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Biomedical subjects

R C Robinson

Publications and source records attributed to R C Robinson.

At least 55 records · Page 3Linked to original sources
PubMed1992

A proteolytically sensitive region common to several rat liver cytochromes P450: effect of cleavage on substrate binding.

Limited proteolysis of rat liver microsomes was used to probe the topography and structure of cytochrome P450 bound to the endoplasmic reticulum. Three cytochromes P450 from two families were examined. Monoclonal antibodies to cytochrome P450 forms 1A1, 2B1, and 2E1 were used to immunopurify these proteolyzed cytochromes P450 from microsomes from rats treated with 3-methylcholanthrene, phenobarbital, and acetone, respectively. Electrophoretic and immunoblot analysis of tryptic fragments revealed a highly sensitive cleavage site in all three cytochromes P450. N-Terminal sequencing was performed on the fragments after transfer onto poly(vinylidene difluoride) membranes and showed that this preferential cleavage site is at amino acid position 298 of P450 1A1, position 277 of P450 2B1, and position 278 of P450 2E1. Multiple sequence alignment revealed that these positions are at the amino terminal of a highly conserved region of these cytochromes P450. The important functional role implied by primary sequence conservation along with the proteolytic sensitivity at its amino terminal suggests that this region is a protein domain. Comparison with the known structure of the bacterial cytochrome P450cam predicts that this proteolytically sensitive site is within an interhelical turn region connected to the distal helix that partially encompasses the heme-containing active site. Substrate binding to the cleaved cytochromes P450 was examined in order to determine whether the newly added conformational freedom near the cleavage site functionally altered these cytochromes P450. Cleavage of P450 2B1 abolished benzphetamine binding, which indicates that the cleavage site contains an important structural determinant for binding this substrate. However, cleavage did not affect benzo[a]pyrene binding to P450 1A1.

Amino Acid Sequence↗
PubMed1992

Dimeric structure and conformational stability of brain-derived neurotrophic factor and neurotrophin-3.

We have examined the molecular structure of the related neurotrophic factors brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) by physical methods, including gel filtration, velocity sedimentation, sedimentation equilibrium, urea gel electrophoresis, fluorescence spectroscopy, and far-ultraviolet circular dichroism. The results of these studies indicate that at physiologically relevant concentrations both recombinant proteins exist as tightly associated dimers. The dimers are stable even in 8 M solutions of urea. In solutions of guanidine hydrochloride, BDNF and NT-3 undergo slow unfolding between 3 and 5 M concentration of denaturant. Circular dichroism spectroscopy revealed approximately 70% beta-sheet and 20% beta-turn content in the native structure of both neurotrophic factors. In this respect, BDNF and NT-3 resemble other polypeptide growth factors whose receptors are also integral protein-tyrosine kinases.

Animals↗
PubMed1992

Bone hole diameter as a function of drill guide length and drilling method in rigid internal fixation.

This study examines drill guide length and drilling method to see which technique produces the most accurate hole diameter. Two hundred twenty-five holes were produced in fresh porcine mandibles and then measured with a micrometer. A long guide, a short guide, and no guide were used with a drill press, a pneumatic drill, and a manual drill. Using a 2-mm drill bit, the overall average diameter was 2.03 mm and the overall range was 1.85 mm to 2.60 mm. Statistical analysis of the data indicates the drilling method and the guide length made no difference in the hole diameter at the 95% level (P = .05).

Analysis of Variance↗
PubMed1992

Stabilization of the structure of horse plasma vitamin D binding protein by disulfide bonds.

