Biomedical subjects
Publications and source records attributed to K Murakami.
Crystals of ILAP (Irpex lacteus aspartic proteinase) have been obtained by the hanging drop method using ammonium sulfate as a precipitant. The crystals are monoclinic, space group P2(1) with cell dimensions a = 54.5 A, b = 79.6 A, c = 37.5 A, beta = 96.8 degrees. The crystals are quite stable to X-rays and diffract beyond 1.9 A resolution. There is one molecule in the asymmetric unit.
Furin, a mammalian homolog of the yeast Kex2 protease, is associated with Golgi membranes and is involved in cleavage of precursor proteins at sites marked by the Arg-X-Lys/Arg-Arg (RXK/RR) motif. We have recently shown that a furin mutant lacking the transmembrane domain can be secreted from cDNA-transfected cells with proteolytic activity for the fluorogenic peptide t-butoxycarbonyl-Arg-Val-Arg-Arg-4-methylcoumarin-7- amide. In this study, we purified and characterized the recombinant furin from the conditioned medium of these cells. Furin was purified as a mixture of 83- and 81-kDa forms and a 96-kDa form. The differences in molecular mass were not due to differences in molecular mass were not due to differences in glycosylation. Moreover, all forms had the same NH2-terminal sequence beginning at the residue after the Arg-Ala-Lys-Arg sequence. These data suggest that the three different forms may be produced by differential COOH-terminal processing of a furin molecule and that mature furin may be autocatalytically produced. Both enzyme preparations showed a pH optimum at 7.0, required Ca2+ for the activity, and showed essentially the same inhibitor profile. These properties resembled those of the Kex2 protease. Both preparations efficiently cleaved fluorogenic peptides with an RXK/RR sequence and moderately cleaved a peptide with an RXXR sequence, but did not cleave dibasic peptides. The sequence requirements determined in vitro were compatible with those determined by expression studies in cultured cells. These data unequivocally demonstrate that furin is an endogenous cellular protease responsible for cleavage of precursor proteins mainly at RXK/RR sites.
Many peptide hormones and neuropeptides are produced from larger, inactive precursors through endoproteolysis at sites usually marked by paired basic residues (primarily Lys-Arg and Arg-Arg), or occasionally by a monobasic residue (primarily Arg). Based upon data concerning processing of prorenin and its mutants around the native Lys-Arg cleavage site expressed in mouse pituitary AtT-20 cells, we present the following sequence rules that govern mono-arginyl cleavages: (a) a basic residue at the fourth (position -4) or the sixth (position -6) residue upstream of the cleavage site is required, (b) at position -4, Arg is more favorable than Lys, and (c) at position 1, a hydrophobic aliphatic residue is not suitable. These rules are compatible with those proposed by comparison of precursor sequences around mono-arginyl cleavage sites. We also provide evidence that precursor cleavages at mono-arginyl and dibasic sites can be catalyzed by the same Kex2-like processing endoprotease, PC1/PC3.
We have previously demonstrated that activin, a member of the TGF-beta family, has a potent mesoderm-inducing activity in Xenopus embryos. In the course of screening for activin-related genes from Xenopus, we have cloned cDNAs for Xenopus homologue of BMP-2, -4 and -7. Northern blot analysis revealed that these BMP genes are maternally encoded and differentially regulated after fertilization. Alkaline phosphatase-inducing assay using the recombinant BMP proteins has shown that at least BMP-2 and -4 have similar activity to mammalian counterparts.
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To examine whether the subpopulation of the rat type 1 angiotensin II (AII) receptor (AT1A) couples with a single or multiple signal transduction pathways, we constructed Chinese hamster ovary (CHO) cell lines producing the recombinant receptor. The expressed AT1A receptor exhibits typical pharmacological characteristics of the AT1 receptor, known to mediate the main physiological function of AII. Addition of AII to the CHO cells induced a rapid, transient increase in intracellular free Ca2+ concentrations ([Ca2+]i) followed by a lower, sustained phase. Nicardipine, a blocker of voltage-dependent L-type Ca2+ channels, attenuated the transient [Ca2+]i response and abolished the sustained phase. The transient phase was also reduced dose-dependently by the phospholipase C inhibitor neomycin. Furthermore, AII inhibited forskolin-evoked cAMP accumulation. These data suggest, although another subpopulation named AT1B is present, that the rat AT1A receptor can independently couple with all three signal transduction pathways known to be induced by AII: i.e., i) activation of phospholipase C resulting in InsP3 generation with a subsequent release of intracellularly stored Ca2+, ii) activation of dihydropyridine-sensitive voltage-dependent Ca2+ channels, and iii) inhibition of adenylate cyclase activity.
