Cloning of PCPTP1-Ce encoding protein tyrosine phosphatase from the rat cerebellum and its restricted expression in Purkinje cells.

Recently, cDNAs encoding brain-specific transmembrane-type protein tyrosine phosphatases (PTPs) with single catalytic domain have been cloned. These include PC12-PTP, PCPTP1, PTPBR7, and PTP-SL, whose cytoplasmic domains had high similarity to STEP, a brain-specific nontransmembrane-type PTP. Based on the high similarity and expression pattern, PCPTP1 seems to be identical with PC12-PTP1 and to be the rat homologue of murine PTPBR7. Here, we report the molecular cloning and expression profile of PCPTP1-Ce, a variant of PCPTP1. Both PCPTP1 mRNA and PCPTP1-Ce mRNA seem to be derived from a single common region gene. Nucleotide and deduced amino acid sequence comparison between PCPTP1-Ce and PCPTP1 revealed that the predicted protein product of PCPTP1-Ce is identical with that translated from the third initiation methionine of the longest ORF of PCPTP1, and that these two clones differ in the 5'-untranslated sequences. Northern blot analyses with specific probes for PCPTP1 and PCPTP1-Ce confirmed our previous observation that PCPTP1-Ce mRNA was almost exclusively expressed in the cerebellum, whereas PCPTP1 was widely expressed in various brain regions dissected including cerebellum. In situ hybridization study demonstrated that PCPTP1-Ce mRNA was exclusively expressed in Purkinje cells of the cerebellum. In contrast, PCPTP1 mRNA was predominantly expressed in granule cells and less in Purkinje cells. Moreover, immunohistochemical analysis using an affinity-purified polyclonal antibody raised against the cytoplasmic region of PCPTP1/PCPTP1-Ce demonstrated that Purkinje cells were strongly immunostained, whereas granule cells were stained only faintly in the cerebellum. These observations clearly demonstrated that PCPTP1-Ce mRNA and its protein products are expressed in Purkinje cells and suggest that PCPTP1-Ce may play an important role in Purkinje cell function in the rat cerebellum.

Cloning and expression of PCPTP1 encoding protein tyrosine phosphatase.

A novel cDNA encoding PTP (protein tyrosine phosphatase) was cloned from PC12h cells and designated as PCPTP1 (gene encoding PC12 protein Tyr phosphatase). The longest open reading frame (ORF) of this clone encodes a 656-amino-acid (aa) protein with a single PTP catalytic domain. Western blot analysis using a polyclonal Ab (antibody) raised against the cytoplasmic region of PCPTP1 detected two products, a major 65-kDa and minor 42-kDa protein, designated PCPTP1-MFI and PCPTP1-MVQ, respectively, in PC12h cells. These two proteins correspond to the products translated from the second and fifth methionine of PCPTP1, respectively. The bacterially expressed GST::PCPTP1-MVQ fusion protein had phosphatase activity with pNPP (p-nitrophenyl phosphate) as a substrate. Alignment of the aa sequence of PCPTP1-MVQ with those of other PTP showed the highest similarity to STEP and LC-PTP/HePTP, with 54 and 51% identity, respectively. Northern blot analysis showed only one 3.9-kb transcript in PC12h cells, indicating that PCPTP1 corresponds to this 3.9-kb transcript. The 3.9-kb PCPTP1 mRNA was detected in the brain and adrenal gland, but not in other non-neuronal tissues in adult rats. Two other transcripts of 3.3 and 1.7 kb were also detected in brain. NGF (nerve growth factor) and glucocorticoid are known to bimodally regulate the cell fate decision of sympathoadrenal precursors like PC12 cells, with NGF promoting the neuronal phenotype and glucocorticoid promoting the chromaffin phenotype. Still, both agents decreased the level of PCPTP1 mRNA in PC12h cells. Therefore, it is likely that the decrease in the level of PCPTP1 mRNA might be associated or correlated with cell differentiation in PC12h cells.

Recombinant soluble CD59 inhibits reactive haemolysis with complement.

Three soluble forms of membrane attack complex inhibitory factor (MACIF or CD59) were prepared using recombinant baculovirus-infected insect cells. They consisted of 70, 77 and 86 amino acids, starting from the amino terminus of naturally occurring CD59, and were designated recombinant (r) CD59 70, 77 and 86, respectively. All three rCD59 lacked a glycosyl-phosphatidylinositol (GPI) anchor, unlike membrane CD59 which has a GPI anchor at the anchor at the carboxyl terminus (77th amino acid). Their activities in inhibiting complement activation were assayed with C5b-7 intermediate cells and C8 and C9 components. The inhibitory activity of rCD59 70 was as high as that of rCD59 77 and twice that of rCD59 86. In addition, it was one-fourth and one-hundredth lower than the activities of urine and erythrocyte CD59, respectively. However, when assayed in the presence of human serum at a final concentration of 50% (v/v), the activities of both urine and erythrocyte CD59 were greatly decreased to to one-tenth of that of rCD59 70. Purified rCD59 70 molecules were all glycosylated, but rCD59 77 and 86 were mixtures of glycosylated and non-glycosylated molecules. The inhibitory activities of rCD59 77 and 86 were the same for the glycosylated and non-glycosylated forms. These results suggest that the soluble rCD59 provide a means for elucidating the biological roles of CD59.

Induction of Two Types of Gene Amplification by a Cloned DNA Fragment and Amplification of a Foreign Gene on the Bacillus subtilis Chromosome by Using the Fragment.

