Methionine adenosyltransferase in man: evidence for multiple forms.

Optimum conditions are presented for the assay of methionine adenosyltransferase (ATP:l-methionine S-adenosyltransferase, EC 2.5.1.6) in human erythrocytes and in human cultured fibroblasts and long-term lymphoid cell lines. The enzymes are similar in having an acidic pH optimum (5.2–5.7) and in being inhibited by more than 10 mm Mg²⁺ in excess of ATP. In contrast, the transferase of human liver, studied previously, is most active at pH 6.8 and requires 100 mm excess Mg²⁺ for greatest activity.Chromatography on DEAE-cellulose has been utilized to demonstrate that the transferases of liver, erythrocytes, cultured fibroblasts, and cultured lymphoid cell lines are different molecular forms. The liver enzyme, which chromatographs mainly as a single sharp peak, is less tightly bound to DEAE-cellulose than are the transferases of the three types of cells. Each type of cell contains a characteristic mixture of active forms of methionine adenosyltransferase; there are two major forms, present in different relative proportions.It is proposed that the liver enzyme is under separate genetic control from the transferases of erythrocytes and cultured cells. This would explain the presence, in two children with persistent hypermethioninemia and demonstrated deficiency of hepatic methionine adenosyltransferase, of normal amounts of transferase activity in erythrocytes, cultured fibroblasts, and cultured lymphoid cell lines.     

Beef muscle troponin: evidence for multiple forms of troponin-T.

A method is described for the purification of troponin from beef skeletal muscle. The resultant preparation differs from the troponin of rabbit skeletal muscle in that it contains at least two forms of the tropomyosin-binding component, Troponin-T: these are designated as the 37 000 and 40 000 dalton forms of Troponin-T on the basis of sodium dodecyl sulphate gel electrophoresis. Either of these Troponin-T forms may be used to reconstitute troponin by mixing with the appropriate amounts of the calcium-binding (Troponin-C) and and actomyosin ATPase-inhibitory (Troponin-I) components. These reconstituted troponins are shown to interact with tropomyosin and also to confer full calcium sensitivity on actomyosin ATPase. Despite the existence of proteolysis in troponin preparations, the experimental evidence indicates that the smaller form of Troponin-T is not derived from the 40 000 dalton species by limited degradation. Although both species of Troponin-T have been found routinely in troponin from beef skeletal muscle, only the larger form is detected in troponin preparations from beef cardiac muscle. Further studies are required in order to clarify the functional significance and differential distribution of these multiple forms of Troponin-T.     

Multiple isoelectric forms of poliovirus RNA-dependent RNA polymerase: evidence for phosphorylation.

Poliovirus-specific RNA-dependent RNA polymerase (3Dpol) was purified to apparent homogeneity. A single polypeptide of an apparent molecular weight of 63,000 catalyzes the synthesis of dimeric and monomeric RNA products in response to the poliovirion RNA template. Analysis of purified 3Dpol by two-dimensional electrophoresis showed multiple forms of 3Dpol, suggesting posttranslational modification of the protein in virus-infected cells. The two major forms of 3Dpol appear to have approximate pI values of 7.1 and 7.4. Incubation of purified 3Dpol with calf intestinal phosphatase resulted in almost complete disappearance of the pI 7.1 form and a concomitant increase in the intensity of the pI 7.4 form of 3Dpol. Addition of 32P-labeled Pi during infection of HeLa cells with poliovirus resulted in specific labeling of 3Dpol and 3CD, a viral protein which contains the entire 3Dpol sequence. Both 3Dpol and 3CD appear to be phosphorylated at serine residues. Ribosomal salt washes prepared from both mock- and poliovirus-infected cells contain phosphatases capable of dephosphorylating quantitatively the phosphorylated form (pI 7.1) of 3Dpol.     

Chromatographic evidence for the existence of multiple forms of cathepsin B1

Preparations of ox spleen cathepsin B1 have been found to give multiple peaks of activity upon chromatography on DEAE-cellulose in NaCl gradients or by equilibrium chromatography in 0.05m-NaCl. CM-cellulose gradient chromatography also shows several cathepsin B1 peaks. This evidence indicates that ox spleen cathepsin B1 can exist in at least three to five forms. All forms have the same molecular weight, are thiol-activated and are inhibited by typical thiol inhibitors. The possible sources of this multiplicity of activity are discussed and a possible physiological role for cathepsin B2 is suggested.     

