Myelin-Associated Calpain II

Anti-chicken muscle calpain (calcium-activated neutral protease) antibody (ACAb) was found to be absorbed by purified human brain myelin when titrated by enzyme-linked immunosorbent assay, suggesting the close association of the protease with myelin. To confirm this, calcium-dependent protease was extracted from myelin membrane and purified on a phenyl Sepharose CL 4B column. It was activated by calcium ion in the millimolar range, and therefore was determined to be calpain II. This enzyme fraction was electrophoresed and immunostained with ACAb, resulting in staining as a single band with apparent molecular weight of 80K. This protease degraded exogenous myelin-associated glycoprotein. From the present results, it is suggested that calpain is bound to myelin membrane and involved in the turnover of myelin proteins.     

Inhibition and binding modes of low-molecular-weight inhibitors of porcine pancreatic alpha-amylase.

Inhibition of porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrase) [EC 3.2.1.1] with maltotriitol (G3OH) and 4-phenylimidazole was investigated by using maltohexaitol (G6OH) and p-nitrophenyl-alpha-D-maltoside (G2PNP) as substrates. When G6OH was the substrate, both G3OH and 4-phenylimidazole behaved as competitive inhibitors. On the other hand, when G2PNP was the substrate, G3OH behaved as a competitive inhibitor, whereas 4-phenylimidazole behaved as a non-competitive inhibitor. Further inhibition study in the presence of both G3OH and 4-phenylimidazole, with G6OH as the substrate, showed that the two inhibitors compete with each other for the active site of the enzyme. Based on a consideration of the productive (reactive) binding modes of G2PNP and G6OH, and a nonproductive (nonreactive) binding mode of G2PNP, it is suggested that the binding sites of the two inhibitors may be partially overlapping around the catalytic site of the enzyme and that the rest of the binding site of each inhibitor lies along the substrate binding cleft of the enzyme.     

The reconstituted NADP photoreducing system by rebinding of the large form of ferredoxin-NADP reductase to depleted thylakoid membranes

The large form of ferredoxin-NADP reductase (FNR-L) was prepared by reassociating the small form of the enzyme (FNR-S) and connectein isolated from spinach leaves. The re-formed FNR-L could be rebound to depleted thylakoids from which most of the "built-in" FNR-L had been extracted. This rebinding of FNR-L brought about good restoration of the diminished NADP photoreducing activity of depleted thylakoids. Although rebinding of FNR-S to the depleted thylakoids took place with or without connectein, restoration of the NADP photoreducing activity required involvement of connectein. It becomes clear that involvement of connectein in the binding of FNR to thylakoids is indispensable for giving the physiological function of NADP photoreducing activity to the flavin enzyme on the surface of thylakoid membranes. It is most likely that FNR-L is the functional entity at the final step of the photosynthetic electron transport system in chloroplasts.     

Structure of the NDH-2 – HQNO inhibited complex provides molecular insight into quinone-binding site inhibitors

Type II NADH:quinone oxidoreductase (NDH-2) is a proposed drug-target of major pathogenic microorganisms such as Mycobacterium tuberculosis and Plasmodium falciparum. Many NDH-2 inhibitors have been identified, but rational drug development is impeded by the lack of information regarding their mode of action and associated inhibitor-bound NDH-2 structure. We have determined the crystal structure of NDH-2 complexed with a quinolone inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). HQNO is nested into the slot-shaped tunnel of the Q-site, in which the quinone-head group is clamped by Q317 and I379 residues, and hydrogen-bonds to FAD. The interaction of HQNO with bacterial NDH-2 is very similar to the native substrate ubiquinone (UQ₁) interactions in the yeast Ndi1–UQ₁ complex structure, suggesting a conserved mechanism for quinone binding. Further, the structural analysis provided insight how modifications of quinolone scaffolds improve potency (e.g. quinolinyl pyrimidine derivatives) and suggests unexplored target space for the rational design of new NDH-2 inhibitors.     

The amino-terminal tails of the core histones and the translational position of the TATA box determine TBP/TFIIA association with nucleosomal DNA.

We establish that the TATA binding protein (TBP) in the presence of TFIIA recognizes the TATA box in nucleosomal DNA dependent on the dissociation of the amino-terminal tails of the core histones from the nucleosome and the position of the TATA box within the nucleosome. We examine TBP/TFIIA access to the TATA box with this sequence placed in four distinct rotational frames with reference to the histone surface and at three distinct translational positions at the edge, side and dyad axis of the nucleosome. Under our experimental conditions, we find that the preferential translational position at which TBP/TFIIA can bind the TATA box is within linker DNA at the edge of the nucleosome and that binding is facilitated if contacts made by the amino-terminal tails of the histones with nucleosomal DNA are eliminated. TBP/TFIIA binding to DNA at the edge of the nucleosome occurs with the TATA box in all four rotational positions. This is indicative of TBP/TFIIA association directing the dissociation of the TATA box from the surface of the histone octamer.     

