Masahiro Kinuta

ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Iγ

Clathrin-mediated endocytosis of synaptic vesicle membranes involves the recruitment of clathrin and AP-2 adaptor complexes to the presynaptic plasma membrane. Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180. Here, we show that the stimulatory effect of ATP and GTPγS on clathrin coat recruitment is mediated at least in part by increased levels of PIP2. We also provide evidence for a role of ADP-ribosylation factor 6 (ARF6) via direct stimulation of a synaptically enriched phosphatidylinositol 4-phosphate 5-kinase type Iγ (PIPKIγ), in this effect. These data suggest a model according to which activation of PIPKIγ by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse.

Cophosphorylation of amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesicles

It has been thought that clathrin-mediated endocytosis is regulated by phosphorylation and dephosphorylation of many endocytic proteins, including amphiphysin I and dynamin I. Here, we show that Cdk5/p35-dependent cophosphorylation of amphiphysin I and dynamin I plays a critical role in such processes. Cdk5 inhibitors enhanced the electric stimulation–induced endocytosis in hippocampal neurons, and the endocytosis was also enhanced in the neurons of p35-deficient mice. Cdk5 phosphorylated the proline-rich domain of both amphiphysin I and dynamin I in vitro and in vivo. Cdk5-dependent phosphorylation of amphiphysin I inhibited the association with β-adaptin. Furthermore, the phosphorylation of dynamin I blocked its binding to amphiphysin I. The phosphorylation of each protein reduced the copolymerization into a ring formation in a cell-free system. Moreover, the phosphorylation of both proteins completely disrupted the copolymerization into a ring formation. Finally, phosphorylation of both proteins was undetectable in p35-deficient mice.

The stimulatory action of amphiphysin on dynamin function is dependent on lipid bilayer curvature

Amphiphysin is a major dynamin‐binding partner at the synapse; however, its function in fission is unclear. Incubation of large unilamellar liposomes with mice brain cytosol led to massive formation of small vesicles, whereas cytosol of amphiphysin 1 knockout mice was much less efficient in this reaction. Vesicle formation from large liposomes by purified dynamin was also strongly enhanced by amphiphysin. In the presence of liposomes, amphiphysin strongly affected dynamin GTPase activity and the recruitment of dynamin to the liposomes, but this activity was highly dependent on liposome size. Deletion from amphiphysin of its central proline‐rich stretch dramatically potentiated its effect on dynamin, possibly by relieving an inhibitory intramolecular interaction. These results suggest a model in which maturation of endocytic pits correlates with the oligomerization of dynamin with either amphiphysin or other proteins with similar domain structure. Formation of these complexes is coupled to the activation of dynamin GTPase activity, thus explaining how deep invagination of the pit leads to fission.

Direct determination of bound sialic acids in sialoglycoproteins by acidic ninhydrin reaction

A simple and rapid method for sialic acid determination in sialoglycoproteins by acidic ninhydrin reaction is described. The method is based on the reaction of sialic acids with an acidic ninhydrin reagent (K. Yao and T. Ubuka (1987) Acta Med. Okayama 41, 237-241). By heating a sample solution containing sialoglycoprotein with the reagent at 100 degrees C for 10 min, a stable color with an absorption maximum at 470 nm was produced. The standard curve was linear in the range of 20 micrograms to 3 mg of fetuin, a sialoglycoprotein, per 3.0 ml of the reaction mixture. The reaction is specific only for sialoglycoproteins among various proteins examined. The acidic ninhydrin method was applied to the determination of sialic acids in sialoglycoproteins in ascites fluids of Ehrlich ascites tumor-bearing mice.

Distribution of dynamins in testis and their possible relation to spermatogenesis

Dynamin 2 and dynamin 3 are highly expressed in testis. However, their functions in the tissue remain unclear. Considering that dynamin 1, neuron-specific isoform of dynamin, plays a pivotal role in endocytosis, functions of dynamin 2 and dynamin 3 in testis must be essential. Cellular expression and subcellular localization of dynamin 2 and dynamin 3 in testis were investigated. Dynamin 2 and dynamin 3 were highly expressed in germ cells and Sertoli cells, constituents of seminiferous tubules. By immunofluorescence it was revealed that dynamin 2 colocalizes with clathrin both at the plasmamembrane and at Golgi in a cell line of Sertoli cells. Immunoreactivity for dynamin 3, on the other hand, appeared as finer puncta, which did not colocalize with clathrin, suggesting that these two dynamins have distinct functions in Sertoli cells. In the klotho deficient mouse testis, which demonstrates disorder in spermatogenesis, expression of dynamin 2 and dynamin 3 was drastically reduced indicating possible association of these proteins with spermatogenesis.

