Crystal structure of the complex of subtilisin BPN' with its protein inhibitor Streptomyces subtilisin inhibitor. The structure at 4.3 Angstroms resolution

The crystal structure of the complex of subtilisin BPN′ (EC 3.4.21.14) with its protein inhibitor (Streptomyces subtilisin inhibitor) was solved at 4.3 Å resolution, thus establishing the following. (1) Two subtilisin BPN′ molecules (2E) associate with one dimeric inhibitor molecule (I₂) to form the complex molecule E₂I₂. (2) The conformation of neither the inhibitor nor subtilisin BPN′ undergoes any detectable change at this resolution upon complex formation. (3) The inhibitor binds to subtilisin to form an antiparallel β-sheet, as in the case of trypsin/ trypsin inhibitor complexes. (4) The scissible bond of the inhibitor is between Met73′ and Val74′, as proposed earlier (Ikenaka et al., 1974). (5) The protein inhibitor and the substrates bind to subtilisin BPN′ in essentially the same way.     

Selective solubilization and purification of cell-surface concanavalin A-binding glycoproteins from Novikoff hepatocellular carcinoma cells

Novikoff hepatocellular carcinoma cells possess cell-surface glycoproteins that bind the lectin, concanavalin A. A subset of Con A-binding plasma membrane glycoproteins was solubilized by addition of n-butanol to a suspension of Novikoff cells. Glycoproteins solubilized into the n-butanol-saturated aqueous phase of the two-phase mixture were purified by sequential chromatography on DEAE-cellulose and Sepharose-conjugated concanavalin A. Glycoproteins specifically bound to the Sepharose-conjugated Con A exhibited apparent M_r = 72,000 to 125,000. The plasma membrane localization of these components was inferred by their isolation from cells surface labeled with NaIO₄/ NaB³H₄. A xenoantiserum, raised against glycoproteins specifically bound to Sepharose-conjugated concanavalin A was employed to identify reactive components in nonionic detergent extracts of Novikoff tumor cells or rat hepatocytes surface labeled using lactoperoxidase-catalyzed iodination (¹²⁵I). Major reactive peptides in extracts of Novikoff cells exhibited apparent M_r = 74,000, 82, 000, 110,000, and 135,000, while those in extracts of hepatocytes possessed apparent M_r = 98,000 and 105,000. The reactivity of the antiserum with extracts of ¹²⁵I-labeled Novikoff cells was abolished by absorption of the antiserum with hepatocytes, indicating that the qualitative differences observed may result from structural modification of one or more cell-surface glycoproteins, rather than the expression of new or inappropriate glycoproteins. This antiserum will provide a useful probe to investigate alterations in the expression or structure of glycoproteins that occur as a consequence of malignant transformation or adaptation of malignant cells to growth in the ascitic form.     

Nucleotide sequence of the genes for β and ε subunits of proton-translocating ATPase from Escherichia coli

The 1855-nucleotide long DNA sequence of part of the gene cluster for the proton-translocating ATPase from $\text{E. coli}$ was determined by the method of Maxam-Gilbert. The sequence covers the genes for the β and ε subunits of F₁ along with the flanking region. The amino acid sequence of these subunits deduced from the nucleotide sequence indicates that the β and ε subunits have 459 and 138 amino acids, respectively. The possible secondary structure of the both subunits was estimated from the deduced primary structures. A possible nucleotide binding site in the β subunit is also discussed on the basis of the primary and secondary structures. The codons used in the genes for all the components of F₁F₀ were different in different genes, suggesting that the amount of each subunit in the F₁F₀ is determined to some extent on a translational level.     

Requirement of the core structure of a complex-type glycopeptide for the binding to immobilized lentil- and pea-lectins

Structural requirements for the binding of oligosaccharides and glycopeptides to immobilized lentil- and pea-lectins were investigated by use of radioactively-labeled glycopeptides and oligosaccharides. The results indicate that an intact 2- acetamido-2-deoxy-β-d-glucopyranosyl residue at the reducing end of a complex-type oligosaccharide is essential for high-affinity binding to lentil lectin-Sepharose but not to concanavalin A-Sepharose and that an asparagine residue is required for the binding of a complex-type glycopeptide to pea lectin-Sepharose. In addition, interaction of a complex-type oligosaccharide with lentil lectin-Sepharose was enhanced by exposure of nonreducing, terminal 2-acetamido-2-deoxy-β-d-glucopyranosyl groups, whereas interaction with pea lectin-Sepharose was enhanced only after exposure of nonreducing, terminal α-d-mannopyranosyl groups.     

