In vitro synthesis of monoamine oxidase of rat liver outer mitochondrial membrane

Monoamine oxidase, an intrinsic protein of outer mitochondrial membrane, was purified from bovine liver and rabbit antibody against the enzyme was prepared. The antibody could react with the monoamine oxidase of rat liver mitochondria. When rat liver RNA was translated $\text{in}\text{vitro}$ using rabbit reticulocyte lysate and monoamine oxidase peptide in the translation products was immunoprecipitated by the antibody, the peptide was detected in the products programmed by the messenger RNA's from total and free polysomes but not that from bound polysomes. The enzyme synthesized $\text{in}\text{vitro}$ had the same apparent molecular size as the mature protein in outer mitochondrial membrane.     

Purification and properties of human liver monoamine oxidase

Human liver monoamine oxidase [monoamine: O₂ oxidoreductase (deaminating), E. C. 1.4.3.4] was purified by a method which does not depend on the isolation of mitochondria, and in which vacuum dialysis, during which the enzyme separates out as a yellow precipitate, is an important step in purification. By this method a final specific activity of 550 and fold purification of 40 was attained. A single peak was obtained with the analytical ultracentrifuge, and a sedimentation constant of 6.78S noted. A single active band was observed by polyacrylamide gel electrophoresis. The enzyme exhibits optimum activity at pH 8.7, with no activity below pH 5.5 or above pH 11.8. Using benzylamine hydrobromide as the substrate, in 0.05 m phosphate buffer (pH 7.4) at 27 °C, the Michaelis constant was found to be 1.7 × 10^?3m. The enzyme, which is quite stable, is a flavo-protein, as shown by absorption and fluorescence spectra. The C-terminal group is glycine. The molecular weight, as determined by SDS polyacrylamide-gel electrophoresis, is 64,000. Repeated attempts to determine the N-terminal group were unsuccessful.     

Dexamethasone selectively increases monoamine oxidase type a in human skin fibroblasts

The effects of several hormones known to affect monoamine oxidase activity $\text{in}\text{vivo}$ have been studied in living human skin fibroblasts grown in culture. Of the hormones tested, the synthetic glucocorticoid dexamethasone caused the greatest increases in activity at physiologic concentrations. Increases of 10–12 fold were observed after 8–9 days of exposure to 5 × 10^?8 M dexamethasone. This increase in activity was accompanied by a change in the relative proportion of the A and B types of activity in fibroblasts, from about 35% A:65% B in control cultures to 90% A:10% B in cultures exposed to dexamethasone. The increase in activity and the shift in the proportion of A and B activities could be accounted for almost exclusively by a specific increase in the number of Type A molecules.     

Biosynthesis of canine fibrinogen: In vitro synthesis of Aα, Bβ and γ precursor chains

An mRNA fraction from dog liver translated with a rabbit reticulocyte protein synthesizing system in the presence of [³⁵S]-methionine produces fibrinogen-related proteins which are immunoprecipitated with rabbit antiserum to dog fibrinogen. Analyses of these radioactive proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography indicate that the three fibrinogen chains (Aα, Bβ and γ) are synthesized separately as larger precursors. The putative pre Aα and pre Bβ chains were characterized by their susceptibility to treatment with thrombin and batroxobin. Thrombin degraded the pre Aα and pre Bβ chains, while batroxobin only acted on the pre Aα chain. The pre γ chain was not degraded by these enzymes.     

Crystal structure of human monoamine oxidase B, a drug target enzyme monotopically inserted into the mitochondrial outer membrane

Monoamine oxidase B (MAO B) is an outer mitochondrial membrane protein that oxidizes arylalkylamine neurotransmitters and has been a valuable drug target for many neurological disorders. The 1.7 angstrom resolution structure of human MAO B shows the enzyme is dimeric with a C-terminal transmembrane helix protruding from each monomer and anchoring the protein to the membrane. This helix departs perpendicularly from the base of the structure in a different way with respect to other monotopic membrane proteins. Several apolar loops exposed on the protein surface are located in proximity of the C-terminal helix, providing additional membrane-binding interactions. One of these loops (residues 99-112) also functions in opening and closing the MAO B active site cavity, which suggests that the membrane may have a role in controlling substrate binding.     

Evidence for participation of the inner membrane in the import of sulfite oxidase into the intermembrane space of liver mitochondria

Sulfite oxidase, a soluble enzyme in mitochondrial intermembrane space, was synthesized as a precursor protein larger than the authentic enzyme when rat liver RNA was translated $\text{in}\text{vitro}$ using reticulocyte lysate. When the $\text{in}\text{vitro}$ translation products were incubated with isolated rat liver mitochondria, the precursor of sulfite oxidase was converted to the size of the mature enzyme. The $\text{in}\text{vitro}$ processed mature enzyme was no longer susceptible to externally added proteases and was extractable by a hypotonic treatment of the mitochondria, suggesting its location in the intermembrane space. When mitochondria were subfractionated, most of the processing activity was recovered in the mitoplast fraction. The import-processing activity of mitochondria was inhibited by CCCP, oligomycin, or atractyloside in the presence of KCN. These results suggest that the import of sulfite oxidase into mitochondrial intermembrane space requires the participation of inner membrane.     