Studies on the mechanism of oxidative phosphorylation. ADP promotion of GDP phosphorylation

The process of ATP or GTP synthesis by bovine heart submitochondrial particles involves the binding of ADP or GDP to 3 exchangeable sites I, II, and III, and only upon substrate occupation of site III does rapid ATP or GTP synthesis take place. The dissociation constants determined for ADP were KADPI less than or equal to 10(-8) M, KADPII approximately 10(-7) M, and KADPIII (equivalent to apparent KADPm), approximately 3 x 10(-6) M in the low Km mode and KADPIII approximately 150 x 10(-6) M in the high Km mode. For GDP, these constants were KGDPI approximately 10(-6)-10(-5) M, KGDPII approximately 10(-4) M, and KGDPIII approximately 10(-3) M when NADH was the respiratory substrate (Matsuno-Yagi, A., and Hatefi, Y. (1990) J. Biol. Chem. 265, 82-88). Because of its low affinity for the above binding sites, GDP at micromolar concentrations does not lead to GTP synthesis. However, as shown in this paper, micromolar [GDP] undergoes phosphorylation in the presence of micromolar concentrations of ADP. Under these conditions, both ATP and GTP are synthesized. GDP inhibits ATP synthesis with KGDPi congruent to 7 microM, while ADP promotes GTP synthesis in a reaction that requires inorganic phosphate (apparent KPim = 2-3 mM) and is inhibited by uncouplers and inhibitors of the ATP synthase complex. The ADP-promoted GTP synthesis exhibited an "apparent" KGDPm = 4 microM and an "apparent" Vmax = 11 nmol of GTP (min.mg of protein)-1. These results were interpreted to mean that (a) micromolar [ADP] occupies sites I and II, allowing site III to bind and phosphorylate GDP, and (b) the KGDPm and Vmax calculated under these conditions represent values for the low Km-low Vmax mode of GTP synthesis, which in the absence of ADP is not detectable because of the positive cooperativity phase of GTP synthesis with the high KGDPII approximately 10(-4) M.     

Studies on Chinese hamster ovary mutants showing multiple cross-resistance to oxidative phosphorylation inhibitors

Several stable Chinese hamster ovary (CHO) mutants were selected after ethylmethane sulfonate mutagenesis for resistance to oligomycin, ruatmycin, venturicidin, or antimycin. These mutants shared a number of common properties. They exhibited cross-resistance to those drugs which act on oxidative phosphorylation, irrespective of the structure and site of action of the drug. All the mutants showed a reduced ability to grow in suspension and to reach high saturation densities. They were also unable to use galactose as a carbon source. The short lag period required for selection (10-15 days), the similarity of the mutation rates for resistance to each of the four drugs, the high variance/mean ratios in fluctuation tests, and the recessive behavior of the resistance marker in hybrids suggest that the mutations responsible for resistance to oxidative phosphorylation inhibitors in CHO cells are coded by nuclear DNA. Segregation experiments indicated no linkage between the oligomycin-resistant marker (OLG) AND Thg (thioguanine resistance). Oxidative phosphorylation, as measured by the rate of respiration coupled to phosphorylation in whole cells remained as sensitive to the drugs in the mutants as in the parental cell line. Glucose transport and the overall Krebs' cycle activities also appeared similar in the mutants and the wild type. All the mutants had an increased rate of lactic acid production (up to twofold), associated with increased specific activities for several glycolytic enzymes when assayed in cell-free extracts.     

Studies on oxidative phosphorylation and ATPase activity of fresh and frozen brain mitochondria

The presence of bovine serum albumin in the incubation medium resulted in increased P:O values with brain mitochondria in limiting concentrations as well as particles prepared in hypertonic sucrose or those exposed to a hypotonic medium.Frozen brain mitochondria retained some of their oxidative phosphorylating ability after storage for several days at −15 °C. The activity was greatly increased by the addition of DPN and nicoti namide to the incubation medium.The ATPase of freshly prepared brain mitochondria was almost completely devoid of 2,4-dinitrophenol-stimulating ability but was greatly activated by Mg⁺⁺.     

Immunodetection of multiple species of retinoic acid receptor alpha: evidence for phosphorylation.