Phosphatidylinositol 4,5-bisphosphate stimulates vesicle formation from liposomes by brain cytosol

As a step toward the elucidation of mechanisms in vesicle budding, a cell-free assay that measures cytosol-induced vesicle generation from liposomes was established. This assay then was used to explore the role of phosphoinositides in vesicle formation. Liposomes incubated with brain cytosol in the presence of ATP and GTP massively generated small vesicles, as assessed both quantitatively and qualitatively by a dynamic light-scattering assay. Both ATP and GTP were required. Vesicle formation was inhibited greatly by the immunodepletion of dynamin 1 from the cytosol, indicating a major contribution of this GTPase in this reaction and suggesting that it mimics endocytic vesicle fission. Increasing the concentration of l-α-phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] but not of l-α-phosphatidylinositol 4-monophosphate or l-α-phosphatidylinositol in the lipid membranes enhanced vesicle formation. Lipid analysis revealed rapid degradation of PtdIns(4,5)P2 to l-α-phosphatidylinositol during the incubation with the reaction reaching a maximum within 5 sec, whereas vesicle formation proceeded with a longer time course. PtdIns(4,5)P2 degradation was independent of vesicle formation and occurred also in the presence of guanosine 5′-O-(thiotriphosphate), where few vesicle formations occurred. These results suggest that PtdIns(4,5)P2 plays a critical role in the early step of vesicle formation, possibly in the recruitment of coats and fission factors to membranes.

High-performance liquid chromatographic determination of taurine and hypotaurine using 3,5-dinitrobenzoyl chloride as derivatizing reagent

A method for the determination of taurine and hypotaurine in biological samples involving the preparation of their 3,5-dinitrobenzoyl derivatives followed by HPLC was established. Taurine and hypotaurine in aqueous media were reacted with 3,5-dinitrobenzoyl chloride in the presence of triethylamine to prepare 3,5-dinitrobenzoyl derivatives. These derivatives were separated on a C18 reversed-phase column and detected by recording the absorbance at 254 nm. Derivatives of taurine and hypotaurine were obtained in yields of 91.4 +/- 3.3 and 85.6 +/- 2.6%, respectively. The calibration graphs for taurine and hypotaurine were linear between 2.5 and 500 microM with correlation coefficients of 0.999. The method was applied to the determination of taurine and hypotaurine in human and rat urine and blood and in rat liver and heart.

Preparation and characterization of S-[2-carboxy-1-1H-imidazol-4-yl)ethyl]glutathione and its derivatives as proposed precursors of S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine, a compound found in human urine

Formation of 3-[(carboxymethyl)thio]-3-(1H-imidazol-4-yl)propanoic acid (I) and S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine (II), compounds found in human urine, has been demonstrated by enzymatic degradation of S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]glutathione (III). Compound (III) was chemically synthesized in 72% yield by incubating the reaction mixture of trans-urocanic acid and 3-fold excess GSH at 65 degrees C for 1 wk, which was accompanied by formation of N-(S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]cysteinyl)glycine (IV) in 15% yield. S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]-N-gamma-glutamylcysteine (V) was produced by partial hydrolysis of compound (III) in HCl. The synthesized compounds were characterized mainly by fast-atom bombardment mass spectrometry and high-voltage paper electrophoresis as well as chemical degradation. Incubation of compound (III) with rat kidney homogenate in a Tris buffer (pH 8), formed compound (II) in 80% yield possibly via compound (IV). Yield of compound (II) was increased by adding glycylglycine to the reaction mixture. However, little degradation of compound (III) occurred in the use of rat liver, brain, heart or spleen homogenate as the enzyme source. Compound (II) was further metabolized to compound (I) by incubation with rat kidney homogenate in a phosphate buffer of pH 7.4. From these results, we suggest that the urinary compounds are products of enzymatic degradation of compound (III) and that GSH may participate in the metabolism of urocanic acid, the first catabolite of L-histidine.

S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine in normal human urine.

SummaryA compound, which had the same mobility on a high-voltage paper electrophoretogram and the sameRF value on a thin-layer chromatogram as those ofS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine (I), was partially purified from human urine by ion-exchange column chromatography. The compound gave a signal at m/z 260 on its FAB mass spectrum, which was assigned as MH+ of compound I. These results suggest that the urinary compound is compound I and it is a physiological precursor of 3-[(carboxymethyl)thio]-3-(1H-imidazol-4-yl)propanoic acid [Kinuta et al., (1991) Biochem J 275: 617–621].

Reaction of S-(2-amino-2-carboxyethylsulfonyl)-l-cysteine with sulfite: Synthesis of S-sulfo-l-cysteine and l-alanine 3-sulfinic acid and application to the determination of sulfite☆☆☆★

Abstract A procedure for the simultaneous preparation of S -sulfo- l -cysteine and l -alanine 3-sulfinic acid is described. The method is based on the quantitative reaction between sulfite and S -(2-amino-2-carboxyethylsulfonyl)- l -cysteine. The yield was 95% for S -sulfo- l -cysteine and 91% for l -alanine 3-sulfinic acid. The reaction was also applied to the quantitative determination of sulfite in biological materials. In this procedure, sulfite reacts with S -(2-amino-2-carboxyethylsulfonyl)- l -cysteine. Separation of the reaction product, S -sulfo- l -cysteine, is done by ion-exchange fractionation, and it is determined with acid ninhydrin reagent 2 ( M. K. Gaitonde, 1967, Biochem. J. 104, 627–663 ). The recovery was 96.8 ± 0.3%.