Catalytic activity and arrangement of subunit polypeptides in rat liver cytochrome c oxidase as studied by proteolysis

Cytochrome c oxidase from rat liver was incubated with various proteinases of different specificities and the enzymic activity was measured after various incubation times. A loss of catalytic activity was found after digestion with proteinase K, aminopeptidase M and a mitochondrial proteinase from rat liver. In each case the decrease in enzymic activity was compared with the changes in intensities of the polypeptide pattern obtained after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The susceptibilities of the subunit polypeptides of the soluble cytochrome c oxidase to proteinases were very different. Whereas subunit I was most susceptible, subunits V–VII were rather resistant to degradation. From the relative inaccessibility of subunits V–VII to proteinases it is likely that these polypeptides are buried in the interior of the enzyme complex.     

Structural and functional degradation of Ca2+:Mg2+-ATPase rich sarcoplasmic reticulum vesicles photosensitized by erythrosin B

Erythrosin B (Red Dye No. 3) and Rose Bengal photosensitize the destruction of the Ca2+:Mg2+-ATPase pump protein in sarcoplasmic reticulum (SR) vesicles with respective quantum efficiencies of (1.53 +/- 0.19) X 10(-3) and (1.25 +/- 0.18) X 10(-3). Damage to vesicle function was assayed by measurements of increases in passive Ca2+ permeability. Rates of passive Ca2+ movement into the SR lumen were increased by dye photosensitization in proportion to radiation absorbed. Active Ca2+ transport into SR vesicles was blocked independent of radiation absorbed by Erythrosin B and Rose Bengal at free concentrations of 0.69 microM and 1.16 microM, respectively. The photochemical lability of the Ca2+ pump protein and alterations in passive and active Ca2+ transport may be dependent on the concentration of the dye in the membrane. The photosensitization results may have implications with respect to the suitability of Erythrosin B usage in vivo, since the brightness of our irradiation source is comparable to that of sunlight at 480 nm.     

Effects of intrastriatal kainic acid injection on [3H]dopamine metabolism in rat striatal slices: evidence for postsynaptic glial cell metabolism by both the type A and B forms of monoamine oxidase

Abstract: Intrastriatal injections of kainic acid (KA) were utilized to investigate the cellular localization of postsynaptic dopamine (DA) metabolism by type A and B monoamine oxidase (MAO) in rat striatum. At 2 days postinjection, maximal degeneration of cholinergic and γ-aminobutyric acid (GABA)ergic neurons was observed and found to be associated with a significant decrease in both type A and B MAO activity. However, over the next 8-day period, when only the process of gliosis appeared to be occurring, a selective return to control of type B MAO activity was seen. When the metabolism of [³H]DA (10^?7 M) was examined in 8-day KA-lesioned rat striatal slices, an increase in [³H]dihydroxyphenylacetic acid (DOPAC) and [³H]homovanillic acid (HVA) formation was observed. The KA-induced elevation of [³H]DOPAC formation (but not [³H]HVA) was abolished by the DA neuronal uptake inhibitor nomifensine. This is consistent with earlier findings suggesting that HVA is formed exclusively within sites external to DA neurons. Experiments with clorgyline and/or deprenyl revealed that the relative roles of type A and B MAO in striatal DA deamination remained unchanged following KA (90% deamination by type A MAO) even though total deamination was substantially enhanced. At high concentrations of [³H]DA (10^?5 M), deamination by type B MAO could be increased to 30% of the total MAO activity; however, this was observed in both control and KA-lesioned striata. These results suggest that KA-sensitive neurons contain type A and/or type B MAO. Moreover, whereas these neurons may metabolize DA, a major portion of postsynaptic DA deamination appears to occur within glial sites of rat striatal tissue. Furthermore, glial cells would appear to contain functionally important quantities of both type A and B MAO.     

N-(1-Methyl)cyclopropylbenzylamine: A novel inactivator of mitochondrial monoamine oxidase

N-(1-Methyl)cyclopropylbenzylamine was synthesized and shown to inactivate pig liver mitochondrial monoamine oxidase. Inactivation is time-dependent, concentration dependent, protected by the substrate and product of the enzyme, and is not reversed by exhaustive dialysis. Unlike N-cyclopropylbenzylamine, the adduct formed between N-(1-methyl)cyclopropylbenzylamine and monoamine oxidase is stable to treatment with benzylamine. A mechanism for the inactivation is proposed.     

