Partial purification of 2,3-oxidosqualene-lanosterol cyclase from hog-liver. Evidence for a functional thiol residue

2,3-Oxidosqualene-lanosterol cyclase is an intrinsic microsomal protein which can be solubilized by ionic (deoxycholate) and nonionic (emulphogene) detergents with good yields. The hog-liver microsomal cyclase was purified approximately 140-fold by chromatography on DEAE-cellulose and hydroxylapatite. The partially purified enzyme was inactivated by N-ethylmaleimide, following pseudo-first order kinetics, indicating that a cysteine residue is essential for activity.     

Purification of 2,3-oxidosqualene cyclase from rat liver.

A rapid and simple purification of milligram amounts of 2,3-oxidosqualene cyclase, an integral membrane enzyme that catalyzes the cyclization of squalene epoxide to lanosterol, is reported. Several nonionic detergents (Triton X-100, Tween 80, Emulphogene, and lauryl maltoside) were evaluated for solubilization of oxidosqualene cyclase from rat liver microsomes. At a detergent concentration of 5 mg/ml, lauryl maltoside was approximately 10 times more effective than Emulphogene in the solubilization of oxidosqualene cyclase; Triton X-100 and Tween 80 were less effective than Emulphogene as judged by the relative specific activities of the solubilized enzyme. Treatment of microsomes with lauryl maltoside resulted in a selective solubilization of the cyclase with concomitant activation of the enzyme. The solubilized enzyme was purified to homogeneity by fast protein liquid chromatography. The purified enzyme consists of a single subunit that has an apparent molecular weight of 65,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme obeys saturation kinetics and the apparent Km of (2,3)-oxidosqualene is 15 microM; the apparent kcat/Km is 200 M-1.min-1. An improved assay of the enzyme that utilizes high performance liquid chromatography methods is also described.     

Preferential cyclization of 2,3(S):22(S),23-dioxidosqualene by mammalian 2,3-oxidosqualene-lanosterol cyclase.

Kinetic studies on the cyclization of 2,3(S)-oxido and 2,3(S):22(S),23-dioxido[14C]squalene catalyzed by liver oxidosqualene-lanosterol cyclase revealed a specificity (in terms of V/Km) of the enzyme for the diepoxide. The specificity ratio was dependent on the enzyme preparation, i.e. purified or microsomal, and was highest (about 5) with the microsomal enzyme in the presence of supernatant protein factors. These results explain why, in the presence of cyclase inhibitors, the squalene epoxides can be channeled into a cholesterol biosynthesis regulatory pathway via 24(S),25-epoxylanosterol and 24(S),25-epoxycholesterol.     

Adenylyl cyclase in yeast. Hydrodynamic properties and activation by trypsin

The adenylyl cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cAMP from ATP, and two RAS polypeptides, responsible for stimulation of cAMP synthesis by guanine nucleotides. We have determined hydrodynamic properties of yeast adenylyl cyclase in taurocholate extracts of wild type and RAS-deficient membranes. In taurocholate extracts of both kinds of membranes, the enzyme is insensitive to guanine nucleotide stimulation; in the presence of 0.5 M NaCl, the taurocholate-solubilized enzyme has a sedimentation coefficient of 12.5 S and a Stokes radius of 11 nm, consistent with a molecular weight of 594,000 for the protein-detergent complex. Treatment of particulate fractions with trypsin (less than 10 micrograms/ml) markedly activates membrane-bound adenylyl cyclase activity, abolishes stimulation by guanine nucleotides, and reduces the sedimentation coefficient of the detergent-solubilized enzyme; higher concentrations of trypsin release a still smaller water-soluble enzyme complex (7.5 S, 6.1 nm Stokes radius, calculated Mr = 190,000) from the membrane. In combination with genetic evidence (Kataoka, T., Broek, D., and Wigler M., (1985) Cell 43, 493-505), our data are consistent with a structural and functional model of yeast adenylyl cyclase in which GTP-activated RAS proteins stimulate cAMP synthesis by relieving an inhibitory constraint on the activity of the CYR1 gene product. This constraint may be mediated by the amino-terminal portion of the CYR1 polypeptide.     

Purification and immunological properties of proton-ATPase complexes from yeast and rat liver mitochondria

Proton-ATPase complexes from yeast and rat liver mitochondria were isolated by a simple method previously employed for the purification of the proton-ATPase complex from chloroplasts. After reconstitution into liposomes, the purified complexes were active in the ATP-Pi exchange reaction, the rate of which was 120 and at least 200 nmol/mg of protein/min for the rat liver and yeast mitochondria ATPases, respectively. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, each complex exhibited 11 to 12 different polypeptides. The isolated ATPase complexes from rat liver and yeast mitochondria, from Swiss chard chloroplasts, and Escherichia coli membranes were reacted with antibodies prepared against the various subunits of ATPase complexes. From all the combinations of antigen-antibody examined, only the antibodies against beta subunit cross-reacted with the corresponding subunit of all the ATPase complexes tested. These results indicate that certain amino acid sequences in the beta subunit have been preserved in all of the proton-ATPase complexes.     