Prion Protein Is Necessary for Latent Learning and Long-Term Memory Retention

1. The cellular prion protein, designated PrP^c, is a key molecule in the prion diseases but its physiological function remains unknown. To elucidate whether PrP^c plays some role in the central nervous system, we established a line of mice in which the PrP gene had been disrupted and subsequently conducted long-term observations.2. Performance in latent learning and passive avoidance was evaluated using water-finding and step-through tests, respectively.3. PrP ^–/– mice showed impaired performance in the water-finding test, indicating a disturbance in latent learning, at 23 weeks of age. In the step-through test, although the PrP ^–/– mice showed normal learning ability and short-term memory retention, they evidenced a significant disturbance in long-term memory retention.4. These results indicate that PrP^c is needed for certain types of learning and memory and that the loss of function of this protein may contribute to the pathogenesis of prion diseases.     

Effect of endurance exercise training on muscle glycogen supercompensation in rats

Nakatani, Akira, Dong-Ho Han, Polly A. Hansen, Lorraine A. Nolte, Helen H. Host, Robert C. Hickner, and John O. Holloszy. Effect of endurance exercise training on muscle glycogensupercompensation in rats. J. Appl.Physiol. 82(2): 711-715, 1997.The purpose of this study was to test the hypothesis that the rate and extent ofglycogen supercompensation in skeletal muscle are increased byendurance exercise training. Rats were trained by using a 5-wk-long swimming program in which the duration of swimming was gradually increased to 6 h/day over 3 wk and then maintained at 6 h/day for anadditional 2 wk. Glycogen repletion was measured in trained anduntrained rats after a glycogen-depleting bout of exercise. The ratswere given a rodent chow diet plus 5% sucrose in their drinking waterad libitum during the recovery period. There were remarkabledifferences in both the rates of glycogen accumulation and the glycogenconcentrations attained in the two groups. The concentration ofglycogen in epitrochlearis muscle averaged 13.1 ± 0.9 mg/g wet wtin the untrained group and 31.7 ± 2.7 mg/g in the trained group(P < 0.001) 24 h after the exercise.This difference could not be explained by a training effect on glycogensynthase. The training induced ~50% increases in muscle GLUT-4glucose transporter protein and in hexokinase activity inepitrochlearis muscles. We conclude that endurance exercise trainingresults in increases in both the rate and magnitude of muscle glycogensupercompensation in rats.

     

Subcellular distribution and properties of kynurenine pyruvate transaminase in rat kidney.

Kynurenine transaminase activity in rat kidney was found in both the mitochondrial and supernatant fractions. These fractions contained (a) kynurenine pyruvate transaminase, which showed a preference for pyruvate as amino acceptor, and had a pH optimum between 8.0 and 8.5, and (b) kynurenine 2-oxoglutarate transaminase, with a preference for 2-oxoglutarate and a pH optimum between 6.0 and 6.5. The apparent Km value of the former enzyme for L-kynurenine was much lower than that of the latter enzyme.     

Production and Characterization of Monoclonal Antibodies Against Myelin-Associated Glycoprotein

Monoclonal antibodies against myelin-associated glycoprotein were generated by fusing mouse myeloma cells with spleen lymphocytes from BALB/c mice immunized with human myelin-associated glycoprotein purified from CNS myelin. Three groups of antibodies were identified: IgG antibodies recognizing the polypeptide moiety and IgG and IgM antibodies recognizing the carbohydrate moiety of the intact molecule. Properties of these antibodies were examined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the immunostaining technique using human CNS and peripheral nerve myelin, and ganglioside fractions isolated from human brain and peripheral nerve, and with immunohistochemical staining of human peripheral nerves. Part of human peripheral blood mononuclear cells was stained with the antibodies against the carbohydrate moiety, but not with IgG antibodies recognizing the polypeptide moiety. Natural killer activity was partially reduced after treatment of human peripheral blood lymphocytes with an IgM antibody and complement in vitro. The possibility that anti-myelin-associated glycoprotein antibodies might play a role in the pathogenesis of demyelinating diseases through modification of natural killer activity is discussed.     

Preparation of fractionated low-molecular-weight poly(S-carboxymethyl-L-cysteine) by ion-exchange chromatography

N-Carboxyanhydride of S-carbobenzoxymethyl-L -cysteine was polymerized by the addition of ethylamine as an initiator at anhydride–initiator ratios A/I = 3, 4, 6, 8, 12, and 16. Acetylation of the amino terminal was carried out with p-nitrophenylacetate. After debenzylation, low-molecular-weight samples of poly(S-carboxymethyl-L -cysteine) were obtained for A/I = 8, 12, and 16. These samples were fractionated by ion-exchange chromatography (DEAE–cellulose). About six fractions of narrow molecular-weight distributions were obtained. Their degrees of polymerization (DP) ranged from 8 to 16, as determined from proton nmr and also estimated based on their elution volumes on the ion-exchange chromatography at constant ionic strengths. This range of DP covered the range of interest concerning the formation of the β-structure in aqueous solutions; a fraction did not form the β-structure, whereas another fraction did. The results of the gel filtration (Biogel P-10) of the fractionated samples were consistent with the estimated DPs.