The VMA1 locus in Saccharomyces cerevisiae contains a nested genetic element, the VDE gene, and expresses two functional proteins. A single VMA1 translational product seems to catalyze a self protein splicing in which an internal domain is excised out to produce a site-specific DNA endonuclease and the N-and C-domains are ligated by a transpeptidation reaction to yield a catalytic subunit of the vacuolar ATPase. Accumulating evidence in the past few years suggests that the VMA1 locus encodes a protozyme (for protos en zym_??_) which has dual roles in life as a protean catalyst for self protein splicing and for self gene homing. Four protozymes that share a common mechanism in protein splicing have been found in four organisms covering three maior nhvlogenic trees.

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The α-amylase inhibitor coded 0.19 (0.19AI) from wheat kernel is a dimeric protein which inactivates exogenous α-amylases. Crystals of 0.19AI were grown at room temperature and pH 7.0 from a protein solution with a low salt concentration. They were trigonal, belonged to space group P3₁ or P3₂, and had unit cell dimensions of α=b=79.3 Å and c=60.8 Å. The crystals diffract X-rays to at least 2.0 A resolution and are stable to X-ray beams. The asymmetric unit appears to contain two 0.19AI dimers. A potential heavy-atom derivative was prepared by soaking the crystal in a HgCl₂ solution.

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Laminin contains multiple oligopeptide motifs to promote cell adhesion and migration. One of these motifs is YIGSR within the Bl chain. We reconstituted the cell-adhesive activity of YIGSR motif by grafting it onto a truncated form of the Staphylococcal protein A (designated tSPA) via cassette mutagenesis. When coated on a polystyrene surface, the YIGSRgrafted tSPA (YIGSR-tSPA) promoted attachment and spreading of mouse melanoma and human rhabdomyosarcoma cells, but not of hamster fibroblasts. The cell-adhesive activity of YIGSR-tSPA was abolished by amino acid substitution or scrambling of the inserted YIGSR sequence. Divalent cations Mn²⁺ and Mg²⁺, but not Ca²⁺, promoted the cell adhesion to YIGSR-tSPA. Interestingly, the YIGSR-tSPA-mediated cell adhesion was barely inhibited by the linear peptide CDPGYIGSR-NH₂, , but was strongly inhibited by the cyclic peptide CDPGYIGSRC and another peptide PEILDVPST, which is a specific inhibitor for integrin α₄β₁. Among various anti-integrin antibodies, anti-α, and anti-β₁ antibodies specifically inhibited the cell adhesion to YIGSR-tSPA. In support of these observations, adhesion of rhabdomyosarcoma cells to intact laminin was also partially inhibited by synthetic PEILDVPST peptide and anti-α₄ antibody. These results, taken together, indicate that the YIGSR motif exerts its cell-adhesive activity through interaction with integrin α₄β₁.

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We have previously reported that fatty liver is easily induced in a novel experimental animal, Suncus murinus (suncus) by withholding food, and that apolipoprotein B (apo B) is not actively synthesized in the liver. In the present paper we describe the effect of starving and refeeding on lipid and lipoprotein metabolism in suncus, in order to explore the mechanisms of induction of fatty liver by starving and of its improvement by refeeding. Starvation induced increase in triglyceride content and decrease in glycogen content of the liver. Although the glycogen content returned to the level before starvation at 12 h after refeeding, the triglyceride content decreased gradually but did not reach the prestarvation level even at 24 h after refeeding in suncus. Plasma lipids, glucose, and insulin levels were decreased by starvation and returned to the levels before starvation between 8 and 24 h after refeeding. On the other hand, the plasma levels of free fatty acid and ketone bodies were elevated significantly by starvation and decreased rapidly by refeeding. These responses to starvation and refeeding, except for the change in hepatic triglyceride, are in common with other experimental animals, suggesting that there are no abnormalities in glucose metabolism or in fatty acid metabolism in suncus. In conclusion, the fatty liver induced by starvation in suncus may be caused by impaired triglyceride transport out of the liver, for which apolipoprotein B is mostly responsible, as reported previously.

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We have isolated two non-overlapping clones containing the genes for subunit Vb of cytochrome c oxidase (COXVb) from a rat genomic library in Charon 4 A using a newly isolated full-length cDNA as a probe. One of the two genomic clones, designated as λCOXVb 741, contained a functional gene (COXVb-1), while the other one, designated as λACOXVb 211, contained an intronless processed pseudogene (COXVb-2). The COXVb-1 gene spans approximately 1.8 kb and consists of four exons interrupted by three introns. The nucleotide sequences of all exons are completely identical to the corresponding sequences of the rat liver and brain COXVb cDNAs, indicating that this gene is actually expressed. The 5'-flanking region of the gene lacks conventional TATA and CAAT boxes, but exhibits strong promoter activity in the chloramphenicol acetyltransferase (CAT) assay. Deletional analysis and gel shift assay of the 5'-flanking region suggested that the binding of nuclear factor Sp-1 could be essential for transcription of the gene. Southern blotting analysis implied the occurrence of multiple COXVb genes in the rat genome. However, the results of the present experiments suggest that only the COXVb-1 gene is expressed in rat tissues.

