Kinetic reaction mechanism for magnesium binding to membrane-bound and soluble catechol O-methyltransferase

Catechol O-methyltransferase (COMT, EC 2.1.1.6) from human brain occurs in both a membrane-bound (MB-COMT) and a soluble form (SOL-COMT). While these enzymes appear to be distinct molecular entities, both catalyze the O-methylation of catecholamines through an ordered reaction mechanism in which S-adenosylmethionine (SAM) is the leading substrate [Rivett, A. J., & Roth, J. A. (1982) Biochemistry 21, 1740-1742; Jeffery, D. R., & Roth, J. A. (1985) J. Neurochem. 44, 881-885]. Both MB-COMT and SOL-COMT require the presence of divalent cations for catalytic activity. This series of experiments provides evidence indicating that magnesium ions bind to both MB-COMT and SOL-COMT in a rapid equilibrium sequence prior to the addition of SAM. An equation is presented that predicts the qualitative results obtained in all kinetic experiments carried out with either MB-COMT or SOL-COMT.     

Crystallization and preliminary X-ray investigation of a recombinant form of rat catechol O-methyltransferase.

Rat catechol O-methyltransferase cDNA was introduced into an E. coli expression vector pKEX14, which utilizes the inducible T7 promoter. Active and soluble recombinant catechol O-methyltransferase was produced in bacteria and purified to electrophoretic homogeneity by chromatographic procedures. The purified enzyme has been crystallized by the method of vapor diffusion using polyethylene glycol as precipitant. The space group is P3(1)21 or P3(2)21 with a = b = 51.3 A and c = 168.5 A and one molecule in the asymmetric unit. The crystals diffract beyond 3.2 A and are suitable for three-dimensional X-ray structure determination.     

Distribution patterns of membrane-bound and soluble enzymes in mitochondrial populations.

Mitochondria were isolated from normal rat liver, kidney, and heart, and from mouse liver and ascites tumor cells, and were dispersed through linear sucrose density gradients in a zonal centrifuge. Distributions of the enzyme activity with respect to mean particle size were constructed. The medians of the distributions for the activities of several enzymes associated with the membranes were significantly different from the medians of the distributions of the soluble mitochondrial enzymes when the mean particle diameters of the fractions were used as the measure of particulate size. On the other hand, when the activity of an outer-membrane enzyme was determined as a function of the area of the mitochondria and the activities of the soluble enzymes were expressed as a function of the volume, the calculated particle diameters corresponding to the respective midpoints of these distributions were in better agreement. This congruence suggests that mitochondria are more nearly homogeneous with respect to the enzyme activities examined than previously proposed. Furthermore, since the distribution of the inner membrane activities was similar to those of the outer membrane enzymes, the area of the inner membrane may be proportional, not to the volume of the mitochondria, but to the area of the outer membrane.     

Expression of anti-tumor recombinant IgG- and IgE-like genes in eukaryotic cells

The tandem of humanized variable VL and VH genes (ScFv fragment 4D5) possessing a high affinity to the HER-2/neu oncogene (the epidermal growth factor receptor expressed in many types of human tumors) was attached through a flexible linker to the second exon of human antibodies of IgG1 or IgE isotypes constant gene. The humanized construct of IgE isotype was generated for the first time. Genes of the recombinant antibodies were cloned into the pCl-neo vector under the control of universal cytomegalovirus (CMV) promoter. Transfected HEK-293 cells efficiently produced antibodies of the corresponding isotypes IgE and IgG1. The results of Western blotting confirmed homogeneity of the expressed antibodies, which had the predicted molecular weight and specifically interacted with the HER-2/neu. The attachment of leader peptide to the 5'-end of the gene resulted in the preferential accumulation of recombinant antibodies in the cultural medium. These results indicate that de novo constructed humanized immunoglobulin genes express functionally active, single-chain recombinant antibodies in eukaryotic cells.     

Expression of anti-tumor recombinant IgG- and IgE-like genes in eukaryotic cells

The tandem of humanized variable VL and VH genes (ScFv fragment 4D5) possessing a high affinity to the HER-2/neu oncogene (the epidermal growth factor receptor expressed in many types of human tumors) was attached through a flexible linker to the second exon of human antibodies of IgG or IgE isotypes constant gene. The humanized construct of IgE isotype was generated for the first time. Genes of the recombinant antibodies were cloned into the pCl-neo vector under the control of universal cytomegalovirus (CMV) promoter. Transfected HEK-293 cells efficiently produced antibodies of the corresponding isotypes IgE and IgG1. The results of Western blotting confirmed homogeneity of the expressed antibodies, which had the predicted molecular weight and specifically interacted with the HER-2/neu. The attachment of leader peptide to the 5′-end of the gene resulted in the preferential accumulation of recombinant antibodies in the cultural medium. These results indicate that de novo constructed humanized immunoglobulin genes express functionally active, single-chain recombinant antibodies in eukaryotic cells.     

