Properties of monoamine oxidase in control and Lesch-Nyhan fibroblasts

Monoamine oxidase activity of the A type was measured in homogenates of cultured human skin fibroblasts. Twenty-four control lines had activities ranging over fifty-fold with an apparent bimodal distribution. Activity in fibroblasts from 20 patients with the Lesch-Nyhan syndrome fell in the low portion of the normal distribution with a mean activity about 50% that of the control mean (p<0.05). In a subgroup of control and Lesch-Nyhan lines with levels of enzyme activity from 0.9 to 179 pmol/min/mg protein, monoamine oxidase was similar with respect to apparent K_m for tryptamine, thermal stability at 56 C, and sensitivity to clorgyline. Thus the lower mean levels of activity observed in the Lesch-Nyhan as compared to control fibroblasts were not associated with other altered properties of the enzyme. The bimodal distribution of enzyme activity suggests that a genetic polymorphism for monoamine oxidase may control levels of activity expressed in fibroblasts.M. R. C. C. was funded by the Dystonia Medical Research Foundation. This work was supported by grants from USPHS—NS12105 and GM20124—and from the National Foundation-March of Dimes.     

Histochemical studies of fibroblasts from patients with Menkes kinky hair disease and Wilson's disease

Summary Cultured fibroblasts from Menkes kinky hair disease patients showed markedly reduced succinate dehydrogenase and amine oxidase activities. Cytochrome oxidase activity, however, was greatly reduced in some cells and almost normal in others. Cultured fibroblasts from patients with Wilson's disease showed moderately reduced succinate dehydrogenase and cytochrome oxidase activities. Amine oxidase activity was only slightly reduced when compared to that of normal. These results indicated that the histochemical phenotype observed in fibroblasts from patients with Menkes kinky hair disease and Wilson's disease were distinctly different from each other and from normal fibroblasts.     

MONOAMINE OXIDASE ACTIVITY IN NORMAL and LESCH-NYHAN FIBROBLASTS

Monoamine oxidase (MAO) activity was studied in cultured skin fibroblasts from 10 Lesch-Nyhan patients, a Lesch-Nyhan variant and 11 controls matched for age. sex and race. Activity (predominantly type A) was measured in cell homogenates using tryptamine as the substrate. For each line activity varied with the conditions of culture. Activity increased 3-10 fold as cultures went from logarithmic to stationary phase of growth. When cultures were confluent, activity was lowered by frequent feedings or the use of fresh medium and serum. Activity for each line remained fairly stable during successive passages, but rose 3-8 fold as cultures became senescent. When comparing activity between control and Lesch-Nyhan lines, cells were cultured under standardized conditions. The mean value of MAO activity in Lesch-Nyhan lines was approximately one fourth of the mean activity in control lines (P < 0.012), In the control population, the distribution of activity appeared to be bimodal. Activities in the Lesch-Nyhan lines fell completely within the lower portion of the control distribution. Cells from a Lesch-Nyhan patient who lacked several of the neurologic symptoms of the disease (including self-mutilation) had an MAO activity 6 fold greater than the control mean. although his hypoxanthine phosphoribosyltransferase activity was <3% of control levels. It appears that: (1) MAO activity is low in fibroblasts from typical Lesch-Nyhan patients: (2) the severity of neurologic symptoms may be correlated with levels of MAO activity: and (3) some interaction between purine and catecholamine metabolism can affect nerve function.     

MULTIPLE FORMS OF MONOAMINE OXIDASE IN DEVELOPING BRAIN: TISSUE AND SUBSTRATE SPECIFICITIES

Brains, hearts and livers from newborn and adult rats were assayed for monoamine oxidase activity using gel electrophoretic techniques. The results suggest that each of the tissues possesses multiple forms (isoenzymes) of monoamine oxidase and that these forms are different for the various tissues. Further, the forms of monoamine oxidase in the neonatal tissues differ from those in the corresponding adult tissue. These different forms of monoamine oxidase have different substrate specificities. Using 5-hydroxy[¹⁴C]tryptamine as substrate, we have demonstrated that the monoamine oxidase patterns appearing on the gel do indeed possess monoamine oxidase activity.     