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Recognition specificity for sialylsugar chains by the hemagglutinin of influenza B viruses isolated in different years from 1940 through 1990 (B/Lee/40, B/Setagaya/3/56, B/Tokyo/ 7/66, B/Kagoshima/1/68, B/Gifu/2/73, B/Kanagawa/3/76, B/Ibaraki/2/85, B/Yamagata/ 16/88, and B/Bangkok/163/90) was studied using 13 gangliosides. Reactivity of the viruses' hemagglutinin binding to gangliosides was determined by using thin-layer chromatography/virus-binding assay, and also by measuring virus binding to eryth-rocytes modified by incubation with gangliosides in terms of the absorbance of hemoglobin released from the infected cells. Eight strains preferentially recognized a novel ganglioside, carrying lacto-series type I and II sugar chains with the Neu5Acα2-6Gal linkage. It was found that B/Gifu/2/73 strain binds to lacto-series gangliosides containing Neu5Acα2-6Gal and Neu5Acα2-3Gal linkages. Other gangliosides studied, including GM4, GM3(α2-3), GM3(α2-6), GM2, GMla, GD3, GD1a, GD1b, and GT1b, were poor receptors.

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Hydrazinolysis of peptide or protein has been used for C-terminal amino acid determination by Akabori et al. (1952). In this study, proteins were reacted with anhydrous hydrazine vapor at 20°C for 16 h. Asparaginyl linkages were cleaved. Asparagine and glutamine were converted to their hydrazides, β-hydrazidyl aspartic acid and γ-hydrazidyl glutamic acid, respectively, even under milder conditions. The former hydrazide cyclizes to a 6-membered ring, asparaginyl bond at the carboxyl side. Other cleavages, including the glycyl-glycine bond, were also observed.

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Daunomycin, an anti-neoplastic agent, is known to be sequestered by acidic organelles in normal and multidrug-resistant cells [Willingham, M. C., Cornwell, M. M., Cardarelli, C. O., Gottesman, M. M., & Pastan, I. (1986) Cancer Res. 46, 5941-5946]. We studied the mechanism of accumulation of daunomycin into acidic organelles using chromaffin granule vesicles and proteoliposomes reconstituted with purified F-type H⁺-ATPase as model systems. Radiolabeled daunomycin was taken up by chromaffin vesicles upon addition of ATP. Its ATP-dependent uptake was stimulated about 1.4- to 1.8-fold by valinomycin plus K⁺, but was inhibited by ammonium chloride (10 mM) and nigericin plus K⁺. Quinidine (5μM), verapamil (5μM), or vanadate (0.5 mM), inhibitors of P-glycoprotein, had no effect on its uptake. Daunomycin was also taken up by liposomes reconstituted with F-type H⁺-ATPase. Furthermore, doxorubicin and vinblastine were taken up by these vesicles, whereas colchicine and rhodamine 123 were not. The accumulations of daunomycin and doxorubicin in acidic organelles of cultured cells were decreased by inhibiting vacuolar ATPase by addition of bafilomycin A₁, or concanamycin A, or by increasing the internal pH by addition of nigericin. Melittin and N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide dissipat ed the ΔpH and inhibited accumulation of daunomycin in the membrane vesicles and acidic organelles in cultured cells. These results indicate that the ΔpH established by vacuolartype ATPase drives the uptake of daunomycin, doxorubicin or vinblastine into acidic organelles, and that no specific transporters are involved in their uptakes.

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To identify nuclear proteins related to muscle tissue specificity, we tried to prepare antibodies recognizing muscle-specific nuclear proteins. Taking advantage of the autoimmunity of some nuclear proteins, we prepared an antiserum against chick muscle nuclear proteins by injecting protein components of the nuclei isolated from chick breast muscles into breast muscle. Three proteins, named p 30, p 32, and p 37, were detected with the antiserum in a two-dimensional SDS-PAGE pattern of the isolated nuclei. P 30 and p 32 were not detected in the nuclei of liver, brain, cardiac muscle, or slow type skeletal muscle (anterior latissimus dorsi). They were detected in those of fast type skeletal muscle (pectoralis major and semitendinosus) and smooth muscle (gizzard) at all developmental stages examined. On serial fractionation of muscle cell nuclei, they were detected in a fraction obtained after DNase I treatment of the sample, suggesting that the proteins weakly bind to chromatin. A homology search of amino acid sequences showed that there is no known protein similar to p 32.

