William F. Benisek

Nucleotide sequence of the gene for the Δ5-3-ketosteroid isomerase of Pseudomonas testosteroni

The structural gene for the delta 5-3-ketosteroid isomerase of Pseudomonas testosteroni has been sequenced by the dideoxy method. The sequence obtained confirms the amino acid (aa) sequence of Benson et al. [J. Biol. Chem. 246 (1971) 7514-7525] at all but 5 aa residues of the 125-aa polypeptide. Amino acid residues 22, 24, 33, and 38, reported to be asparagines by Benson et al., are found to be encoded by aspartic acid codons. Amino acid residue 77, reported to be a glutamine by Benson et al., is encoded by a glutamic acid codon. The identification of aa 38 as aspartic acid, coupled with its presence in the active site, as indicated by previous affinity and photoaffinity-labeling studies and confirmed independently by x-ray crystallographic studies, strengthens the hypothesis that Asp-38 is the aa responsible for the 4 beta to 6 beta proton transfer which is part of the enzymatic reaction.

Recent Studies on Steroid Isomerases From Pseudomonas Testosteroni and Pseudomonas Putida

In 1955, Paul Talalay reported the occurrence and partial purification of a AS-3-ketosteroid isomerase (EC 5.3.3.1) in Pseudomonas testosteroni grown in the presence of testosterone.’ By 1959, the isomerase was available in essentially pure crystalline preparations.’ The reaction catalyzed by the isomerase has been shown to consist of an allylic isomerization of steroidal 5-ene-3-ones to their conjugated 4-ene-3-one isomers with concomitant 4p to 68 intramolecular proton transfer. This reaction is shown in FIGURE 1. The enzyme has been the object of numerous investigations of its structure and function. Much of this work has been the subject of a recent review.’ AS-3-Ketosteroid isomerase has attracted interest because of its rather large turnover number, its moderate size, and the fact that it is obtainable in a pure state in large quantities. In addition, because it binds steroids as substrates or competitive inhibitors, albeit with less affinity than mammalian intracellular steroid receptors, some effort has been made to elucidate the structural bases for steroid binding by the isomerase. The primary structure of the constituent polypeptide chain has been reported by Benson et al.4 and a crystallographic study of its threedimensional structure is reported to be ~ n d e r w a y . ~ Below a concentration of approximately 2 mg/ml, the testosteroni isomerase is a dimer of identical 13,394 dalton subunits. Each subunit consists of a single polypeptide chain. The dimer probably has two independent functioning steroid binding although Vincent et a1.* have reported that, in their experiments, the dimer exhibits a “half-of-the-sites’’ binding stoichiometry. Considerable variation of the steroid structure can be tolerated without exceeding the ability of the enzyme to bind the steroid. In a recent comprehensive survey, Weintraub et al.9 found that a wide variety of estrogens, androgens, and progestins were competitive inhibitors of the catalytic process. Many of the enzyme’s competitive inhibitors are steroidal A4-3-ones, the products of the enzymatic reaction when it is conducted in the thermodynamically favored direction. Over the past several years, we have sought to exploit the inherent photochemical reactivity of the A4-3-one competitive inhibitors of the enzyme in an effort to identify, by photochemical modification techniques, important functional groups of the steroid binding active sites of this enzyme.’&’* The most detailed work has been conducted

An inexpensive computerized enzyme kinetics system based on a Gilford spectrophotometer and an Apple IIe microcomputer

A microcomputer-controlled data acquisition system for spectrophotometric enzyme kinetics measurements has been assembled. The system uses an Apple IIe computer which is interfaced to the binary coded decimal output of a Gilford spectrophotometer. No analog-to-digital converter had to be purchased. A BASIC program which collects timed absorbance readings every 500 ms, plots the data in real time, performs a linear regression of the data to measure the reaction rate, and calculates the enzyme activity concentration is given in full. Details describing the interfacing of the computer to the spectrophotometer are presented which will permit other laboratories to readily assemble their own systems using this hardware. Kinetic data acquired by the system are highly reproducible and agree well with data processed much more slowly by manual techniques from strip chart recordings.

Catalysis of the isomerization of Δ5-3-ketosteroids to Δ4-3-ketosteroids by primary amines: Evidence for an imine intermediate☆

Abstract The isomerization of 5-androstene-3,17-dione and 17β-hydroxy-5-androstene-3-one to 4-androstene-3,17-dione and 17β-hydroxy-4-androstene-3-one, respectively, is catalyzed by primary amines. In the case of the isomerization catalyzed by glycylglycine the reaction proceeds through an intermediate which absorbs maximally at 275 nm. Based on spectral similarities to appropriate model compounds and structural analysis of the intermediate after its reduction by sodium borohydride, the intermediate has been tentatively identified as the Δ 4 -3-imine.

Allotype-dependent stimulation of peripheral blood and synovial lymphocytes by IgG3 in rheumatoid arthritis.

The immunopathologic process of rheumatoid arthritis (RA) is primarily expressed in the synovium where rheumatoid factor (RF) synthesis is concentrated. We hypothesized that RF synthesized by rheumatoid synovial cells (RSC) may be driven via a T cell-mediated immune response developed against IgG3 epitopes. To identify and characterize specific RSC RF epitopes and T cell antigens, two 28 amino acid peptides homologous with the C-terminus of IgG1 (P1) and IgG3 [G3m(5)] (P3) were synthesized and used in RF-binding studies and lymphocyte proliferation assays. Our results indicate that (i) the C-terminus of the CH3 domain contains epitopes for IgG3-reactive RSC RF; (ii) IgG3-reactive RSC RF binds primarily to IgG3 [G3m(5)]; (iii) P3 stimulated proliferation of T lymphocytes from both RA peripheral blood and RSC; and (iv) RF production was enhanced by P3 in selected RA cell cultures. These observations suggest that the C-terminus of IgG3 allotype G3m(5) may be important in T cell activation and RF production in RA.