Association between platelet monoamine oxidase activity and hematocrit in childhood autism

Platelet monoamine oxidase activity was strongly, negatively associated with hematocrit and hemoglobin in male autistic children, unlike the nonsignificant positive relation in normal male controls. Platelet monoamine oxidase activity and hematocrit were also negatively related, although with less strength, in other childhood psychiatric patients and in male relatives of autistic children.     

Multiple forms of monoamine oxidase in chronic schizophrenia

Reduced monoamine oxidase was found in the sera of fourty five schizophrenic patients with enzyme level (10 units), only five of these patients show enzyme level identical with minimum range of normal controls. Michaelis constants of schizophrenic patients show great difference from that of the normal controls. Our result suggests that schizophrenic patients possess a qualitatively different monoamine oxidase with an altered molecular structure. Four and five multiple forms were found in sera of normal human and schizophrenic patients respectively, using sepharose 6B, indicated that these different forms were of different molecular weight. Each multiple form is identified by cellulose acetate electrophoresis as they attached to different fraction of serum proteins.     

Pathological changes in platelet histamine oxidases in atopic eczema

Increased plasma histamine levels were associated with significantly lowered diamine and type B monoamine oxidase activities in platelet-rich plasma of atopic eczema (AE) patients. The diamine oxidase has almost normal cofactor levels (pyridoxal phosphate and Cu(2+)) but the cofactor levels for type B monoamine oxidase (flavin adenine dinucleotide and Fe(2+)) are lowered. The biogenic amines putrescine, cadaverine, spermidine, spermine, tyramine and serotonin in the sera, as well as dopamine and epinephrine in EDTA-plasma were found to be normal. It is unlikely, therefore, that these amines are responsible for the decreased activities of monoamine and diamine oxidase in these patients. The most likely causative factors for the inhibition of the diamine oxidase are nicotine, alcohol, food additives and other environmental chemicals, or perhaps a genetic defect of the diamine oxidase.     

OCCURRENCE AND PROPERTIES OF MONOAMINE OXIDASE IN ADRENERGIC NEURONS

—Monoamine oxidase activity of peripheral organs of various species has been examined after surgical, chemical and immunological sympathectomy to assess the proportion of enzyme activity in adrenergic neurons and in extraneuronal cells. Significant falls in monoamine oxidase activity of vas deferens, submaxillary gland, iris and spleen were seen after sympathetic denervation although not in heart, small intestine and kidney. It was suggested that a correlation exists between the extent of the fall in monoamine oxidase activity after sympathectomy and the density of sympathetic innervation of the control organ. Studies of monoamine oxidase activity in vas deferens after inhibition with clorgyline suggested multiple forms of monoamine oxidase. Differences in inhibitor sensitivity, substrate specificity and thermal inactivation of monoamine oxidase in normal and denervated vas deferens were found and it was suggested that differences exist in the properties of the neuronal and extraneuronal monoamine oxidase.     

Increased incorporation of aspartate and decreased incorporation of orotate in fibroblasts from Lesch-Nyhan patients as revealed by autoradiography

While it has been known for many years that tissues from Lesch-Nyhan patients are deficient in hypoxanthine-guanine phosphoribosyl transferase, it has been demonstrated more recently that erythrocyte lysates show an increase in the levels of enzymes which participate in “de novo” synthesis of pyrimidines. Also, we were able to demonstrate, using autoradiography, an increased rate of incorporation of aspartate into the nucleus of intact cultured fibroblasts from a Lesch-Nyhan patient. We have now confirmed this finding. In this paper we show increased incorporation of aspartate into the nucleus of cells from three other patients. Interestingly, the incorporation of orotate is decreased in the fibroblasts from the four patients studied. The incorporation of leucine is the same in Lesch-Nyhan fibroblasts as in normal fibroblasts. The significance of these findings is briefly discussed.     