Vitamin D binding protein (DBP) was isolated from horse plasma in a four-step procedure that involved Affi-Gel Blue affinity chromatography, gel filtration, hydroxylapatite chromatography, and anion exchange high-pressure liquid chromatography. The yield of DBP from 80 mL of plasma was 6-7 mg. Horse plasma DBP closely resembles other plasma DBPs, being a tryptophan-free protein of Mr 53,000. It is able to bind to and block the polymerization of monomeric actin. The secondary structure of DBP was calculated from circular dichroism measurements to be 39% alpha-helix, 42% beta-sheet, and 19% random coil. Circular dichroism and fluorescence studies revealed that the disulfide bonds of DBP contribute substantial structural stabilization to the molecule with respect to thermal denaturation. The thermal stability of DBP can be used to advantage. Incorporation of a brief treatment at 70 degrees C into the preparative scheme enables omission of one chromatographic step, without detectable alteration of the purified product.

Actins↗
PubMed1991

Interactions among cytochromes P-450 in the endoplasmic reticulum. Detection of chemically cross-linked complexes with monoclonal antibodies.

The quaternary structure of rat liver cytochrome P-450 within microsomal membranes from 3-methyl-cholanthrene-treated rats was examined by a novel chemical cross-linking-monoclonal antibody approach. Complex formation among the different forms of P-450 was probed by cross-linking of membrane proteins followed by immunopurification with a monoclonal antibody (mAb) to P-450c, the major 3-methylcholanthrene-inducible form. Subsequent immunoblot analysis of the immunopurified proteins with this mAb indicated that P-450c formed complexes with other microsomal proteins. Immunoblots with mAbs to different P-450s were carried out to identify the P-450s that were cross-linked to P-450c. This approach detected specific cross-linking of P-450c to P-450 2a. Immunoinhibition experiments suggest that P-450 2a further metabolizes the primary phenols produced by P-450c-catalyzed hydroxylation of benzo[a]pyrene. Complex formation among membrane-bound enzymes has implications for their catalytic efficiency and an approach combining cross-linking and monoclonal antibody-based characterization of cross-linked proteins will be useful for elucidating such membrane protein macrostructures.

Animals↗
PubMed1990

Synthetic peptide antigens elicit monoclonal and polyclonal antibodies to cytochrome P450 IA2.

Two peptide sequences from cytochrome P450 IA2 were synthesized, coupled to ovalbumin and used as antigens to generate anti-peptide monoclonal and polyclonal antibodies. Antisera to both peptides reacted with rat IA2 but not the structurally similar IA1 form as determined by enzyme-linked immunosorbent assay. However, antisera to both peptides detected both rat IA2 and IA1 on immunoblots. In addition immunoblots of human liver microsomes revealed that both antisera recognized human IA2, but not IA1. Monoclonal antibodies generated against one of the peptides recognized rat IA2 and IA1 but did not detect human IA2. These results demonstrate the utility of anti-peptide antisera as a practical approach for the generation of P450 specific antibodies.

Amino Acid Sequence↗
PubMed1990

Developmental regulation of hepatic testosterone hydroxylases: simultaneous activation and repression of constitutively expressed cytochromes P450 in senescent rats.

The aging process is generally associated with marked decreases in the activities of numerous enzymes as well as lower levels of sex hormones such as testosterone. We therefore examined testosterone metabolism in liver microsomes from individual 3- and 24-month-old male rats. Although the old rats exhibited lower 16 alpha-, 6 beta-, and 2 alpha-hydroxylase activities than the young rats, the old rats had a higher 7 alpha-hydroxylase activity. Immunoquantitation of P450a, a known 7 alpha-hydroxylase, showed that the level of this protein was elevated in the old rats, and was correlated with 7 alpha-hydroxylase activity. The mRNA for P450a was measured with a cDNA probe and its level was fivefold higher in the old rats, whereas levels of mRNA coding for a 6 beta-hydroxylase P450 were markedly decreased. The increased expression of cytochrome P450a demonstrates that the observed common decrease in cytochrome P450-catalyzed activities with senescence is not a universal phenomenon. Thus, constitutive expression of specific cytochrome P450 genes is repressed or activated in senescent rats.

Aging↗
PubMed1989

Age-related changes in the iron spin state of testosterone-binding rat liver microsomal cytochromes P-450.