Isolation and characterization of des(Ala-Lys)calmodulin in porcine brain

A calmodulin-like protein -des(Ala-Lys)calmodulin- was isolated from porcine brain extract, and was characterized in comparison to porcine brain calmodulin. Des(Ala-Lys)calmodulin was distinguishable from calmodulin by its slightly faster mobility in 10% polyacrylamide gels without sodium dodecyl sulfate. The protein gave an amino acid composition very similar to calmodulin, and contained one ?-N-trimethyllysyl residue. Comparative peptide mapping of calmodulin and des(Ala-Lys)calmodulin by high performance anion-exchange liquid chromatography, and the subsequent analyses of the isolated peptides, have indicated that des(Ala-Lys)calmodulin lacks the Ala(147)-Lys (148) sequence at the C-terminus of calmodulin. The content of des(Ala-Lys)-calmodulin was about one-tenth of calmodulin.     

Characterization and solubilization of the cytochalasin B binding component from human placental microsomes

Human placental microsomes exhibit uptake of d-[³H]glucose which is sensitive to inhibition by cytochalasin B (apparent K_i = 0.78 /gm M). Characterization of [³H]cytochalasin B binding to these membranes reveals a glucose-sensitive site, inhibited by d-glucose with an ED₅₀ = 40 mM. The glucose-sensitive cytochalasin B binding site is found to have a K_d = 0.15μM by analysis according to Scatchard. Solubilization with octylglucoside extracts 60–70% of the glucose-sensitive binding component. Equilibrium dialysis binding of [³H]cytochalasin B to the soluble protein displays a pattern of inhibition by d-glucose similar to that observed for intact membranes, and the measurement of an ED₅₀ = 37.5 mM d-glucose confirms the presence of the cytochalasin B binding component, putatively assigned as the glucose transporter. Further evidence is attained by photoaffinity labelling; ultraviolet-sensitive [³H]cytochalasin B incorporation into soluble protein (M_r range 42 000-68 000) is prevented by the presence of d-glucose. An identical photolabelling pattern is observed for incorporation of [³H]cytochalasin B into intact membrane protein, confirming the usefulness of this approach as a means of identifying the presence of the glucose transport protein under several conditions.     

Isolation, partial chemical and biological characterization of thymone B

A new approach to the study of the molecular arrangements of proteins in membranes is described. Irradiation with visible light of native erythrocytes or washed erythrocyte membranes suspended in buffers containing a) riboflavin, fluorescein or fluorescein coupled to dextran and b) ³H-labelled tryptophan resulted in incorporation of radioactivity into the membrane proteins. Polyacrylamide gel electrophoresis of solubilized membranes followed by radioactivity measurements of the separated membrane proteins revealed that in native erythrocytes the protein components known to be located at the exterior cell surface, Band 3 and the major sialoglycoproteins became specifically labelled, whereas in washed lysed cells all of the major membrane proteins were labelled.     

Antigenic subunit of the polypeptide antigenic complex of the Melvin strain of Neisseria gonorrhoeae.

An antigenic subunit of molecular weight 66,000 daltons has been isolated from the antigenic complex of the Melvin strain of $\text{Neisseria gonorrhoeae}$. Incubation of the complex in 8M urea at room temperature for four hours resulted in the dissociation of the subunit from the complex. It was separated from the complex by chromatography of the incubation mixture on a Sepharose 6B column in 50 mM ammonium bicarbonate pH 8.5 without 8M urea and further purified by affinity chromatography. This communication reports on a newly isolated antigenic protein devoid of LPS present in the bacteria.     

Genetic studies of the lac repressor. VI. The B116 repressor: an altered lac repressor containing amino acid specified by both the trp and lacI leader regions.

Human erythrocytes were treated with the diazanium salt of oligodeoxythymidylic acid 5′-p-aminophenylphosphate, a reagent unable to penetrate the plasma membrane. The oligomers, covalently linked to the cell surface, were extended by treatment with terminal deoxynucleotidyl transferase in the presence of deoxythymidine triphosphate. The cells then hybridized readily to columns of polyriboadenylic acid-agarose. We expect this technology to be of value for cell sorting, for the isolation of proteins accessible at the surfaces of cells, and for the synthesis of a variety of DNA-protein polymers.     