Renin, a key enzyme controlling blood pressure, is produced mainly in the kidney. To identify the transcriptional regulatory elements of the mouse Ren-1c gene, the promoter regions were fused to the CAT reporter gene and transfected into embryonic kidney-derived 293 cells and four extrarenal cell lines, HeLa, HepG2, HT1080 and NIH3T3 cells. Transient transfection assay showed that sequences from -365 to +16 of the renin gene could direct transcription of the CAT hybrid gene only in 293 cells. Deletion analysis identified two transcriptionally active regions; the renin upstream-promoter element (RU-1 element; position -224 to -138) and the renin proximal-promoter element (RP-2 element; position -75 to -47). Although the RU-1 element functioned as an activator, depending on its orientation, it failed to trans-activate the renin promoter when the RP-2 element was deleted. By contrast, the proximal element alone exhibited a weak trans-activator property. Gel shift assay identified RU-1 element-binding factors in both 293 and HeLa cells, whereas 293 cell-dominant factors were shown to bind only to RP-2 element. Therefore, both RU-1 and RP-2 elements were found to be necessary for efficient CAT expression from the renin promoter in 293 cells, suggesting that activation of the Ren-1c promoter requires combined action between cell type-dominant and ubiquitous nuclear factors.
A cytosolic protein factor(s) is involved in the import of precursor proteins into mitochondria. PBF (presequence binding factor) is a protein factor which binds to the precursor form (pOTC) of rat ornithine carbamoyltransferase (OTC) but not to the mature OTC, and is required for the mitochondrial import of pOTC. The precursors for aspartate aminotransferase and malate dehydrogenase as well as pOTC synthesized in a reticulocyte lysate were efficiently imported into the mitochondria. However, the precursors synthesized in the lysate depleted for PBF by treatment with pOTC-Sepharose were not imported. Readdition of the purified PBF to the depleted lysate fully restored the import. pOTC synthesized in the untreated lysate sedimented as a complex with a broad peak of around 9 S, whereas pOTC synthesized in the PBF-depleted lysate sedimented at an expected position of monomer (2.5 S). When the purified PBF was readded to the depleted lysate, pOTC sedimented as a complex of about 7 S. In contrast to most mitochondrial proteins, rat 3-oxoacyl-CoA thiolase is synthesized with no cleavable presequence and an NH2-terminal portion of the mature protein functions as a mitochondrial import signal. The thiolase synthesized in the PBF-depleted lysate could be efficiently imported into the mitochondria, and readdition of PBF had little effect on the import. The thiolase synthesized in the untreated, the PBF-depleted, or the PBF-readded lysate sedimented at an expected position of monomer (2.5 S). These observations provide support for the existence of PBF-dependent and -independent pathways of mitochondrial protein import.
We have produced human angiotensinogen in Chinese hamster ovary (CHO) cells. The expression products were purified to homogeneity by a single column chromatography and its 17 amino-terminal sequences were identical to those of the native protein. We demonstrated the recombinant human angiotensinogen to be a substrate for human renin.
Four distinct cDNAs for activin receptor designated as XSTK2, 3, 8 and 9 have been cloned from a Xenopus laevis cDNA library. The protein structures deduced from the cDNAs have shown that they all have a putative extracellular ligand-binding domain, a single transmembrane domain and cytoplasmic Ser/Thr kinase domain, except that XSTK2 is extremely similar to the XSTK3 gene but lacks a carboxyl-terminal part of the kinase motif. Northern blot analysis showed that all transcripts are maternally inherited. The levels of transcript for XSTK2, 3 and 8 appeared to fluctuate during early development while those for XSTK9 maintain constant.
We acquired dynamic images over the whole liver by inversion recovery snap shot FLASH method after a bolus intravenous injection of Gadolinium-DTPA. Each nodule of hepatoma in the liver showed early enhancement and gradually turned to show low intensity. In two cases of hepatoma, small intrahepatic metastases, which were not detected by US, CT and spin-echo image of MRI, were suspected as high intensity nodules on early phase. Also recurrent areas after TAE were enhanced on early phase. This method is practical for improving the detection of lesions and is useful for evaluating the recurrence after TAE.