In our previous work we cloned on λ Charon4A phage vector a 6.4-kb EcoRI fragment, which had the transforming activity of inducing gene amplification with a 16-kb repeating unit on the Bacillus subtilis chromosome by competence transformation; those transformants were selected as tunicamycin resistant (Tm(r)) transformants. We found that this DNA fragment can induce another type of gene amplification with a 8.7-kb repeating unit by transformation when we use an amyE07 mutant as a recipient cell and select both Tm(r) and amyE(+) transformants. In the latter (8.7-kb type) gene amplification, the copy number was increased up to 20 in contrast to about 10 copies in the former 16-kb type. We replaced a part of the 6.4kb EcoRI fragment with a cat gene as a model of a foreign gene, and succeeded in the amplification of the cat gene on the chromosome by the latter type of transformation.

The proton motive force lowers the level of ATP required for the in vitro translocation of a secretory protein in Escherichia coli.

The role of the electrochemical potential difference of proton (delta mu H+) in protein translocation across the membrane of Escherichia coli was examined in detail using an efficient in vitro assay system (Yamada, H., Tokuda, H., and Mizushima, S. (1989) J. Biol. Chem. 264, 1723-1728). Delta mu H+ reduced the level of ATP necessary for the efficient translocation of OmpF-Lpp, a chimeric model secretory protein. The apparent Km value of the translocation reaction for ATP was lower by 2 orders of magnitude in the presence of delta mu H+ than in its absence. The membrane potential and delta pH, both of which are components of delta mu H+, independently lowered the apparent Km value of the translocation reaction for ATP. An ATP-generating system also lowered the level of ATP required for translocation in the absence of delta mu H+ but not in its presence. It is proposed that ADP formed during protein translocation lowers the affinity of the putative translocation machinery for ATP and that the removal of ADP from the secretory machinery, a possible critical step in the translocation reaction, is stimulated in the presence of either delta mu H+, an ATP-generating system, or a higher concentration of ATP.

Reconstitution of translocation activity for secretory proteins from solubilized components of Escherichia coli.

The protein translocation system of Escherichia coli was solubilized and reconstituted, using the octylglucoside dilution method, into liposomes prepared from E. coli phospholipids. SecA, ATP, phospholipids and membrane proteins were found to be essential for the translocation of a model secretory protein, uncleavable OmpF-Lpp. Phospholipids were found to play roles not only in liposome formation but also in the stabilization of membrane proteins during the octylglucoside extraction. The effects of IgGs specific to five distinct regions of the SecY molecule on protein translocation into proteoliposomes were examined. IgGs specific to the amino- and carboxyl-terminal regions of the SecY molecule strongly inhibited the translocation activity, indicating the participation of SecY in the translocation. Generation of a proton motive force due to the simultaneous reconstitution of F0F1-ATPase was also observed in the presence of ATP. An ATP-generating system consisting of creatine phosphate and creatine kinase significantly enhanced the formation of the proton motive force and the protein translocation activity of the proteoliposomes. Collapse of the proton motive force thus generated partially inhibited the translocation.

Molecular cloning of the protocatechuate 4,5-dioxygenase genes of Pseudomonas paucimobilis.

We determined the nucleotide sequence of a 1.9-kilobase fragment of Pseudomonas paucimobilis SYK6 chromosomal DNA that included genes encoding protocatechuate 4,5-dioxygenase, the enzyme responsible for the aromatic ring fission of protocatechuate. Two open reading frames of 417 and 906 base pairs were found that had no homology with previously reported sequences, including those encoding protocatechuate 3,4-dioxygenase. Since both open reading frames were indispensable for the enzyme activity, they should encode the subunits of protocatechuate 4,5-dioxygenase. We named these genes ligA and ligB. Protocatechuate 4,5-dioxygenase was efficiently expressed in Escherichia coli with the aid of the lac promoter, and the polypeptides of the ligA and ligB gene products were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid sequencing.

In vitro analysis of the process of translocation of OmpA across the Escherichia coli cytoplasmic membrane. A translocation intermediate accumulates transiently in the absence of the proton motive force.

The proton motive force (delta mu H+) plays an important role, although it is not absolutely essential, in the in vitro translocation of secretory proteins, such as OmpA, across the cytoplasmic membrane of Escherichia coli (Yamada, H., Tokuda, H., and Mizushima, S. (1989) J. Biol. Chem. 264, 1723-1728). The transient accumulation in membrane vesicles of a possible translocation intermediate of OmpA was observed in the absence of delta mu H+. The intermediate was detected on a polyacrylamide gel as a proteinase K-resistant band corresponding to a molecular weight of 26,000. The intermediate did not possess the signal peptide. The appearance of this band was inhibited in the absence of ATP or the presence of adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP) and enhanced upon the addition of SecA. Upon the addition of NADH that energizes the membrane, the intermediate was converted to the translocated form of OmpA, even in the presence of AMP-PNP. These results suggest different requirements of ATP and delta mu H+ for the early and late stages of the translocation reaction. The SecA requirement for the early stage of the translocation has also been suggested. In addition to this band, two other bands were observed at higher positions on the gel, when the translocation reaction was performed in the absence of delta mu H+. Although these two bands also represented the mature form of OmpA, which was partly protected from the proteinase K treatment by the membrane vesicles, the accumulation was not transient. These bands did not appear when the translocation reaction was performed in the presence of dithiothreitol. Together with other evidence, the above observations suggest that OmpA, which has an intramolecular disulfide bridge, cannot undergo the translocation unless delta mu H+ is imposed.