Design,synthesis, and pharmacological evaluation of 2-amino-5-nitrothiazole derived semicarbazones as dual inhibitors of monoamine oxidase and cholinesterase: effect of the size of aryl binding site

A series of 2-amino-5-nitrothiazole derived semicarbazones were designed, synthesised and investigated for MAO and ChE inhibition properties. Most of the compounds showed preferential inhibition towards MAO-B. Compound 4, (1-(1-(4-Bromophenyl)ethylidene)-4-(5-nitrothiazol-2-yl)semicarbazide) emerged as lead candidate (IC₅₀?=?0.212?µM, SI?=?331.04) against MAO-B; whereas compounds 21 1-(5-Bromo-2-oxoindolin-3-ylidene)-4-(5-nitrothiazol-2-yl)semicarbazide (IC₅₀?=?0.264?µM) and 17 1-((4-Chlorophenyl) (phenyl)methylene)-4-(5-nitrothiazol-2-yl)semicarbazide (IC₅₀?=?0.024?µM) emerged as lead AChE and BuChE inhibitors respectively; with activity of compound 21 almost equivalent to tacrine. Kinetic studies indicated that compound 4 exhibited competitive and reversible MAO-B inhibition while compounds 21 and 17 showed mixed-type of AChE and BuChE inhibition respectively. Docking studies revealed that these compounds were well-accommodated within MAO-B and ChE active sites through stable hydrogen bonding and/or hydrophobic interactions. This study revealed the requirement of small heteroaryl ring at amino terminal of semicarbazone template for preferential inhibition and selectivity towards MAO-B. Our results suggest that 5-nitrothiazole derived semicarbazones could be further exploited for its multi-targeted role in development of anti-neurodegenerative agents.

A library of 2-amino-5-nitrothiazole derived semicarbazones (4–21) was designed, synthesised and evaluated for in vitro MAO and ChE inhibitory activity. Compounds 4, 21 and 17 (shown) have emerged as lead MAO-B (IC₅₀:0.212?µM, competitive and reversible), AChE (IC₅₀:0.264?µM, mixed and reversible) and BuChE (IC₅₀:0.024?µM, mixed and reversible) inhibitor respectively. SAR studies disclosed several structural aspects significant for potency and selectivity and indicated the role of size of aryl binding site in potency and selectivity towards MAO-B. Antioxidant activity and neurotoxicity screening results further suggested their multifunctional potential for the therapy of neurodegenerative diseases.     

In vitro synthesis of a putative precursor of serine: pyruvate aminotransferase of rat liver mitochondria

Serine:pyruvate aminotransferase [EC 2.6.1.51] of rat liver, an enzyme induced by glucagon in mitochondria, was synthesized in cell-free protein synthesizing systems derived from nuclease-treated rabbit reticulocyte lysate and wheat germ extract as a putative precursor which was approximately 2,000 daltons larger than the subunit of mature enzyme. The hepatic level of translatable messenger RNA coding for the putative precursor was approximately 40 times higher in rats received a glucagon administration 3.5 h before sacrifice than in control animals.     

Rendomab B4, a monoclonal antibody that discriminates the human endothelin B receptor of melanoma cells and inhibits their migration

Metastatic melanoma is an aggressive cancer with a poor prognostic, and the design of new targeted drugs to treat melanoma is a therapeutic challenge. A promising approach is to produce monoclonal antibodies (mAbs) against the endothelin B receptor (ETB), which is known to be overexpressed in melanoma and to contribute to proliferation, migration and vasculogenic mimicry associated with invasiveness of this cancer.

We previously described rendomab-B1, a mAb produced by DNA immunization. It is endowed with remarkable characteristics in term of affinity, specificity and antagonist properties against human ETB expressed by the endothelial cells, but, surprisingly, had poor affinity for ETB expressed by melanoma cells. This characteristic strongly suggested the existence of a tumor-specific ETB form. In the study reported here, we identified a new mAb, rendomab-B4, which, in contrast to rendomab-B1, binds ETB expressed on UACC-257, WM-266-4 and SLM8 melanoma cells. Moreover, after binding to UACC-257 cells, rendomab-B4 is internalized and colocalizes with the endosomal protein EEA-1. Interestingly, rendomab-B4, despite its inability to compete with endothelin binding, is able to inhibit phospholipase C pathway and migration induced by endothelin. By contrast, rendomab-B4 fails to decrease ERK1/2 phosphorylation induced by endothelin, suggesting a biased effect on ETB.

These particular properties make rendomab-B4 an interesting tool to analyze ETB-structure/function and a promising starting point for the development of new immunological tools in the field of melanoma therapeutics.     

Immunological relationships among subunits of glutathione S-transferases A, AA, B and ligandin and hybrid formation between AA and ligandin by guanidine hydrochloride

Immunological properties of ligandin(Lig) and glutathione S-transferase(GST)-A, -AA and -B were investigated for elucidating their subunit relationships. By using either anti-Lig or -AA antibody, GST-B made a clear common precipitin line with Lig or AA in double immunodiffusion and the activity was inhibited intermediately between Lig and AA, whereas Lig and AA reacted very weakly with antibodies to each other. A hybrid between Lig and AA formed by guanidine hydrochloride treatment was identified immunochemically to be GST-B. GST-A had no immunological relationship with any of other three forms.