception of one tryptic peptide, a very close sequence homology between the murine and human transferrin receptors.
    European Journal of Biochemistry 06/1985; 148(3):485-91. · 3.58 Impact Factor
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    ABSTRACT: The murine plasma cell alloantigen PC-1 is selectively expressed on B lymphocytes in their terminal phase of differentiation into antibody-secreting cells. Previous work on an analytical scale has shown that PC-1 consists of two apparently identical disulfide-bonded polypeptides, each of Mr 115,000. In this paper, we describe the generation of a monoclonal antibody to PC-1 and its use in the preparative isolation of PC-1 by affinity chromatography. Final purification to apparent homogeneity was achieved by preparative polyacrylamide gel electrophoresis. It was estimated that NS-1 myeloma cells possess 1 to 4 X 10(5) PC-1 monomers per cell on their surface. The yield of PC-1 after purification was approximately 10(5) monomers per cell. Purified PC-1 was digested with trypsin, and the resulting peptides were separated by reversed-phase high-performance liquid chromatography. Purified peptides were sequenced with a gas-phase sequencer.
    The Journal of Immunology 02/1985; 134(1):443-8. · 5.52 Impact Factor
  • European Journal of Biochemistry 01/1985; 153:629 - 637. · 3.58 Impact Factor
  • B Grego, M T Hearn
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    ABSTRACT: The chromatographic behaviour on alkylsilicas of a variety of hormonal proteins is described. Optimization of resolution and recovery of these protein hormones, which included porcine relaxins, human chorionic gonadotropin, human placental lactogen, pituitary derived growth hormone and adenohypophyseal glycoprotein hormones, was achieved by manipulation of both mobile and stationary phase parameters. With standard stainless-steel analytical columns (10-30 cm X 0.4 cm) packed with meso- or macro-porous n-alkylsilica supports these proteins can be readily fractionated at the semi-preparative level with separation times generally under 90 min using elution systems directly compatible with subsequent methods of primary structure determination or biological functional analysis. The effects of changes in several experimental parameters on peak symmetry, retention and recovery are described.
    Journal of Chromatography A 01/1985; 336(1):25-40. · 4.61 Impact Factor
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    ABSTRACT: The receptor for transferrin is one of the major surface proteins of proliferating lymphocytes and other cells. It binds ferrotransferrin from serum and endocytoses it into an acidic nonlysosomal intracellular compartment where iron is released, but in which apotransferrin remains tightly bound to its receptor. Recycling of the apotransferrin-receptor complex to the cell surface is associated with a return to neutral pH and concomitant loss of affinity of apotransferrin for its receptor. Apotransferrin is then free to leave the cell and initiate a new cycle. We have exploited this cycle in a novel method for the purification of the receptor for transferrin. Murine myeloma cells were lysed in nonionic detergent, and the lysate passed over a column of ferrotransferrin-agarose at pH 7.4. After washing with sodium acetate at pH 5.0, iron was removed with sodium citrate pH 5.0 and desferrioxamine. Upon returning the pH to neutrality, the receptor was eluted and found to be homogeneous by SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. The degree of purification was estimated to be at least 3,000-fold, and the calculated yield was 10 to 20%. The purified receptor was capable of binding to transferrin. The receptor was digested with trypsin, and the resulting peptides were separated by reversed-phase high performance liquid chromatography in NH4HCO3. Selected peptides were rechromatographed in 0.1% trifluoroacetic acid, and their amino acid sequences were determined.
    The Journal of Immunology 01/1985; 133(6):3220-4. · 5.52 Impact Factor
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    ABSTRACT: Procedures for the purification of native and phosphorylated human growth hormone (hGH), S-carboxymethylated hGH, and hGH tryptic peptides, based exclusively on reversed-phase chromatography have been developed. Combinations of several volatile ion-pairing systems with small- and large-pore n-alkylsilicas were exploited in a general strategy, which allowed high recoveries of various hGH-related polypeptides from enzymatic incubations, as well as rapid desalting of samples following chemical modifications of the native protein, such as reductive alkylation in 6 M guanidine hydrochloride. The influence of the elution conditions on retention behaviour of phosphorylated hGH and its tryptic peptides in reversed-phase high-performance liquid chromatography is discussed.
    Journal of Chromatography A 09/1984; 297:21-9. · 4.61 Impact Factor
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    ABSTRACT: This paper explores the influence of mobile-phase and temperature effects on the gradient elution reversed phase chromatographic behavior of proteins. Using LiChrospher SI 500, bonded with n-butyl groups and a gradient in 1-propanol, 10 mM H3PO4, rapid separation and high mass recovery were obtained for a series of globular proteins. This protein separation and recovery are compared to those obtained when acetonitrile and acetonitrile plus 10 mM H3PO4 are used as eluting gradient solvents. In general, acetonitrile yielded lower recovery than 1-propanol, particularly for the more hydrophobic proteins, e.g., ovalbumin. For all three gradient solvents, little difference was observed in retention or recovery when the n-alkyl chain of the bonded phase varied. On the other hand, relative to the n-alkyl phases, a significantly lower retention of all proteins was found on more hydrophilic phases, e.g., cyano and nonendcapped n-butyl, when acetonitrile was the organic modifier, while in the case of 1-propanol, no retention difference was observed. Thus, column comparisons depend on the protein/mobile-phase combinations examined. The role of column temperature was also studied, and it was found that for certain proteins dramatic changes in peak shape occurred as a function of temperature. The influences of ionic strength and salt type were also studied. Protein mass recovery was shown to decrease with an increase in salt concentration; moreover, perchlorate was shown to have a larger effect in this regard than phosphate. In addition, salt concentration and type were found to influence peak shape greatly in certain cases. The results indicate the strong influences of mobile phase and temperature on chromatographic behavior, and some of the options available when this behavior is not satisfactory. Several protein separations are presented illustrating the power of the reversed phase approach.
    Analytical Biochemistry 08/1984; 140(1):223-35. · 2.58 Impact Factor

Publication Stats

558 Citations
146.18 Total Impact Points

Institutions

  • 1986–1990
    • Ludwig Institute for Cancer Research Australia
      Melbourne, Victoria, Australia
    • Royal Melbourne Hospital
      Melbourne, Victoria, Australia
  • 1987
    • University of Western Australia
      Perth City, Western Australia, Australia
  • 1985–1987
    • Ludwig Institute for Cancer Research
      La Jolla, California, United States
  • 1983–1985
    • Saint Vincent Hospital
      Worcester, Massachusetts, United States
    • University of Auckland
      Окленд, Auckland, New Zealand
  • 1984
    • Northeastern University
      Boston, Massachusetts, United States
  • 1983–1984
    • St. Vincent's Hospital Melbourne
      Melbourne, Victoria, Australia
  • 1981
    • University of Otago
      • Department of Biochemistry
      Dunedin, Otago, New Zealand