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The effects of lipid peroxidation on the SH reactivity of the proteins in porcine intestinal brush-border membranes were examined using a fluorogenic thiol reagent, N- [7-dimethylamino-4-methylcoumarinyl] maleimide (DACM) in relation to lipid organization. Changes in the lipid organization were assessed by measurement of the rate of incorporation of 1, 6-diphenyl-1, 3, 5-hexatriene (DPH) into the membrane lipids and the fluorescence anisotropy of DPH-labeled membranes. Treatment of the membranes with an oxygen-radicalgenerating system, i.e., ascorbicacid/Fe²⁺/tert-butyl hydroperoxide (t-BuOOH), resulted in decrease in the rate of DACM incorporation into the SH groups of the membrane proteins (DACM-labeling) and the amount of DACM labeled to the SH groups with a decrease in the lipid fluidity, depending on the formation of thiobarbituric acid-reactive substances and conjugated diene. Pretreatment of the membranes with diphenylamine effectively prevented the ascorbic acid/Fe²⁺/ t-BuOOH-induced decreases in the DACM-labeling and DPH incorporation rates, whereas neither superoxide dismutase, catalase, sodium benzoate, nor mannitol showed a protective effect. The contribution of the lipid fluidity to the SH reactivity to DACM of the proteins in the membranes with different levels of lipid peroxidation was further examined using a lipid fluidizer, benzyl alcohol. The results showed that the DPH incorporation rate increased in proportion to increasing concentrations of the alcohol regardless of the peroxidation level of the membranes, whereas the susceptibility of the SH reactivity of the membrane proteins as to benzyl alcohol transitionally changed as the membranes were peroxidized to levels greater than 400 nmol conjugated diene/mg protein. Based on these results, we proposed the possibility that the SH reactivity of the membrane proteins changes in a different manner before and after a critical level of lipid peroxidation, and that the SH reactivity of the membrane proteins in highly peroxidized membranes is not dependent on the lipid fluidity per se.

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3α-Hydroxysteroid dehydrogenase (3α-HSD) [EC 1.1.1.213]² plays important multifunctional roles in metabolizing steroid hormones, polycyclic aromatic hydrocarbons, and prostaglandins and also in transforming the steroid nucleus for the biosynthesis of bile acids from cholesterol in liver. To gain insight into the details and physiological functions of 3α-HSD in the bile acid biosynthetic pathway, cDNA clones of 3α-HSD were isolated from rat liver γ phage cDNA libraries by using specific antibodies to 3α-HSD purified from rat liver. Transfection of the 3α-HSD cDNA in Simian COS7 cells resulted in the expression of an immunoreactive protein to the antibodies against the purified enzyme, and the transfected cells exhibited activities for not only 7α-hydroxy-5β-cholestan-3-one, the intermediate of bile acid biosynthesis, but also steroid hormones and 9, 10-phenanthrenequinone. Northern blot analysis on poly(A)+RNA by selective use of different cDNA fragments of the 5'-untranslated region, the coding region, and the 3'-untranslated region as probes revealed three hybridizable bands, 3.6, 2.7, and 2.5 kb, in liver and four bands, 3.6, 2.7, 2.5, and 1.8 kb, in ovary. Of these, the 2.7- and 1.8-kb bands were predominant in liver and ovary, respectively. Northern hybridization analysis also revealed that the coding region of the various sizes of mRNA seemed to be common. Southern blot analysis of genomic DNA by the selective use of the cDNA fragments as probes indicated that the various mRNA species were derived from a single gene, probably due to an alternative splicing mechanism. Inhibition experiments with the specific antibodies and immunoblotting studies indicated that the enzymes expressed in liver and ovary were similar in size and catalytic properties, and immunochemically indistinguishable. Northern blotting of poly(A)+RNA in liver using the cDNA fragment of the coding region as a probe showed that the major 2.7-kb mRNA level at 10:00 a. m. was approximately twofold higher in female rat than in male rat, whereas the mRNA level at 10:00 p. m. was nearly identical in both female and male rat liver. The activity for 7α-hydroxy-5β-cholestan-3-one and the immunoreactive protein level did not show a significant sex difference although their levels were slightly higher at night than in daytime. Among various treatments tested, dexamethazone and 6-n-propyl-2-thiouracil modulated the levels of mRNA for 3α-HSD, indicating that glucocorticoid and thyroid hormone may play a role in the regulation of 3α-HSD expression.