Subcellular distribution of soluble and membrane-bound Leu-, Arg- and Asp-beta-naphthylamide-hydrolysing activities in rat brain

Subcellular distribution of soluble and membrane-bound Leu-, Arg- and Asp-beta-naphthylamide hydrolysing activities (arylamidase activity) was studied from left and right rat brains, each including hemisphere, cerebellum and brain stem. Both soluble Leu- and Arg-beta-naphthylamide hydrolysing activities showed the highest levels in the synaptosomal fraction. However, the microsomal fraction presented the highest levels when membrane-bound activity was assayed. When we used Asp-beta-naphthylamide as substrate, there were no differences among fractions in the membrane-bound activity, and the highest soluble activity was present at the mitochondrial level. Two different patterns in the subcellular distribution of enzymatic activity were observed: One of them was the result of the use of Leu- or Arg-beta-naphthylamide as substrate and the other when Asp-beta-naphthylamide was employed. No differences between left and right brains in soluble or membrane-bound activities were found.     

Isolation and various properties of soluble and membrane-bound acid RNAse from rat brain lysosomes

Preparations of soluble (I) and membrane-bound (II) acid RNAse with Mr 68,000 and 72,000 Da, respectively, and purified about 2000-fold were isolated from lysosome-rich fractions of rat brain large hemispheres. RNAase II differed from RNAase I by a lower temperature stability. The pH optimum (pH 5.8-6.1), temperature optimum and substrate specificity of RNAase I and II appeared to be identical. The Km values of RNAases I and II for poly(U) are 166 and 160 micrograms/ml; those for RNA--1200 and 1250 mu k/ml, respectively. RNAases I and II extensively hydrolyze soluble, polymeric RNA, rRNA from brain and yeast and poly(U) but do not influence poly(C), poly(A), poly(G), tRNA and DNA. Monovalent cations (K+, Na+, NH4+) activate both RNAase forms.     

Kinetics and inhibition studies of catechol O-methyltransferase from the yeast Candida tropicalis.

The Kms for esculetin and S-adenosyl-L-methionine for catechol O-methyltransferase from the yeast Candida tropicalis were 6.2 and 40 microM, respectively. S-Adenosyl-L-homocysteine was a very potent competitive inhibitor with respect to S-adenosyl-L-methionine, with a Ki of 6.9 microM. Of the catechol-related inhibitors, purpurogallin, with a Ki of 0.07 microM, showed the greatest inhibitory effect. Sulfhydryl group-blocking reagents, such as thiol-oxidizing 2-iodosobenzoic acid and mercaptide-forming p-chloromercuribenzoic acid, provided evidence for sulfhydryl groups in the active site of the enzyme. Yeast catechol O-methyltransferase is a metal-dependent enzyme and requires Mg2+ for full activity. Zn2+ and Mn2+ but not Ca2+ were able to substitute for Mg2+. Mn2+ showed optimal enzyme activation at concentrations 50- to 100-fold lower than those of Mg2+.     

Characterization and biosynthesis of soluble and membrane-bound carbonic anhydrase in brain

Carbonic anhydrase from both the cytoplasmic and membrane fractions of the forebrains of rats was characterized with respect to enzymatic activity, immunoreactivity, and in vitro biosynthesis. A procedure for the rapid purification of both membrane-bound and soluble brain carbonic anhydrase is presented that permits retention of full enzymatic activity. Both forms of the enzyme were found to show specific activities of approximately 5500 Units/mg protein when CO2 hydrating activity was determined. In addition, they exhibited similar esterase activity when assayed with p-nitrophenyl acetate. The membrane-bound form, although requiring detergent for extraction from membranes, was freely soluble in aqueous buffers after purification. The molecular weights of both soluble and membrane-bound carbonic anhydrase are 30,000 daltons, and mixing experiments failed to show any significant differences with respect to size. The two forms also exhibit isoelectric points of 7.2. However, the two proteins were found to differ in two respects. Complement fixation indicated that antibodies to soluble carbonic anhydrase had a higher affinity for the soluble form than for the membrane-bound form. The failure to observe any precursor-product relationship between these two proteins with pulse chase studies and the establishment that carbonic anhydrase-like proteins are synthesized on both free polysomes and the rough endoplasmic reticulum indicated that these proteins are synthesized by two separate mechanisms. In vitro synthesis on both free and bound polysomes was determined by two independent methods using different antibodies and different analytical procedures. The basis for these findings and their physiologic importance are discussed.     