The spontaneous azaguanine-resistant mutants of diploid human fibroblasts

Summary The range of incidences of azaguanine-resistant colonies in cultures of fibroblasts from 16 unrelated humans was 0.4×10^-6 to 19×10^-6 and the mean value was 4.1×10^-6. A fluctuation test showed that most or all of the mutant colonies derived from mutations that occurred during in vitro proliferation of the fibroblasts and before exposure to azaguanine. The estimated rate of spontaneous mutation was 0.45×10^-6 to 1.8×10^-6 per cell generation. At least ten independent mutants, comprising two general classes, were studied. Class I mutants were a minority and resembled cells from boys having the Lesch-Nyhan syndrome: they had very little HG-PRT activity, showed maximum resistance to azaguanine and could not utilize hypoxanthine for growth. At least 90% of the mutants were in Class II: their apparent HG-PRT activities ranged between normal and Lesch-Nyhan amounts, they were partially sensitive to azaguanine and they could utilize hypoxanthine. Some Class II mutants resembled cells cultured from a family having an X-chromosomal mutant gene that does not cause the Lesch-Nyhan syndrome but does confer resistance to azaguanine, although the quantity of HG-PRT activity is apparently normal and hypoxanthine can be utilized. Electrophoretic differences between the HG-PRT activities of normal and mutant strains were not detected but other qualitative alterations were observed in some mutants.Paper No. 1558 from the Laboratory of Genetics.Supported by N.I.H. Grants GM-06983 and GM-15422 and by a grant from the Food Research Institute of The University of Wisconsin, Madison, Wisconsin.Supported by Grant He 753-1 from Die Deutsche Forschungsgemeinschaft.     

Monoamine Oxidases A and B as Components of a Membrane Complex

Abstract: The activities of monoamine oxidase A (MAO A) and monoamine oxidase B (MAO B) represent two independent types of substrate binding site, as indicated by experiments with selective inhibitors and also by substrate competition. We have tried to determine whether A and B active sites of human brain and liver MAO are located on physically separable enzyme forms or as subunits in large membrane-bound complexes. MAO was extracted from several sources by a procedure that was designed to give solubilized enzyme in high-speed supernatants, with ratios of MAO A/MAO B activities similar to those in initial crude homogenates. This solubilized enzyme gave gel filtration profiles that suggested the presence of large molecular complexes. Affinity binding experiments indicated that both MAO A and B activities may occur on the same complexes in tissues that initially contain both activities. These complexes were broken down to enzymatically active subunits by treatment with either low concentrations of sodium dodecyl sulfate, with phospholipase A₂, or with a combination of both agents. Results of this study support a concept of MAO as part of a membrane unit in which A and B are two distinct enzymes embedded in a phospholipid structure. The enzymatic activity of MAO A is critically dependent on associated phospholipids, whereas that of MAO B is not.     

Oxygenation of mitochondrial membranes by the reticulocyte lipoxygenase. Action on monoamine oxidase activities A and B

Incubation of isolated rat liver mitochondria with the pure rabbit reticulocyte lipoxygenase caused a time-dependent inactivation of the monoamine oxidase activities A and B. Furthermore, a conversion of the monoamine oxidase into a diamine oxidase was observed. The inactivation kinetics for both monoamine oxidase activities A and B showed a biphasic behaviour; a reversible short-term inhibition during the first 5 min of incubation was followed by an irreversible inactivation of the enzyme. The kinetic studies suggest that the slow irreversible inactivation of the monoamine oxidase activities is due to secondary reactions subsequent to the initial attack of the lipoxygenase on the mitochondrial outer membrane. During the interaction of the lipoxygenase with the mitochondria, only about 1.5% of the polyenoic fatty acids present in the mitochondrial membranes were oxygenated. The predominant products formed during the interaction of the lipoxygenase with the mitochondrial membranes are (13S)-hydro(pero)xy-9Z,11E-octadecadienoic acid and (15S)-hydro(pero)xy-5,8,11,13(Z,Z,Z,E)-eicosatetraenoic acid.     