The aging process is accompanied by decreased drug metabolism as well as lower levels of sex hormones such as testosterone. We examined the age-dependence of liver microsomal cytochromes P-450 from young (3 months) and old (24 months) male rats by absorption and ESR spectroscopy. Spectral perturbations by testosterone were used to identify testosterone-specific P-450 forms. Absorption difference spectra indicated that testosterone induced a greater conversion of P-450 to the high spin form in young rats than in old rats. ESR signals corresponding to total low spin P-450 were of higher intensity in the young rats and were increased by testosterone. Testosterone also interconverted one low spin P-450 species to another. These results demonstrate age-related differences in the types and amounts of testosterone-specific P-450's in rats.

Aging↗
PubMed1989

An outbreak of Mycobacterium bovis infection in fallow deer (Dama dama).

In an outbreak of Mycobacterium bovis infection in fallow deer in South Australia, 3 herds related by recent movement of deer were infected. From these 3 infected herds, 47 of 51 animals were tuberculosis at necropsy. A range of lesions was seen most of which differed from classical bovine tuberculosis in that pus was a white liquid, fibrous encapsulation was not marked and calcification was rare. Histopathology was of classical tuberculosis. M. bovis was cultured from lesions and M. avium-intracellulare was cultured from one deer with no visible lesions. The source of M. bovis infection has not been determined.

Animals↗
PubMed1989

Human liver cytochrome P-450 related to a rat acetone-inducible, nitrosamine-metabolizing cytochrome P-450: identification and isolation.

A monoclonal antibody (MAb) to a rat acetone-inducible and nitrosamine-metabolizing form of microsomal cytochrome P-450, P-450ac, detected a related P-450 in human liver microsomes by both immunoblot and competitive radioimmunoassay. This MAb was also used to immunopurify microsomal cytochromes P-450 from both human liver and acetone-treated rats; these were electrophoretically homogeneous with apparent molecular weights of 56,200 and 53,000 daltons, respectively. The structures of the cytochromes P-450 were compared by peptide mapping and amino-terminal sequence analyses. They differed in their peptide maps but displayed amino-terminal sequence similarity in their first 19 residues. This report thus demonstrates the utility of MAbs to rat cytochromes P-450 for detection, identification and structural characterization of human P-450s.

Acetone↗
PubMed1986

Structural comparison of monoclonal antibody immunopurified pulmonary and hepatic cytochrome P-450 from 3-methylcholanthrene-treated rats.

A pulmonary cytochrome P-450 was purified from lung microsomes of 3-methylcholanthrene (MC)-treated rats by immunoaffinity chromatography using a monoclonal antibody to MC-induced rat liver cytochrome P-450. The isolated pulmonary cytochrome P-450 was MC-inducible and had an apparent molecular weight of 57 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight, as well as the NH2-terminal sequence of the first nine amino acids of the pulmonary cytochrome P-450, was identical to that of an epitopically related MC-induced rat liver cytochrome P-450. In addition, partial proteolysis of both cytochromes P-450 yielded indistinguishable peptide patterns on SDS-Page. Treatment of rats with MC, therefore, induces a pulmonary cytochrome P-450 which is structurally identical to the MC-induced hepatic enzyme by several criteria.

Amino Acid Sequence↗
PubMed1985

Preparation of catalytically active cytochromes P-450 by antigen exchange on monoclonal antibody based immunoadsorbents.

Catalytically active cytochromes P-450 have been prepared by monoclonal antibody (MAb) directed immunopurification using an antigen-exchange technique. Immunoaffinity-purified cytochromes P-450 that require denaturants for efficient desorption from the immunoaffinity matrix, although significantly lacking in catalytic activity, were found to retain epitopic structural integrity as probed by radioimmunoassay using MAbs to 3-methylcholanthrene and phenobarbital-induced rat liver cytochromes P-450. These denatured cytochromes P-450 were capable of displacing from the immunoaffinity matrices epitopically related cytochromes P-450 that retained aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase activities. Such epitope-specific exchange of denatured for native antigen on a solid-phase matrix containing a MAb may be generally applicable to preparation of proteins with the retention of activity.