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We studied the degranulation reaction of electropermeabilized human neutrophils induced by 1, 2-didecanoyl-3-sn-phosphatidic acid (PA₁₀). PA₁₀ dose-dependently induced the release of β-glucuronidase, an enzyme of azurophil granules, but did not induce the release of lactoferrin, a protein of specific granules. The enzyme release by PA₁₀ absolutely required Ca²⁺, ATP, and Mg²⁺ and the concentrations for the half-maximal response were 2.5 μM, 60 μM, and 0.25 mM, respectively. Although Ca²⁺ alone at concentrations higher than 10 μM induced the release of both β-glucuronidase and lactoferrin, the extents of the release were far less than that of the β-glucuronidase release by PA₁₀. Phorbol myristate acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol induced the release of lactoferrin alone at concentrations of Ca²⁺ below 0.5 μM while they induced the release of both β-glucuronidase and lactoferrin at higher Call concentrations, indicating that the degranulation induced by PA₁₀ is not mediated by diacylglycerol which might be formed from PA. The degranulation reactions induced by PA¹⁰ and PMA were dose-dependently inhibited by staurosporine and calphostin C, protein kinase C inhibitors, although no direct activation of protein kinase C by PA₁₀, was observed. The extent of the β-glucuronidase release by PA₁₀ was not enhanced by the addition of PMA. Propranolol, which inhibits protein kinase C as well as phosphatidic acid phosphohydrolase, strongly inhibited the degranulation reactions induced by PA₁₀ and PMA. Ethanol, a metabolic modulator of phospholipase D, and cyclic AMP did not affect the degranulation reactions by PMA and PA₁₀. These observations suggest that selective enzyme release from azurophil granules of the permeabilized neutrophils might be induced through the activation of protein kinase C or some other protein kinase sensitive to the protein kinase C inhibitors and that phospholipase D may not be involved in the PMA-induced release.

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Addition of the alkyllysophospholipid ET16-OMe, a putative antitumor drug, to the culture medium of human vascular endothelial cells resulted in apoptotic cell death. The death was characterized as programmed cell death since the process was inhibited by the addition of an inhibitor of protein synthesis. The mechanism responsible for apoptosis induced by alkyllysophospholipid has unique characteristics, as compared to those of apoptosis induced by other antitumor drugs, since the drug caused fragmentation of dying cells and its effect could be overcome by the presence of a survival factor, namely, fibroblast growth factor.

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The expression of neutral glycosphingolipids and gangliosides was investigated in mouse and rabbit skeletal muscle by means of biochemical and immunochemical techniques. Neutral glycosphingolipids from muscle of the inbred rabbit strain used in this study showed a simple TLC pattern, comprising mainly monohexosylceramide. In addition to this compound, lactosylceramide, lacto-N-neotetraosylceramide, globoside and Forssman GSL were detected in mouse muscle. The major ganglioside in both species was G_M3; G_M3(Neu5Ac) and G_M3(Neu5Gc) were found in a 3:1 ratio in mouse muscle, whereas the absence of G_M3(Neu5Gc) is characteristic of rabbit muscle. As a general structural feature of all muscle G_M3 gangliosides investigated, aC₁₈ fatty acid and C₁₈ sphingosine were the major components besides minor C₂₂ and C_24:1 fatty acids of the respective ceramide portions, as revealed by positive and negative ion FAB-MS. α2-3 sialylated lacto-N-neotetraosylceramide (sialylparagloboside) was expressed in both species, whereas the α2-6 sialylated isomeric compound was found only in mouse muscle. Minute quantities of ganglio-series G_M1 G_D1a, G_D1b, and G_T1b were detected in muscles from both species. Glycosphingolipid expression could be confirmed immunohistochemically by examining transverse and longitudinal cryosections of skeletal muscle samples. The results provide the basis for the investigation of muscle specific glycosphingolipids that might modulate membrane protein functions in muscle.

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To obtain information on the role of proteasomes in the immune system, we examined the effect of a major immunomodulatory cytokine, gamma interferon (IFN-γ), on the expressions, structures, and functions of proteasomes. IFN-γ greatly increased the levels of the mRNAs encoding LMP 2 and LMP 7, putative immuno-proteasome subunits encoded by genes within the class II MHC region, and these two subunits synthesized were assembled completely into the proteasomal multi-subunit complex in various types of human cells. The subunit organization of the proteasome changed in response to IFN-γ stimulation, due to assembly of newly synthesized subunits through up-and down-expressions of at least 6 proteasome genes including LMP 2/LMP 7 without change in the structure of pre-existing proteasomes. Interestingly, IFN-γ dramatically stimulated the trypsin-like and chymotrypsin-like activities of the multifunctional proteasome and depressed the peptidylglutamyl-peptide-hydrolyzing activity, without affecting the activity for ATP-, ubiquitindependent proteolysis. These results indicate that IFN-γ modifies not only the structural organization of the proteasome, but also its functions. Based on these findings, we discuss the role in the antigen processing/presentation pathway of proteasomes with functional diversity acquired through alteration of their subunit assembly in response to IFN-γ stimulation.