A long-chain neutral phospholipid, dioleoylphosphatidylcholine, was found to support protein kinase C activation by cis-fatty acid and diacylglycerol (DAG). This effect of phosphatidylcholine (PC) is totally dependent on the presence of cis-fatty acid; PC greatly stimulates the cis-fatty acid-induced protein kinase C activity, but it does not activate protein kinase C at all, even in the presence of DAG, if cis-fatty acid is absent. DAG, however, plays a modulatory role in the presence of Ca2+; it further enhances the PC-potentiated cis-fatty acid activation of protein kinase C. Although the activities of all three protein kinase C subtypes tested (types I, II and III) are supported by this PC mechanism, type III is most sensitive to the DAG effect, and it is activated synergistically by cis-fatty acid and DAG. The potency of PC to support the synergistic activation of this subtype is equivalent to that of phosphatidylserine (PS). There are several differences, however, between PC- and PS-supported synergism observed in type III protein kinase C: (1) Ca(2+)-sensitivity is different; PC requires higher concentrations of Ca2+ (10-20 microM-Ca2+) than those required for PS (micromolar Ca2+); (2) PC/cis-fatty acid/DAG-induced autophosphorylation of protein kinase C subtypes (types I, II and III) is very weak, whereas PS/cis-fatty acid/DAG strongly stimulate autophosphorylation of these subtypes under the conditions at which both PC and PS systems fully activate the protein kinase C in terms of histone phosphorylation. These observations suggest that a neutral phospholipid such as PC may also participate in the activation and differential regulation of protein kinase C.
The effect of highly purified recombinant human renin (rh-renin), expressed in Chinese hamster ovary cells, on mean blood pressure (MBP) was evaluated in pithed spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Intravenous bolus injection of rh-renin produced dose-dependent increases in MBP in pithed SHR and WKY. The pressor response to rh-renin in pithed SHR was about 3 times as potent as that in pithed WKY. Intravenous infusion of rh-renin produced dose-dependent progressive increases in MBP during the first 40 min, reaching plateaus and thereafter MBP was maintained up to 120 min. This hypertensive response to rh-renin was antagonized by renin inhibitors, YM-21095 and KRI-1314, which inhibited the reaction between rh-renin and tetradecapeptide competitively, with Ki values of 5.1 x 10(-10) and 4.3 x 10(-9) M, respectively. In rh-renin-infused pithed SHR, the hypotensive effect of YM-21095 was 37 times as potent as that of KRI-1314. These results suggest that rh-renin can stimulate the rat renin-angiotensin system, thereby producing hypertension. Moreover, the rh-renin-infused rat model could be useful to evaluate the effect of renin inhibitor.
We have recently demonstrated that the Arg-X-Lys/Arg-Arg sequence is a signal for precursor cleavage catalyzed by furin, a mammalian homologue of the yeast precursor-processing endoprotease Kex2, within the constitutive secretory pathway. In this study, we further examined sequence requirements for the constitutive precursor cleavage by expression of various prorenin mutants with amino acid substitutions around the native Lys-Arg cleavage site in Chinese hamster ovary cells. The results delineate the following sequence rules that govern the constitutive precursor cleavage. (a) A basic residue (Lys or Arg) at the 4th (position -4) or 6th (position -6) residue upstream of the cleavage site besides basic residues at positions -1 and -2 is necessary. (b) At position -2, a Lys residue is more preferable than Arg. (c) At position -4, an Arg residue is more preferable than Lys. (d) At position 1, a hydrophobic aliphatic amino acid is not suitable.
In order to investigate the molecular events which take place during gastrulation, extracts from developing Xenopus embryos were analyzed for temporal peptide distribution by high performance liquid chromatography. One peptide peak which became increasingly dominant after gastrulation was purified and partially characterized. The amino acid sequence of enzymic digests showed the peptide is extremely similar to mammalian thymosin beta 4. The peptide was capable of binding actin monomers like mammalian counterparts. Cloning of the Xenopus thymosin beta 4 cDNA showed that only three amino acid substitutions occurred between amphibian and bovine. Northern blot analysis revealed the mRNA is maternally present at a low level and the transcript becomes abundant after gastrulation, supporting the distribution of the peptide.
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We used the polymerase chain reaction to identify a mouse testis cDNA that represented another member of a growing class of mammalian endoproteases involved in the processing of precursor proteins. This cDNA encoded a 655-residue protein, designated PC4, containing a bacterial subtilisin-like catalytic domain closely related to those of the recently characterized precursor-processing endoproteases, furin, PC1/PC3, PC2, and Kex2. Within this domain, the amino acid sequence of PC4 was 70, 58, 55, and 45% identical with those of mouse furin, mouse PC1/PC3, mouse PC2, and yeast Kex2, respectively. Northern blot analysis indicated that the PC4 mRNA was detectable only in the testes after the 20th day of postnatal development. Moreover, this message was mainly expressed in the round spermatids. These data suggest that PC4 represents a prime candidate for a precursor-processing endoprotease in the testicular germ cells and that its gene expression is regulated during spermatogenesis.
A new method was developed to display MR angiographic images. Nine MR angiographic images rotated along the cranio-caudal axis with ten incremental degree were constructed. These images were printed from left-anterior-oblique view to give a stereoscopic effect. With the stereoscopic viewing of these images, multiple stereoscopic images with different viewing directions were observed at once. Anatomical relationships of the vessels were more clearly understood than usual stereoscopic viewing. This multi-directional stereo viewing method is useful to display not only the MR angiographic images but also other multi-viewing techniques.