Determination of the affinity of monoclonal 19 S IgM rheumatoid factor for IgG by modified immunoassay (ELISA)

In rheumatoid arthritis (RA), the pathogenicity of IgM rheumatoid factor (RF), an autoantibody whose antigen is IgG, is still unclear although RF-IgG complexes appear to be important mediators of immune injury. The polyclonality of RF in RA makes it difficult to characterize certain qualitative properties such as specificity and affinity which may be very important in determining pathogenicity. Monoclonal IgM RF can be used to circumvent this problem. Monoclonal RF secreting cells can be produced via hybridizations with RA B lymphocytes fused with mouse or human myeloma cell lines. Another source of monoclonal RF is the sera of patients with Waldenströms macroglobulinemia (WM). One particular WM IgM RF (Kas) was chosen for our experiments to measure affinities and specificities to eight different monoclonal IgGs (three IgG1s, three IgG3s, one IgG2, and one IgG4). 19 S IgM RF, a pentavalent molecule, was mildly reduced with DTT to make 7 S univalent fragments (7 S IgM RF). 7 S IgM RF was incubated with each of the different IgGs at several different concentrations. These mixtures were allowed to come to equilibrium. An aliquot was then used to determine the amount of free 7 S IgM RF by ELISA. By plotting the reciprocal of the fraction of bound RF versus the reciprocal of the concentration of free antigen at equilibrium, different affinities were determined. The results of these determinations compare favorably with published IgM RF affinities determined by more traditional methods. This method can also be used with proteolytic digest fragments of IgG and short synthetic peptides of the IgG molecule to better locate the antigen binding site. The technique may also help us to determine whether there are select clones of RSC producing RF with different affinities that could complex to a particular type of IgG which, in vivo, could produce greater inflammatory tissue damage. Furthermore, this methodology should be useful in the study of other autoimmune diseases characterized by pathogenic autoantibodies of differing affinities.

Preparative fractionation of peptide mixtures by two-dimensional chromatography and electrophoresis on paper: Location of peptides by contact printing

Abstract A technique is described for locating peptides on paper electrophoretograms and two-dimensional maps which avoids exposure of a major portion of the peptides to chemical reagents.

Identification of serotonin from rabbit upper stomach as a stimulant of in vitro gallbladder contraction

Using an in vitro rabbit gallbladder bioassay, the distribution and identification of bioactive substances in rabbit gastrointestinal tract were investigated. Comparison of the bioactivities of tissue extracts before and after cholecystokinin was removed by affinity chromatography demonstrated that the distributions of cholecystokinin and non-cholecystokinin substances were different. While cholecystokinin bioactivity per g of tissue was highest in the duodenum, non-cholecystokinin bioactivity was greatest in the upper stomach. The biochemical properties of the non-cholecystokinin substance in the upper stomach could not be distinguished from those of serotonin. These included molecular weights of 176, identical ultraviolet spectra, similar nuclear magnetic resonance spectra, and co-chromatography in HPLC. By weight, serotonin had 1/6th of the bioactivity of cholecystokinin octapeptide. We conclude that the principal gallbladder-contracting substance in rabbit upper stomach is serotonin.

Binding of steroids to P.testosteroniΔ5-3-ketosteroid isomerase: Measurement of the number of binding sites by equilibrium dialysis

Abstract The binding of progesterone, 17 β -estradiol and 19-nortestosterone acetate to the Δ 5 -3-ketosteroid isomerase from Pseudomonas testosteroni has been investigated by the technique of equilibrium dialysis. Under the conditions used, all three steroids formed 2:1 complexes with each molecule of enzyme dimer (M.W. = 26,788). No evidence of any cooperative binding phenomena was obtained. The dissociation constants of the enzyme steroid complexes at 25°C were: progesterone, 2.2 μ M ; estradiol, 2.5 μ M ; 19-nortestosterone acetate, 9.2 μ M .

Modifications of Δ5-3-ketosteroid isomerase induced by ultraviolet irradiation in the presence of the solid-phase photoaffinity reagent Δ6-testosterone agarose

The nature of the products formed during the photoinactivation of Δ5-3-ketosteroid isomerase in the presence of the solid-phase photoaffinity reagent Δ6-testosterone succinyl agarose has been investigated after ultraviolet irradiation. The polypeptide products eluted from the agarose phase by sodium cholate, sodium dodecyl sulfate, and pH 10.5 triethylamine buffer have been characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis, pH 4–6 gel isoelectric focusing, and amino acid analysis. The amino acid compositions of the cholate eluted and SDS eluted products are found to be similar to that of native isomerase, whereas the covalently bound polypeptide eluted by pH 10.5 triethylamine possesses a distinetly different composition. Digestion of the covalently bonded isomerase polypeptide with trypsin yields an agarose-bound peptide fraction that has been characterized by its amino acid composition. This composition is different from that of the undigested covalently bound polypeptide and suggests that the site of covalent attachment lies somewhere between residues 28 and 45 of the isomerase polypeptide.