The temperature-dependence of adenylate cyclase from baker''s yeast.

The Michaelis constant of membrane-bound adenylate cyclase increased from 1.1 to 1.8 mM between 7 and 38 degrees C (delta H = 13 kJ/mol). Over this temperature range, the maximum velocity increased 10-fold, and the Arrhenius plot was nearly linear, with an average delta H* of 51 kJ/mol. The temperature-dependence of the reaction rate at 2 mM-ATP was examined in more detail: for Lubrol-dispersed enzyme, Arrhenius plots were nearly linear with average delta H* values of 45 and 68 kJ/mol, respectively, for untreated and gel-filtered enzymes; for membrane-bound enzyme, delta H changed from 40 kJ/mol above about 21 degrees C to 62 kJ/mol below 21 degrees C, but this behaviour does not necessarily indicate an abrupt, lipid-induced, transition in the reaction mechanism.     

Comparative properties of amplified external and internal invertase from the yeast SUC2 gene

Saccharomyces cerevisiae external and internal invertases have been amplified by introducing the normal and modified SUC2 genes into yeast multicopy plasmids, which were then used to transform a yeast strain resistant to repression by glucose. Amino acid compositional analysis of these enzymes, in addition to end group sequencing, confirmed the DNA sequence data of Taussig and Carlson (Taussig, R., and Carlson, M. (1983) Nucleic Acids Res. 11, 1943-1954), indicating that both enzymes were encoded in the same gene. Comparison of the properties of carbohydrate-containing external invertase and its nonglycosylated internal form revealed that although the carbohydrate did not appear to influence the conformation of the peptide backbone, as determined by circular dichroism analyses, its presence considerably enhanced the ability of guanidine HCl-denatured external invertase to be renatured relative to internal invertase. The Mr of the internal enzymes was found to be greatly dependent on pH with the enzyme being a monomer at pH 9.4, a dimer at pH 8.3, and an apparent octamer at pH 4.9.     

Characterization and partial purification of squalene-2,3-oxide cyclase from Saccharomyces cerevisiae.

The membrane nature of squalene oxide cyclase from Saccharomyces cerevisiae was investigated by comparing properties of the enzyme recovered from both microsomes and the soluble fraction of the yeast homogenate. The "apparent soluble" form and microsomal form of the enzyme were both stimulated by the presence of mammalian soluble cytoplasm and corresponded to one another in response to detergents Triton X-100 and Triton X-114. The observed strong dependence of the enzyme activity on the presence of detergents and the behavior of the enzyme after Triton X-114 phase separation were peculiar to a lipophilic membrane-bound enzyme. A study of the conditions required to extract the enzyme from microsomes confirmed the lipophilic character of the enzyme. Microsomes, exposed to ipotonic conditions to remove peripheral membrane proteins, retained most of the enzyme activity within the integral protein fraction. Quantitative dissociation of the enzyme from membranes occurred only if microsomes were treated with detergents (Triton X-100 or octylglucoside) at concentrations which alter membrane integrity. The squalene oxide cyclase was purified 140 times from yeast microsomes by (a) removal of peripheral proteins, (b) extraction of the enzyme from the integral protein fraction with octylglucoside, and (c) separation of the solubilized proteins by DEAE Bio-Gel A chromatography. Removal of the peripheral proteins seemed to be a key step necessary for obtaining high yields.     

Comparative electrostatic analysis of adenylyl cyclase for isoform dependent regulation properties

The enzyme adenylyl cyclase (AC) plays a pivotal role in a variety of signal transduction pathways inside the cell, where it catalyzes the cyclization of adenosine triphosphate (ATP) into the second‐messenger cyclic adenosine monophosphate (cAMP). Among other roles, AC regulates processes involved in neural plasticity, innervation of smooth muscles of the heart and the endocrine system of the pancreas. The functional diversity of AC is manifested in its different isoforms, each having a specific regulation pattern. There is an increasing amount of data available concerning the regulatory properties of AC isoforms, however little is known about the interactions on a structural level. Here, we conducted a comparative electrostatic analysis of the catalytic domains of all nine transmembrane AC isoforms with the aim of detecting, verifying and predicting the binding sites of molecular regulators on AC. The results provide support for the positioning of the binding site of the inhibitory protein G_iα at a pseudo‐symmetric position to the stimulatory G_sα binding site. They also provide a structural interpretation of the Gβγ interaction with ACs 2, 4, and 7 and suggest a new binding site for RGS2. Comparison of the small molecule binding sites on AC shows that overall they have high electrostatic similarity, but regions of electrostatic differences are identified. These could provide a basis for the development of novel compounds with isoform‐specific modulatory effects on AC. Proteins 2016; 84:1844–1858. © 2016 Wiley Periodicals, Inc.