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The dynamic properties of nucleic acids in five different types of intact supramolecular systems, namely, chicken erythrocyte chromatin, the wild type and a deletion mutant of the λ phage, lipid-containing phage PM2, and Alteromonas espejiana ribosomes, were investigated by means of ³¹P solid-state NMR. The nucleic acids in the different supramolecular systems showed unique dynamic properties, which are closely connected with their functions. The total anisotropy of the phosphorus chemical shift (Δσ=σ₃₃-σ₁₁) of the ribosomes was 210 ppm at 5°C. This anisotropy was much larger than those of any DNA complexes, suggesting the highly rigid structure of ribosomal RNA. In contrast, 160 ppm was the largest chemical shift anisotropy at 5°C for B-form DNA in the supramolecular systems. This flexibility would be essential for DNAs to exert their functions. The involvement of a condensation protein in the PM2 phage was supported by the chemical shift anisotropy. The spin-lattice relaxation time in the proton rotating frame [ T₁ρ(H)] of the nucleic acids became shorter with the increase in the effective field in the rotating frame for all systems examined, showing that the motions of the nucleic acids effective for the relaxation are in the slow motional regime or in the range of ω_Iτ_c=1 at 5°C. The motional state of DNA of the λ phage was found to change at about 20°C on the basis of the temperature dependence of the spin-lattice relaxation time of phosphorus (T₁). X-ray scattering experiments also showed a change in the organization of the λ phage particle at about 20°C. The dynamic state of the viral particle was suggested to play an important role in the infection mechanism of bacteriophages.

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Recombinant human bone morphogenetic protein-2 (rhBMP-2) was expressed in silkworm larvae, and a milligram quantity of the protein was purified and characterized. The expressed rhBMP-2 was biologically active in terms of induction of alkaline phosphatase activity in MC3T3-El cells and ectopic bone formation in mice. On SDS-polyacrylamide gel electrophoretic analysis, the purified protein showed a 16 kDa band under reducing conditions and a 30 kDa band under non-reducing conditions. The silkworm-expressed rhBMP-2 was glycosylated and susceptible to endo-β-N-acetylglucosaminidase F (endo F) and endo H, but resistant to endo D. Deglycosylated rhBMP-2 treated with endo F retained its biological activity. These results suggest that rhBMP-2 exists as a dimer and disulfide bond(s) are responsible for the dimerization. Moreover, sugar chains have no direct effect on the biological activity of the protein. The availability of a quite large amount of rhBMP-2 has allowed us to study the biological function of this interesting factor in detail.

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Transcription of the two clustered genes, faoA and faoB, for the multienzyme complex for fatty acid β-oxidation (HDT) from the Gram-negative bacterium, Pseudomonas fragi, was investigated. Northern blot hybridization and primer extension analysis indicated that these genes comprised an operon, giving a 3.6 kb mRNA. Transcription of the 3.6 kb faoAB mRNA was induced by palmitic acid. Using the lacZ gene as a reporter gene for a promoter activity search, the essential region for induction of the faoAB gene transcription by palmitic acid was mapped at the 331 bP region just upstream of the transcription initiation site. Deletion of the 136 bP SalI-NheI fragment just downstream of the transcription initiation site caused the constitutive expression of the faoA-lacZ fusion protein in P. fragi, suggesting that there is a regulatory element interacting with the transcription repressor.

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α2-Plasmin inhibitor (α₂PI) deficiency Okinawa results from defective secretion of the inhibitor from the liver and appears to be a direct consequence of the deletion of Glu¹³⁷ in the amino acid sequence of α₂PI. To examine the effects of replacing the amino acid occupying position 137 or deleting its neighboring amino acid on α₂PI secretion, we used oligonucleotide-directed mutagenesis of α₂PI cDNA to change the codon specifying Glu¹³⁷ or delete a codon specifying its neighboring amino acid. The effects were determined by pulse-chase experiments and by enzyme-linked immunosorbent assay of media from transiently transfected COS-7 cells. Replacement of Glu¹³⁷ with an amino acid other than Cys had little effect on α₂PI secretion. In contrast, deletion of an amino acid in a regio spanning a sequence of less than 30 amino acids including positions 127 and 137 severely impaired the secretion. The results suggest that structural integrity of the region, ratherthan its component amino acids, is important for the intracellular transport and secretion of α₂PI.

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The cyclic AMP-response element (CRE), a transcriptional enhancer, is regulated by CREB (CRE-binding protein) which is the leucine zipper protein phosphorylated by protein kinase A in response to cAMP signal. The highly homologous protein CREM (CRE-modulator) is thought to modulate CREB-stimulated transcription, and is also involved in transcriptional control during spermatogenesis. In this paper, we report two types of cDNAs of human CREM (hCREM), type 1 and type 2; type 1 is a group of human counterparts of the mouse CREMα and type 2 is a novel form having a distinct 5' exon which is unrelated to any species of the CREB and CREM isoforms so far described. This unique 5' region of type 2 hCREM may suggest its independent expression from type 1 CREM. The specific 5' region of type 2 hCREM consisted of 88 bp, containing an initiation codon for translation, but no possible phosphorylation site, suggesting different roles from type 1 CREM. Both type 1 and 2 hCREMs are expressed in lymphoid and non-lymphoid cell lines. Their excess expression by transfection induced suppression of cAMP-mediated activation of transcription, suggesting their negative regulation of CRE-mediated transcription.