Kinetic mechanism of monoamine oxidase A

Steady-state kinetic data for monoamine oxidase A in crude extracts suggest an exclusively ping-pong mechanism, in contrast to those for monoamine oxidase B, which indicate alternate mechanisms involving either a binary or ternary complex. In this study, with use of purified monoamine oxidase A, steady-state data for the inhibition by D-amphetamine of the oxidation of primary amines indicate the possibility of a ternary complex mechanism for monoamine oxidase A also. Stopped-flow studies demonstrate that the rate of reoxidation of reduced enzyme is enhanced by substrates but not by the product, 1-methyl-4-phenylpyridinium. Thus, for the A enzyme, the ternary complex with substrate, but not product, is reoxidized at a faster rate than the free, reduced enzyme. For both the A and B forms of monoamine oxidase, the mechanism is determined by competition between alternate pathways on the basis of the relative rate constants and dissociation constants.     

Significance of multiple forms of brain monoamine oxidase in situ as probed by electron spin resonance.

Spin-labeled hydroxyamphetamine, a competitive reversible inhibitor of brain monoamine oxidase, has been shown to be useful as an electron spin resonance (ESR) probe of the microenvironment of the active sites of the possible monoamine oxidase multiple forms. The ESR spectrum of spin-labeled hydroxyamphetamine was strongly quenched upon binding to the enzyme. The conformation of the active site of rat brain monoamine oxidase existing in various physical states, i.e. monoamine oxidase in situ (intact brain mitochondria), crude solubilized monoamine oxidase (MAOS) and isolated monoamine oxidase fractions (MAOa and MAOb) were critically and systematically examined. Nonlinear least squares regression analyses have been used to fit the binding data (obtained at room temperature with varying spin-labeled hydroxyamphetamine concentrations) to three groups of independent noninteracting ligand-binding models. A Gibbs-Helmholtz relationship was applied to the interpretation of the measured apparent association constant K as a function of temperature ranging from 4-50 degrees with increments of 2 degreesmfrom the extracted intensive parameters, k (intrinsic association constant) and deltaF (intrinsic free energy), as well as the apparent heat, deltaH, it was clear that the microenvironment of the binding sites existing in the more purified enzyme fractions MAOa and MAOb were similar to those found in the crude solubilized enzyme. More importantly, they correlated well with the conformation of the sites characterized in situ. The data suggested that the microenvironment of this multienzyme system was unperturbed in spite of the treatment due to the isolation process. In terms of the composition of binding sites, MAOa appeared to be heterogeneous while MAOb appeared to be more homogeneous. Since the isolated fractions MAOa and MAOb possessed marked different substrate specificities, these observations directly implied that monoamine oxidase multiple forms do exist in situ. The extracted extensive parameters, n (specific binding activity, nanomoles/mg of protein), as well as the measured characteristic transition temperatures, indicated that the relative abundance of the sites which directly affected substrate specificities was indeed altered. The consistency of the characteristic transition temperatures of 21 degrees and 38 degrees for the case of intact membrane preparations was particularly significant. A tenable hypothesis is that the manipulation in the composition of the monoamine oxidase binding forms through intimate lipid-protein interactions, which has been amply demonstrated in many biomembrane systems to be functionally important might be the underlying regulatory mechanism in vivo.     