Purification and characterization of rat heart and brain catechol methyltransferase.

In an effort to detect the similarities and differences in the properties of rat heart, brain and liver catechol methyltransferase (S-adenosyl-L-methionine:catechol O-methyltransferase, EC 2.1.1.6), we have determined the cellular distribution of this enzyme activity and extensively purified the soluble and microsomal enzymes present in these tissues. Purification of soluble heart (688-fold) and brain enzymes (240-fold) were achieved using an affinity chromatographic system. The properties of these enzymes were compared with respect to their molecular weights, substrate specificities, inhibitor specificities and immunological properties. The characteristics of the enzyme active sites were investigated using various methyl acceptor substrates and various analogs of S-adenosylmethionine as methyl donors. A series of analogs of S-adenosylhomocysteine was also evaluated as inhibitors of these enzymes. The immunological properties of the purified soluble and microsomal enzymes from heart and brain were investigated using an antibody isolated from rabbits which had been immunized with the soluble rat liver enzyme. In general the properties of catechol methyltransferases isolated from heart and brain were similar to the properties of the enzyme isolated from liver. Some minor differences in substrate and inhibitor specificities were observed which might suggest slight differences in the active sites of these enzymes.     

A quantitative immunoelectronmicroscopic study on soluble, membrane-associated and membrane-bound lysosomal enzymes in human intestinal epithelial cells

Summary We have used quantitative immunoelectronmicroscopy to compare thein situ localization of acid -glucosidase, lysosomal acid phosphatase, -hexosaminidase and glucocerebrosidase in intestinal epithelial cells of the human duodenum. Differences between these four lysosomal enzymes were observed with respect to their presence at the apical cell surface. Transport to the apical membrane seems to be a more important intracellular route for lysosomal acid phosphatase and acid -glucosidase than it is for -hexosaminidase. The membrane associated lysosomal enzyme glucocerebrosidase is not transported to the microvilli. The studies emphasize that lysosomal enzyme transport pathways are enzyme and cell type specific.     

Hormone responsiveness of plasma membrane-bound enzymes in normal and regenerating rat liver.

The hormonal responsiveness of plasma membrane-bound enzymes (Na-+-K-+)-ATPase and adenylate cyclase has been investigated in normal and regenerating rat liver. (Na-+-K-+)-ATPase basal activity is not affected by surgery and only slightly affected by partial hepatectomy; its response to epinephrine and cyclic AMP is decreased only 15 h after hepatectomy. Adenylate cyclase activity of plasma membranes from untreated animals is stimulated by parathyroid hormone and thyroxine; partial hepatectomy increased basal activity as well as the stimulation exerted by the aforementioned hormones, when glucagon and epinephrine sensitivity is essentially unaltered.     

Isolation and characterization of cytosolic and membrane-bound deubiquitinylating enzymes from bovine brain.

The deubiquitinylating enzymes (DUBs), that release free ubiquitin (Ub) from its precursors or ubiquitinylated proteins, are known to comprise of a large protein family in eukaryotes, but those in mammalian tissues remain largely unknown. Here we report the existence of unexpectedly large species of DUBs in both soluble and membrane-bound fractions of bovine brain, based on their ability to cleave (125)I-labeled Ub-fused alphaNH-MHISPPEPESEEEEEHYC (designated as Ub-PESTc). Two cytosolic enzymes, tentatively called sDUB-1 and sDUB-2, with molecular masses of about 30 kDa were purified to near homogeneity by Ub-Sepharose affinity chromatography. sDUB-1 and sDUB-2 corresponded to UCH-L3 and UCH-L1/PGP 9.5, respectively. Intriguingly, the particulate fraction of the brain homogenate was found to also contain strong activities against (125)I-Ub-PESTc, which can be solubilized by treatment with 5% n-heptyl-beta-D-thioglucoside and 1% Nonidet P-40, but not by washing with 1 M NaCl. From the solubilized material, two new 30-kDa, membranous DUBs (called mDUB-1 and mDUB-2) were purified to apparent homogeneity by Ub-Sepharose chromatography. Two other Ub-aldehyde sensitive DUBs, designated as mDUB-3 and mDUB-4, were also partially purified by conventional chromatographic operations. These mDUBs differed from each other in substrate specificity and exhibited different characteristics from the sDUBs, revealing that they are a new type of membrane-bound DUB. These results indicate the presence of divergent DUBs in mammalian brain, which may contribute to regulation of numerous pivotal cellular functions mediated by the covalent modification of Ub.     