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Vasoactive intestinal peptide (VIP), a neuropeptide coupled with adenylate cyclase, was found to induce neurite extension of PC12h cells. Neurites appeared within 1 h after addition of VIP and extended for at least 24 h. The half-maximal concentration for the effect of VIP was 50 nM. In addition to the morphological change, VIP induced expression of VGF protein, a neuron-specific protein associated with neuronal differentiation. Western blotting with anti-phosphotyrosine antibody showed that VIP stimulated tyrosine phosphorylation of two proteins of 42 and 44 kDa, which may be two isoforms of MAP kinase, erkl and erk 2. Activation of MAP kinases was confirmed by ion-exchange chromatography on a Mono Q column, from which VIP-induced kinase activity was co-eluted with MAP kinase-immunoreactivity. Tyrosine-phosphorylation of MAP kinases was also stimulated by forskolin or dibutyry l cAMP, indicating that activation of MAP kinases by VIP might be mediated by cAMP. These results suggest that VIP-induced differentiation of PC 12 cells is associated with cAMP-dependent activation of MAP kinases.

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The primary structure of the aspartate aminotransferase (AspAT) of an archaebacterium, Methanobacterium thermoformicicum strain SF-4, has been determined by cloning and sequencing of the gene for the enzyme. The gene had a consensus promoter and a ribosome binding sequence of methanogens in the 5' untranslated region, followed by an open reading frame starting with ATG and terminating with TGA. The deduced amino acid sequence was identical with the partial amino acid sequences of the enzyme including the N-terminal sequence, and the deduced molecular weight of 41, 684 was virtually identical to that reported earlier for this enzyme [Tanaka, T., Yamamoto, S., Taniguchi, M., Hayashi, H., Kuramitsu, S., Kagamiyama, H., & Oi, S. (1992) J. Biochem. 112, 811-815]. The gene was expressed in Escherichia coli by inserting it into an expression vector just downstream of the lacZ promoter, and this verified that the cloned gene really encodes the Methanobacterium AspAT. The primary structure of the Methanobacterium AspAT showed extremely low homology, 5%, with AspATs of eubacteria, eukaryotes, and a thermoacidophilic archaebacterium, Sulfolobus solfataricus. On the other hand, the Methanobacterium AspAT showed remarkable amino acid sequence homology, 31.5%, with rat serine:pyruvate aminotransferase and, 13.5%, with E. coli phosphoserine aminotransferase. Thus, the Methanobacterium AspAT apparently belongs to subgroup IV of the aminotransferases [Mehta, P. K., Hale, T. I., & Christen, P. (1993) Eur. J. Biochem. 214, 549-561], but not to subgroup I, in which all the AspATs known so far are included.

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Bacteroides fragilis SBT3182 produced two sialidases which differ in molecular weight on SDS-PAGE. These sialidases, a 50 kDa and a 55 kDa enzymes, were purified separately and their properties were compared. Both enzymes preferentially hydrolyze sialyl α 2-8 linkage rather than α 2-3 and α 2-6 bonds. The K_m values for Neu 5 Acα 2-3 lactose, Neu 5 Acα 2-6lactose, and colominic acid, which is a homopolymer of N-acetylneuraminic acid linked by α 2-8 bonds, were identical between the two enzymes. These enzymes had K_m value of 1.0-1.2mM for Neu 5 Acα 2-3 lactose and 1.3-1.5mM for Neu 5 Acα 2-6 lactose, which are in the ranges reported for other sialidases. However, the Km values for colominic acid (0.03-0.04mM) were lower than those of other sialidases, indicating that sialidases from B. fragilis SBT 3182 show high affinity for the sialyl α 2-8 linkage. The two sialidases also had identical N-terminal amino acid sequences and did not reveal any homology to known sialidases. PAS-staining suggested that these two sialidases were glycoproteins. In the lectin analysis, the 50 kDa enzyme was stained with Con A, DBA, and UEA-I while the 55 kDa sialidase was stained only with Con A. This suggested that the difference in molecular weight may be due to the carbohydrate composition. When the 50 kDa enzyme was incubated with UEA-I, which is a lectin specific for α-fucose residue, the activity decreased by 20%.

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Denaturations of ribonuclease A, lysozyme, and cytochrome c by guanidine hydrochloride (GdnHC1), urea, and GdnHCl-urea mixture were studied at constant temperature and pH to assess the functional dependence of denaturational free energy change (ΔG_D) on denaturant concentration over an extended GdnHCl concentration range. Conventional analysis of GdnHC1-induced transition curve exhibits a linear plot of ΔG_D versus [GdnHCl] in the transition zone. To extend ΔG_D measurements beyond this narrow concentration range, GdnHCl-induced unfolding was measured in the presence of different concentrations of urea. ΔG_D values from these measurements were corrected for the effect of urea on the free energy change using the appropriate relation. The corrected ΔG_D data were mapped onto the ΔG_D versus [GdnHCl] plot. For each protein, the dependence of free energy change on denaturation was found to be linear over the full GdnHCl concentration.