Interactions of imidazoline ligands with the active site of purified monoamine oxidase A

The two forms of monoamine oxidase, monoamine oxidase A and monoamine oxidase B, have been associated with imidazoline-binding sites (type 2). Imidazoline ligands saturate the imidazoline-binding sites at nanomolar concentrations, but inhibit monoamine oxidase activity only at micromolar concentrations, suggesting two different binding sites [Ozaita A, Olmos G, Boronat MA, Lizcano JM, Unzeta M & García-Sevilla JA (1997) Br J Pharmacol121, 901-912]. When purified human monoamine oxidase A was used to examine the interaction with the active site, inhibition by guanabenz, 2-(2-benzofuranyl)-2-imidazoline and idazoxan was competitive with kynuramine as substrate, giving K(i) values of 3 microM, 26 microM and 125 microM, respectively. Titration of monoamine oxidase A with imidazoline ligands induced spectral changes that were used to measure the binding affinities for guanabenz (19.3 +/- 3.9 microM) and 2-(2-benzofuranyl)-2-imidazoline (49 +/- 8 microM). Only one type of binding site was detected. Agmatine, a putative endogenous ligand for some imidazoline sites, reduced monoamine oxidase A under anaerobic conditions, indicating that it binds close to the flavin in the active site. Flexible docking studies revealed multiple orientations within the large active site, including orientations close to the flavin that would allow oxidation of agmatine.     

Early changes in mitochondrial monoamine oxidase activity of rat liver during 2-acetylaminofluorene feeding

The activities of mitochondrial type A and B monoamine oxidase were determined in the liver of rats fed a diet containing 2-acetylaminofluorene (AAF). Three days after the initiation of AAF-feeding, there was a significant decrease of type B monoamine oxidase activity without affect on type A enzyme. The decreased activity of type B monoamine oxidase, which reached a minimum after three weeks, was sustained for as long as AAF-feeding was continued. Sex-related difference in response to AAF was seen in the rat with respect to the onset and the intensity of the decreased type B monoamine oxidase activity, male rats being more sensitive to the carcinogen than female rats. In contrast to the in vivo effect, AAF showed a potent inhibitory effect on type A monoamine oxidase, rather than on type B enzyme, when added in vitro. The pI₅₀ values were estimated to be 7.5 against type A monoamine oxidase and 4.1 against type B enzyme, respectively. The in vitro inhibition of both types of monoamine oxidase by AAF was competitive. The K_i values for AAF were calculated to be 9.51 · 10^?9 M for type A monoamine oxidase and 1.30 · 10^?5 M for type B enzyme, respectively. In accordance with the potent inhibitory effect of AAF on type A monoamine oxidase in vitro, a single administration of the carcinogen, at a dose of 50 mg/kg, resulted in a marked and temporal decrease of the enzyme activity in the mitochondria of male rat liver. Recovery of the decreased type B monoamine oxidase activity was slow, and the enzyme activity did not return to control levels, even if rats were fed the basal diet for 2 or 4 weeks after the cessation of AAF-feeding.     

A biochemically distinct form of cytochrome oxidase (COX) deficiency in the Saguenay-Lac-Saint-Jean region of Quebec.

We report the results of biochemical and molecular investigations on a group of patients from the Saguenay-Lac-Saint-Jean region of Quebec who have an unusual form of cytochrome oxidase deficiency and Leigh disease. This group can be distinguished from the classical presentation of cytochrome oxidase deficiency with Leigh disease, by the severity of the biochemical defect in different tissues. The activity in skin fibroblasts, amniocytes, and skeletal muscle of cytochrome oxidase is 50% of normal, while in kidney and heart it is close to normal values. Brain and liver, on the other hand, have very low activities. The defect in activity appears to result from a failure of assembly of the cytochrome oxidase complex in liver, but levels of mRNA for both mitochondrially encoded and nuclear-encoded subunits in liver and skin fibroblasts were found to be the same as those in controls. The cDNA sequence of the liver-specific cytochrome oxidase subunits VIa and VIIa were determined in samples from patient liver and skin fibroblasts and showed normal coding sequence.     

On hepatic mitochondrial monoamine oxidase activity in lipid deficiency.