Functional and structural comparisons of cysteine residues in the Val108 wild type and Met108 variant of human soluble catechol O-methyltransferase

Catechol O-methyltransferase (COMT) plays an important role in the inactivation of biologically active and toxic catechols. This enzyme is genetically polymorphic with a wild type and a variant form. Numerous epidemiological studies have shown that the variant form is associated with an increased risk of developing estrogen-associated cancers and a wide spectrum of mental disorders. There are seven cysteine residues in human S-COMT, all of which exist as free thiols and are susceptible to electrophilic attack and/or oxidative damage leading to enzyme inactivation. Here, the seven cysteine residues were systematically replaced by alanine residues by means of site-directed mutagenesis. The native forms and cysteine/alanine mutants were assayed for enzymatic activity, thermal stability, methylation regioselectivity, and reactivity of cysteine residues to thiol reagent. Our data showed that although there is only one encoding base difference between these two COMT forms, this difference might induce structural changes in the local area surrounding some cysteine residues, which might further contribute to the different roles they might play in enzymatic activity, and to the different susceptibility to enzyme inactivation.     

Influence of dietary fat on the activities of subcellular membrane-bound enzymes from different regions of rat brain

The effect of different dietary fats with varying degrees of unsaturation and essential fatty acid composition, which are commonly consumed in India, on the activity of some important membrane-bound enzymes was assessed in different brain regions of rat. Four groups of male CFY weanling rats were fed nutritionally adequate diets containing groundnut, coconut, safflower or mustard oil as fat source at 20% level for 16 weeks. The synaptosomal, microsomal and myelin membranes were prepared from three brain regions, viz., cerebrum, cerebellum and brain stem from each group. The activities of Na⁺, K⁺-ATPase, Mg²⁺-ATPase and acetylcholinesterase were assayed and the fatty acid composition was determined in these subcellular membrane fractions. The safflower oil-fed group showed higher Na⁺, K⁺-ATPase activity in most membrane fractions.than the coconut or mustard oil-fed groups. The Mg²⁺-ATPase activity was found to be similar amongst all groups in all the brain regions. The synaptosomal acetylcholinesterase activity was distinctly higher in coconut and groundnut oil-fed groups when compared to safflower or mustard oil consuming groups. Alterations in the activities of these subcellular membrane-bound enzymes are expected to exert a significant impact on the electrophysiological and metabolic functions of brain. Results of the present study show that depending on the nature of dietary fat the fatty acid composition of subcellular membranes is altered, which in turn could regulate the activity of membrane-bound enzymes that are vital for brain function.     

Quantitation of intracellular membrane-bound enzymes and receptors in digitonin-permeabilized cells

The distribution of membrane-bound receptors and enzymes between the cell surface and the cell interior can be determined without solubilization or gross disruption of cell organelles in the presence of the nonionic detergent digitonin. This steroid glycoside permeabilizes cells, releases cytoplasmic proteins with subunit molecular weights up to 200,000, and allows exogenous molecules to gain access to intracellular receptors. All cell types examined were affected similarly by digitonin. Permeabilization was complete within 2 min at 0°C and did not require the continued presence of digitonin. A characteristic amount of protein (～50%) was lost between 0.02 and 0.08% ($\text{w}\text{v}$) digitonin. Three independent systems were examined: the insulin receptor in 3T3 fibroblasts and the asialoglycoprotein receptor and the $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}\text{-ATPase}$ in rat hepatocytes. In each case an increase in the specific activity of enzyme/receptor occurred over a range of detergent concentration in which the retention of cell protein was constant and virtually no solubilization of membrane-bound activity occurred. The binding of ¹²⁵I-asialo-orosomucoid to rat hepatocytes at 0°C in the presence of digitonin was linear with cell number and kinetically indistinguishable from binding to intact cells. Receptors exposed by digitonin were shown to be intracellular by light microscopic examination of permeabilized cells first treated with antiserum to the receptor and then with a second antibody horseradish peroxidase conjugate. The use of digitonin has many advantages over procedures which require total cell disruption or solubilization to assess intracellular receptors. The technique has already been valuable in studies on recycling and endocytosis mediated by the asialoglycoprotein receptor (P. H. Weigel and J. A. Oka (1983)J. Biol. Chem.258, 5095–5102) and should also be useful in studies with other membrane-bound receptors and enzymes in other cell types.