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An extramitochondrial acetyl-CoA hydrolase [EC 3. 1. 2. 1] in the rat liver, which is stimulated by ATP and inhibited by ADP, is known to be extremely cold-labile. During subcellular fractionations at low temperatures (2-4°C), most of the enzyme activity was lost; however, most could be recovered by rewarming at 37°C in the presence of a high concentration of potassium phosphate. This enabled us to measure the activities of cold-treated samples. The majority of the ATP-stimulated and ADP-inhibited acetyl-CoA hydrolase activity in rat livers was detected in the cytosolic fraction and small amounts were detected in the peroxisomal fraction. The activity of peroxisomal ATP-stimulated acetyl-CoA hydrolase was not noticeably increased after clofibrate-treatment. However, the cytosolic activity greatly increased after clofibrate treatment. The activity in the isolated peroxisomal fraction per g of liver was about 5% of that in the cytosolic fraction of liver from the control and about 2% in that from clofibrate-treated rats. Besides having similar nucleotide (ATP and ADP) sensitivity and cold lability, the enzyme protein in the peroxisomal fraction migrated to the same position as the cytosolic acetyl-CoA hydrolase based on Western blot analysis with antibody against purified acetyl-CoA hydrolase from rat liver cytosol. These results suggest that the peroxisomal enzyme and cytosolic enzyme may be the same entity.

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We prepared phospho-tyrosyl glutamine synthetase (P-GS) with suppressed activity from a highly adenylylated glutamine synthetase and applied it to the assay of protein-tyrosine phosphatase (PTPase) present in non-malignant rat liver cells (BRL) by RSV-transformation. The maximum PTPase activity toward P-GS was observed at neutral pH (pH 7.5-8.0) in the soluble and particulate fractions prepared from both BRL and RSV-transformed (RSV-BRL) cells. At low activity levels (<about 0.3 U), the PTPase activity in each fraction was proportional to the sample protein concentration (A₂₈₀) and the specific activity of PTPase in the soluble fraction of BRL cells was about twofold higher than that in the soluble fraction of BRL cells, while those in particulate fractions of BRL and RSV-BRL cells were almost the same as each other. Soluble fractions of BRL and RSV-BRL were subjected to molecular-sieve and anion-exchange chromatographies. One major PTPase activity, with an M_r of about 40, 000 (40 k), was detected in the BRL soluble fraction, and two were detected in the RSV-BRL soluble fraction with M_rs of about 40 k and 60 k. The 40 k PTPases in BRL and RSV-BRL had the same profiles on anion-exchange chromatography, but the 60 k PTPase in RSV-BRL cells showed a different profile. We suggest that the RSV-transformation of BRL cells induced the appearance of the 60 k PTPase in the soluble fraction.

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P450 2C2 as well as P450 2E1 [Fukuda, T. et al. (1993) J. Biochem. 113, 7-12] catalyzed the hydroxylation of medium chain fatty acids, although the regioselectivity of substrates of the former contrasted with that of the latter. Whereas P450 2E1 hydroxylated C₉-C₁₈ fatty acids at the ω-1 position and to a much lesser extent at the ω and ω-2 positions, P450 2C2 hydroxylated C₉-C₁₃ fatty acids at different positions dependent on the chain length of fatty acids. Among the fatty acids used as the substrate, undecanoate was hydroxylated at the ω-1 position almost exclusively by P450 2C2. The proportion of ω-hydroxylated products produced by P450 2C2 was markedly increased with decreasing chain length of fatty acids, while the hydroxylation positions were enlarged to the ω-3 position with tridecanoate. When the conserved Thr at the putative distal helix was replaced with Ser, the substrate regioselectivity of the two P450s was affected in different manners. The mutation of P450 2C2 did not change the hydroxylation positions of C₉-C₁₂ fatty acids, but caused a significant decrease in the proportion of the ω-1 hydroxy analog in the total products. In sharp contrast to P450 2C2, the mutated P450 2E1 gave additional products to those with the wild-type P450, and the number of different products increased with increasing chain length of the fatty acids. Thus, the products of palmitate hydroxylation were identified as ω-1, ω-2, ω-3, ω-4, ω-5, ω-6, and ω-7 monohydroxy isomers using gas chromatography-electron impact mass spectrometry. From these findings, (i) P450 2C2 shows the substrate selectivity of undecanoate 10-hydroxylation, whereas P450 2E1 has the activity of fatty acid ω-1 hydroxylation, and (ii) P450 2E1 is speculated to have a larger substrate pocket near the distal heme surface than P450 2C2 and the γ-methyl group of the conserved Thr may contribute to the limitation of the hydroxylation position in different ways in the two P450s.