Animals↗
PubMed1985

Monoclonal antibody directed isolation and amino-terminal sequence analysis of phenobarbital induced rat liver cytochromes P-450.

Cytochrome P-450 was isolated from liver microsomes of phenobarbital treated rats by an essentially single step immunopurification with a monoclonal antibody (MAb). The amino terminal sequence of the isolated cytochrome P-450 displayed a microheterogeneity of isozymes related to previously identified phenobarbital induced forms, indicating that each of these isozymes possess the MAb-specific epitope. This monoclonal antibody-based approach to isolation and subsequent identification of cytochrome P-450 may serve to classify different isozymes by their content of epitopes that bind to different MAbs.

Amino Acid Sequence↗
PubMed1985

Monoclonal antibodies to cytochrome P-450 immunopurify a 45-kDa protein from a human lymphoblastoid cell line.

Monoclonal antibodies (MAbs) to rat liver cytochromes P-450 have previously been used for successful immunopurification of cytochromes P-450 from animal tissues. We now report application of this MAb-based immunopurification technique to the human lymphoblastoid AHH-1 cell line. Immunopurification carried out with 3 different MAbs each yielded a 45-kDa polypeptide. The purified protein contains an MAb-specific epitope present on cytochromes P-450, and may therefore be a human cytochrome P-450.

Animals↗
PubMed1985

Monoclonal antibody-directed determination of cytochrome P-450 types expressed in a human lymphoblastoid cell line.

Cytochrome P-450-dependent aryl hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase activities of a cloned line of human lymphoblastoid AHH-1 cells are inhibited by a monoclonal antibody (MAb 1-7-1) prepared to a 3-methylcholanthrene-induced rat liver cytochrome P-450. The monoclonal antibody inhibition determined that a single MAb 1-7-1-sensitive type of cytochrome P-450 is responsible for all of AHH expression in both the basal and benz[a]anthracene-induced cells. Partial inhibition by the MAb 1-7-1, however, indicates that at least two forms of cytochrome P-450 catalyze 7-ethoxycoumarin O-deethylase in both the basal and the induced cells, one form of which is identical to the MAb-sensitive cytochrome P-450 responsible for all of the AHH. Thus, a single cloned cell line is capable of expressing two classes of cytochromes P-450, and the observed multiplicity of cytochrome P-450 in animal tissues does not necessarily depend on cell heterogeneity. A sensitive MAb 1-7-1-based radioimmunoassay also directly demonstrates the presence in these cells of a MAb 1-7-1-specific type of cytochrome P-450 as well as its elevation in the induced cells. These MAb-based methods thus can determine the contribution of specific MAb-defined types of cytochromes P-450 to the cellular metabolism of specific xenobiotics.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1984

Alterations in leukocyte aryl hydrocarbon hydroxylase activity associated with treatment and age in psoriasis patients and healthy individuals.

We have examined the relationship between topical psoriasis treatment and the activity of the mixed function oxidase, aryl hydrocarbon hydroxylase (AHH), in peripheral blood monocytes and lymphocytes from 20 patients with psoriasis. These data were compared to monocyte and lymphocyte AHH activity measured in 20 normal subjects. AHH activity was determined in cells induced with benzanthracene and in uninduced control cells. Monocyte and lymphocyte AHH activity in six untreated psoriasis patients was similar to that in the healthy controls. AHH activity in either uninduced or induced monocytes showed an increase with age in both the healthy and the untreated psoriatic subjects. Lymphocytes from the healthy subjects showed an age-related decline in enzyme activity. Fourteen patients with psoriasis receiving topical tar and/or topical corticosteroid therapy had significantly higher (P less than 0.05) levels of basal and induced monocyte and lymphocyte AHH activity than the healthy controls. AHH activity is age-related and appears to be controlled differently in monocytes and in lymphocytes. AHH activity in circulating monocytes and lymphocytes may be stimulated by topical tar and/or steroid therapy of psoriasis.

Administration, Topical↗