Mitsuo Tagaya

Association of N‐ethylmaleimide‐sensitive factor with synaptic vesicles

N‐Ethylmaleimide‐sensitive factor (NSF) mediates docking and/or fusion of transport vesicles in the multi‐pathways of vesicular transport. NSF was highly expressed in brain and adrenal gland. Immunostaining of cerebellum with an anti‐NSF monoclonal antibody showed that NSF is predominantly localized in the molecular layers and the glomeruli of the granule cell layers. This distribution coincided well with that of synaptophysin, a marker protein of synaptic vesicles. Purification and immunoprecipitation revealed that NSF is associated with brain synaptic vesicles. The present results suggest that NSF is associated with synaptic vesicles without Ca2+ influx.

Correlation between phospholipase A2 activity and intra-Golgi protein transport reconstituted in a cell-free system

A wide variety of phospholipase A2 inhibitors blocks intra‐Golgi protein transport reconstituted in a cell‐free system. Phospholipase A2 activity detectable under the protein transport assay conditions is actually inhibited by the inhibitors. There is a good correlation between the inhibition of protein transport and that of phospholipase A2 activity. Prolactin secretion from GH3 cells is also blocked by a membrane‐permeable phospholipase A2, inhibitor, suggesting the physiological relevance to inhibition of protein transport in vitro by phospholipase A2 inhibitors.

Identification of α‐subunit Lys201 and β‐subunit Lys115 at the ATP‐binding sites inEscherichia coli F1‐ATPase

Binding of about 1 mol of adenosine triphosphopyridoxal to Escherichia coli F1‐ATPase resulted in the nearly complete inactivation of the enzyme [(1987) J. Biol. Chem. 262, 7686–7692]. About two thirds of the label was bound to the α‐subunit, and the rest to the β‐subunit. The present study revealed that Lys201 in the α‐subunit and Lys155 in the glycinerich region of the β‐subunit are the major sites labeled with this reagent. Thus, these two residues might be located close to the γ‐phosphate of the bound ATP.

Adenosine di-, tri- and tetraphosphopyridoxals modify the same lysyl residue at the ATP-binding site in adenylate kinase

Adenosine diphosphopyridoxal modifies Lys‐21 in adenylate kinase which is located in a glycine‐rich loop [(1987) J. Biol. Chem. 262, 8257–8261]. We presently report that adenosine tri‐ and tetraphosphopyridoxals modify the same lysyl residue more rapidly than the diphospho compound does. However, susceptibilities of the Schiff bases between the labels and the lysyl residue to sodium borohydride considerably differ in the modifications with the three reagents. These observations seem to be ascribable to the mobility of the ε‐amino group of Lys‐21 in the active‐site region of the enzyme.

Phosphorylase Isozymes of Higher Plants

The relationship among structure, function and regulation in phosphorylase is discussed on the basis of structural comparison between the rabbit muscle and potato tuber enzymes. The two forms of phosphorylase, each of which has similar structural and catalytic properties, are shown to be present in both leaf and tuber of potato. Tapioca leaf also contains the two forms of similar properties. Thus, higher plant phosphorylases are classified into two types of isozymes.

Affinity labeling of the essential lysyl residue at the active site of enzymes by using nucleotide polyphosphate pyridoxal

We have discovered a new type of affinity labeling reagents for the nucleotide-binding site of protein by introducing an active site-directing moiety to pyridoxal 5′-phosphate. Uridine diphosphopyridoxal almost completely inactivated glycogen synthase in a time-dependent manner (Kinact=25 µM;k0=0.22 min−1). The inactivation was pronouncedly protected by UDP-Glc and UDP, but not by the allosteric activator Glc-6-P. The addition of cysteamine to the inactivated enzyme restored the original activity, whereas the treatment of the inactivated enzyme with NaBH4 resulted in the fixation of the label to the enzyme protein. A peptide containing the label was isolated after proteolysis, and sequenced as E-V-A-K*-V-G-G-I-(Y). Adenosine polyphosphopyridoxal considerably inactivated lactate dehydrogenase in a time-dependent manner. The degree of inactivation was dependent on the number of phosphate groups; 64% (N=2), 51% (N=3), and 35% (N=4) at a reagent concentration of 1 mM for 30 min. The inactivation was protected by NADH, but not by pyruvate. Although the inactivation was not completed, the reagent was stoichiometrically incorporated into enzyme protein concomitantly with the inactivation. Affinity chromatographic analysis of the inactivated enzyme of Blue-Toyopearl revealed the presence of several protein species. The ratio of those species changed according to the stage of inactivation.

Base-catalyzed reactivation of glucogen phosphorylase reconstituted with a coenzyme-substrate conjugate and its analogues

Glycogen phosphorylase reconstituted with pyridoxal (5′)diphospho(1)‐α‐D‐glucose (PLDP‐Glc) is catalytically inactive but slowly converted to the active enzyme through the cleavage of the pyrophosphate linkage. A similar reaction occurs more rapidly on PLDP‐Gal and ‐Xyl but not on PLDP‐Man. Values of pK a for all the reactions are about 8.3, suggesting the participation of a common basic residue in these reactions. Based on the present and other results, it is presumed that Tyr‐573 or Lys‐574 acts as the base abstracting the proton from 2‐hydroxyl group of the glucosyl moiety of PLDP‐Glc.

Direct phosphate-phosphate interaction between coenzyme and substrate in the catalytic reaction of glycogen phosphorylase

Glycogen phosphorylase contains firmly bound pyridoxal 5′-phosphate (PLP), and catalyzes the reversible transfer of a glucosyl moiety between glucose-1-phosphate (G-1-P) and α-1,4-glucan. X-ray crystallographic studies revealed that PLP is located in a pocket where the phosphate group of PLP is pointed toward the G-1-P binding site. We have synthesized pyridoxal(5′)diphospho(1)-α-d-glucose, as a model compound for the phosphate-phosphate interaction between PLP and G-1-P, and reconstituted the enzyme with this compound. The resulting enzyme is catalytically inactive in itself, but, in the presence of glucan, the glycosyl moiety of this compound is transferred to the glucan forming a new α-1,4-glucosidic linkage along with the production of pyridoxal 5′-diphosphate. This glucosyltransfer is similar to the normal catalytic reaction in various aspects, although the rate is smaller in the order of three. AMP accelerates the transfer about 24 times compared with the reaction in its absence. We have more recently used pyridoxal(5′)triphospho(1)-α-D-glucose to reconstitute the enzyme. In the presence of glucan, the compound bound to enzyme is gradually degraded to pyridoxal 5′-triphosphate. This reaction is essentially dependent on AMP, and proceeds several times more slowly than the glucosyltransfer from the diphospho compound. These results provide evidence for the direct phosphate-phosphate interaction between the coenzyme and the substrate in the normal enzyme reaction, and seem to reflect a rather wide allowance in regard to this interaction.