Polyclonal and monoclonal antibodies were raised against synthetic peptides (or fusion protein) corresponding to cDNA-deduced amino acid sequences unique to the human and mouse retinoic acid (RA) receptor alpha 1 (hRAR-alpha 1 and mRAR-alpha 1, respectively). Two rabbit polyclonal antibodies directed against either the F region fused to DHFR [RP alpha (F)] or the D2 region [RP alpha (D2)] were selected. Using either immunocytochemistry, Western blotting analysis, or immunoprecipitation, they were found to be specific for human and mouse RAR-alpha 1 proteins produced by COS-1 cells transiently transfected with vectors expressing the RAR-alpha 1 cDNA. Three mouse monoclonal antibodies directed against either the F region [(Ab9 alpha (F) and Ab12 alpha (F)] or the A1 region [Ab10 alpha 1(A1)] recognized transiently expressed human and mouse RAR-alpha 1 proteins, when either immunocytochemistry or immunoprecipitation was used. In addition, Ab9 alpha (F) and Ab12 alpha (F), but not Ab10 alpha 1(A1), revealed the RAR-alpha 1 proteins by Western blotting analysis. Ab9 alpha (F) was also able to "supershift" RAR-alpha 1 protein-RARE oligonucleotide probe complexes in gel retardation assays. All these antibodies recognized also the transiently expressed mRAR-alpha 2 isoform, with the exception of Ab10 alpha 1 (A1), which is specific for the A1 region of RAR-alpha 1. These antibodies have enabled us to detect the presence of mRAR-alpha as multiple species in mouse embryo and adult tissue extracts as well as in embryonal carcinoma (EC) cells. Moreover, we found that one of these species (51 kDa) was phosphorylated in EC cells. This phosphorylation was not affected by RA treatment, but appeared to be dependent on the differentiation state of the EC cells.     

Influence of NAD-linked dehydrogenase activity on flux through oxidative phosphorylation.

1. We have examined systematically the relationship between the percentage reduction of cardiac mitochondrial NAD and the flux through oxidative phosphorylation, as measured by O2 uptake. Reduction of NAD was varied by varying the concentration of palmitoyl-L-carnitine, pyruvate, 2-oxoglutarate or glutamate in the presence of malate as the oxidizable substrate. 2. In the presence of ADP (State 3 respiration) there was a substantially linear positive relationship between O2 uptake and the percentage reduction of NAD. Coupled respiration in the absence of ADP also showed an increase with increasing NADH, with the exact shape of the relationship being variable. 3. When pyruvate and 2-oxoglutarate dehydrogenase activity were increased by increasing medium Ca2+ concentration within the range 5 nM to 1.23 microM, at non-saturating substrate concentrations, there was again a positive relationship between O2 uptake and the reduction of NAD; however, rates of O2 uptake tended to be higher at given values of NAD reduction when the incubation medium contained Ca2+. This is taken to indicate an activation by Ca2+ of the enzymes of phosphorylation or of the respiratory chain, in addition to the dehydrogenase activation. 4. When carboxyatractyloside plus ADP were used to generate 50% State 3 rates of O2 uptake with pyruvate or 2-oxoglutarate, sensitivity to Ca2+ was retained. However, when oligomycin plus 1 mM-ADP and 1 mM-ATP were used to generate 50% State 3, no such dependence was seen. 5. The results are interpreted to indicate a substantial role for substrate dehydrogenation in the overall regulation of oxidative phosphorylation when substrates are available at near-physiological concentrations.     

A requirement for ubiquinone in ATPase activity and oxidative phosphorylation.

The suggestion that a rapidly sedimenting rough endoplasmic reticulum fraction in close association with mitochondria, is the preferred site of cytochrome P-450 synthesis has been examined. The rate of cytochrome P-450 synthesis in the different subcellular fractions has been evaluated $\text{in}\text{vivo}\text{and}\text{in}\text{vitro}$, using the immunoprecipitation technique. The results indicate that the conventional microsomal fraction (100,000 X g sediment) is the major site of cytochrome P-450 synthesis and that the rapidly sedimenting rough endoplasmic reticulum fraction associated with mitochondria is not a preferred site for the hemoprotein synthesis.     

Studies on esterolytic activity of alternative complement component factor B

Alternative complement component factor B was purified from human plasma and its esterolytic activity on various α-naphthylester derivatives was examined. Of these substrates, LeuAlaArg-naphthylester was the most susceptible. The K_m value of factor B for this substrate was about 5·10⁻⁴ M. 6-Amidino2-naphthyl-4-guanidinobenzoate inhibited the esterolytic activity of factor B. After activation of factor B, the esterolytic activity did not increase and the decayed form, Bb, cleaved the substrate to the same extent as factor B.