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A glycosylated form of human interleukin 6 (hIL-6) has been produced in Chinese hamster ovary (CHO) cells transfected with a cDNA clone for human IL-6. Recombinant hIL-6 was purified from a culture supernatant of the transfected CHO cells, and used for structural characterization. The complete amino acid sequence, composed of 185 amino acid residues, was determined and is identical to that predicted from the cDNA sequence. However, a recombinant hIL-6 species lacking two amino acid residues (Ala-Pro) from the N-terminus was also found. Two disulfide bonds are formed, between Cys45 and Cys51 and between Cys74 and Cys84. Recombinant hIL-6 carries one O-linked carbohydrate chain, and Thr139 is fully O-glycosylated. A portion of recombinant hIL-6 protein carries one N-linked sialooligosaccharide chain, and the N-glycosylation occurs at Asn46. The structure of the N-linked sugar chains was estimated by a combination of sugar mapping and glycosidase digestion. The major structure of the N-linked sugar chain predicted was of a fucosylated biantennary or triantennary complex type. Fucosylated triantennary sugar chains with one or two N-acetyllactosaminyl repeats were also found. The structure of the O-linked sugar chain was determined by 500 MHz ¹H-NMR to be NeuAca2-3Galβ1-3(NeuAcα2-6) Gal-NAcol.

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When myofibrils prepared from chicken leg muscle were treated with a solution containing 0.1mM CaCl₂ and 30 μg/ml of leupeptin, α-connectin, which exists as a longitudinal thin fil Pament in a sarcomere, was split into β-connectin and a 1, 200-kDa subfragment. The native subfragment was successfully purified without using any denaturant: It was extracted with 1M KI solution from the Ca-treated myofibrils and purified by TSKgeI G6000PW column chromatography. About 10mg of the subfragment was yielded from 100g of starting muscle. Using immunofluorescence microscopy and immunoelectron microscopy, we show here that polyclonal antibodies against the 1, 200-kDa subfragment bind to the Z-disk and the epitope, which is about 0.34 μm apart from the Z-disk at a sarcomere length of 2.6 μm; the 1, 200-kDa subfragment constitutes the proximal region of connectin filaments. Purified α-actinin decorated α-connectin and the 1, 200-kDa subfragment on nitrocellulose blots of myofibrillar proteins separated by SDS-PAGE. Therefore, we conclude that connectin filaments are anchored to the Z-disk by the binding of the 1, 200-kDa subfragment to α-actinin.

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In order to study the metabolism of spermidine in mammals, radioactive spermidine was injected intraperitoneally into a rat and urine was collected for analysis. Incorporation of radioactivity into putreanine, isoputreanine, spermidic acid, and N-aminopropylpyrrolidin-2-one was confirmed by ion-exchange chromatography, thin layer chromatography, and paper electrophoresis, the highest radioactivity being observed in the non-polar and acidic fraction of the collected urine. A radioactive compound was purified from the non-polar and acidic fraction, and identified as 2-oxo-1-pyrrolidinepropionic acid by comparison of its behavior on ion-exchange chromatography and thin layer chromatography with that of authentic 2-oxo-1-pyrrolidinepropionic acid, and recrystallization with the authentic compound. Acid hydrolysis of the radioactive compound produced radioactive spermidic acid, confirming the identification. To examine the interconversion between isoputreanine and N-aminopropylpyrrolidin-2-one, these compounds were deuterated and then intraperitoneally injected into a rat. Analysis of 24-h urine by gas-chromatography-mass-spectrometry indicated no interconversion between the two metabolites of spermidine under these conditions. An intracerebroventricular injection of radioactive spermidine into a rat showed that radioactivity was also incorporated into the metabolites of spermidine in the brain, and oxidative deamination of the aminopropyl moiety of spermidine was thought to be dominant in the central nervous system and vice versa in peripheral organs.

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A substance that exhibited a tryptophan-like fluorescence peak at 354 nm on excitation at 295 nm at neutral pH was isolated from human urine. This compound was determined by visible-light absorption spectroscopy, fluorescence spectroscopy, ¹H and ¹³C NMR spectroscopies, and FAB-MS to be 1-(1', 2', 3', 4', 5'-pentahydroxypentyl)-1, 2, 3, 4-tetrahydro-2-carboline-3-carboxylic acid. This compound, named tetrahydropentoxyline, is a new type of hydrophilic tetrahydro-β-carboline, and its elution position was between those of 4-pyridoxic acid and kynurenic acid on C₁₈ reversed-phase HPLC. The amount of tetrahydropentoxyline excreted in the urine of normal subjects [n=21; age, 45 (SD 20) years] was about 5.2 (SD 1.0) mg per day.

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