A marked decrease in liver mitochondrial monoamine oxidase activity was noticed in rats fed a fat-free diet as compared with that of their controls. In lipid-deprived rats, the specific activity of this enzyme was very low towards different substrates studied. The activity of kynurenine 3-monooxygenase, which like monoamine oxidase is localized on the mitochondrial outer membrane, was similarly depressed under conditions of lipid deprivation. On the other hand no major changes were observed in the activity of the inner membrane enzyme, kynurenine amino-transferase. Mitochondria from fat-free diet-fed rats were deficient in essential fatty acids whereas no appreciable variations were found in the relative proportions of phospholipids in comparison with those of control mitochondria. Mitochondrial monoamine oxidase activity of the deficient rats retained its sensitivities to inhibitor drugs like clorgyline and deprenyl. No changes were noticeable in the substrate specificity of monoamine oxidase in these rats. When we switched the fat-free diet-fed rats to a diet supplemented with a source of essential fatty acids, there was an elevation in the activities of both monoamine oxidase and kynurenine 3-monooxygenase, their levels approaching those of the control rats.     

Presence of two active X chromosomes in hybrids between normal human and SV40-transformed fibroblasts from patients with the Lesch-Nyhan syndrome

Skin fibroblasts (LNSV) derived from a hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient patient with the Lesch-Nyhan syndrome, who has glucose-6-phosphate dehydrogenase (G6PD) type A, were transformed with SV40 and hybridized with WI38 human diploid fibroblasts derived from a female embryo which have normal HGPRT and G6PD type B activities. The hybrid clones selected in hypoxanthine, aminopterin and thymidine (HAT) medium, were essentially tetraploid and contained three X and one Y chromosomes. These hybrids contained HGPRT, types A and B and the AB heteropolymeric form of G6PD enzymes which were indicative that in these cells X linked genes of both parental cells were fully active. Hybrids back-selected in medium containing 8-azaguanine (8-AG) contained only two X chromosomes. They had no HGPRT activity and they contained only G6PD type A enzyme. It is concluded that the hybrid cells which grew in the presence of 8-AG retained the X chromosome of the LNSV parental cell and apparently the inactive X of the WI 38 cell.     

Partial hypoxanthine-guanine phosphoribosyl transferase deficiency with full expression of the Lesch-Nyhan syndrome

Summary A patient with the full clinical expression of the classical Lesch-Nyhan syndrome is presented with a residual hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity of 5–10% in erythrocyte lysate and about 30% in fibroblast lysate. The activities of other erythrocyte enzymes of purine metabolism were typical for a classical Lesch-Nyhan patient. The effects of allopurinol therapy on the excretion of urinary purine metabolites were studied by a newly developed isotachophoretic technique.The unusually high residual activity of HGPRT in erythrodytes and fibroblasts of the patient enabled the enzymologic characterization of the mutant enzyme: in fibroblasts the affinities for the substrates hypoxanthine and guanine were normal. However, there was an increased apparent K _m for phosphoribosylpyrophosphate (PRPP), a complete absence of product inhibition by IMP and GMP, and a decreased heat stability. Addition of PRPP did not stabilize the mutant enzyme. In addition to the altered properties of the fibroblast enzyme, the K _m of the erythrocyte enzyme for hypoxanthine was also increased.Immunoprecipitation experiments revealed the presence of an approximately normal amount of material cross-reacting with anti-human HGPRT antiserum. However, it appeared that this cross-reacting material had a decreased stability. When intact erythrocytes were incubated with radiolabeled purine bases, no formation of IMP or GMP could be detected, despite the relatively high residual activity of HGPRT in the hemolysate. The results fit the following hypothesis: as a consequence of a structural mutation affecting the PRPP-site of the enzyme and a decreased heat stability, the activity of the mutant enzyme under in vivo conditions is virtually zero.In the erythrocytes of the patient's mother a normal HGPRT-activity was found. However, the activity in her fibroblasts was lower than normal, while a decreased heat stability and an intermediate behavior towards IMP could be shown.Hair root analysis of several members of the patient's family confirmed the heterozygosity of the mother, whereas no other heterozygotes could be detected. The family anamnesis did not show other cases of Lesch-Nyhan syndrome. These findings were taken as evidence that the patient described in this paper might represent a mutation orginating from the gametes in either of the maternal grandparents.