A cytolysin, theta-toxin, preferentially binds to membrane cholesterol surrounded by phospholipids with 18-carbon hydrocarbon chains in cholesterol-rich region.

We have previously suggested the existence of two distinctive states of cholesterol in erythrocyte and lymphoma cell membranes as revealed by high- and low-affinity binding sites for theta-toxin of Clostridium perfringens [Ohno-Iwashita, Y., Iwamoto, M., Mitsui, K., Ando, S., & Nagai, Y. (1988) Eur. J. Biochem. 176, 95-101; Ohno-Iwashita, Y., Iwamoto, M., Ando, S., Mitsui, K., & Iwashita, S. (1990) Biochim. Biophys. Acta 1023, 441-448]. To understand factor(s) which determine membrane cholesterol heterogeneity, we analyzed toxin binding to large unilamellar liposomes composed of cholesterol and phospholipids (phosphatidylcholine/phosphatidylglycerol = 82:18, mol/mol). Liposomes containing phospholipids with 18-carbon hydrocarbon chains at both positions 1 and 2 of the glycerol have both high- and low-affinity toxin-binding sites with Kd values similar to those of intact erythrocytes, whereas liposomes with hydrocarbon chains containing 16 or fewer carbons at either position 1 or 2 have only low-affinity toxin-binding sites. The cholesterol/phospholipid ratio, in addition to the length of phospholipid hydrocarbon chain, also determines the number of toxin-binding sites, indicating that at least these two factors determine the topology of membrane cholesterol by creating distinctively different affinity sites for the toxin. Since theta-toxin binding detects specific populations of membrane cholesterol that are not detectable by the measurements of susceptibility to cholesterol oxidase and cholesterol desorption from membranes, the toxin could provide a unique probe for studying the organization of cholesterol in membranes.     

A case of renin producing leiomyosarcoma originating in the lung.

A 54-year-old woman was referred to our hospital for the treatment of a tumor of the right chest wall. Clinical examination revealed hypertension, hypokalemia, metabolic alkalosis, hyperaldosteronism and hyperreninemia. Computed tomography and an abdominal echogram indicated a tumor in the right phrenic area and two tumors in the retroperitoneum near the pancreas head. After the surgical resection of these tumors, the primary reninism was diminished. The pathological diagnosis of these tumors was leiomyosarcoma. Plasma active and inactive (trypsin-activated) renin activities (PRA) were 85.7 and 38.9 ng angiotensin I/ml/h, respectively. These PRA did not respond to either postural stimulation or suppression by the volume expansion. Active and inactive renin activities in a right phrenic area tumor were 208 and 32 ng angiotensin I/mg protein /h, respectively. Those of an abdominal tumor were 196 and 30 ng angiotensin I/mg protein/h, respectively. Renin mRNA identical in molecular size to that of the human kidney was identified by northern blot analysis. This is the first case report of renin producing leiomyosarcoma derived from the lung, which is characterized by relatively lower plasma prorenin concentrations.     

An Activation of Synaptosomal Na+, K+-ATPase by a Novel Dibenzoxazepine Derivative (BY-1949) in the Rat Brain: Its Functional Role in the Neurotransmitter Uptake Systems

In search of factors mitigating the final outcome of ischemic and epileptic brain damage, we tested a novel dibenzoxazepine derivative (BY-1949), as the compound has been shown to be effective under these two conditions. First, using rat brain, we assessed whether or not BY-1949 affects the Na+,K(+)-ATPase activity. Although in vitro applications of either BY-1949 or its three major metabolites did not cause any apparent effects, both acute and chronic oral administrations of the compound (10 mg/kg) invariably increased the Na+,K(+)-ATPase activity in the synaptosomal plasma membranes by increasing Vmax values. Second, it was shown by this study that the drug treatment caused marked increases in the uptake of both glutamic acid and gamma-aminobutyric acid into the synaptosomes. These results suggest that the activity against ischemic/epileptic brain damage by BY-1949 is explicable, at least partly, in terms of improvement of ionic derangements across the neural membranes via Na+,K(+)-ATPase activation.     

Secretion of endothelin-1 in human endothelial cell line but not in B cell line by transfection of preproendothelin-1 cDNA.

Stable transformants with preproendothelin-1 (preproET-1) cDNA were established for the study of the regulation of endothelin-1 (ET-1) biosynthesis in human cells. ET-1, a potent vasoconstrictor peptide, is produced by endothelial cells and is secreted into the blood at a low level. Human preproET-1 cDNA was introduced into two immortal human cell lines, t-HUE2, an endothelial cell line, and Raji, a B cell line, with Ecogpt selection. Several stable transformants of t-HUE2 expressed extraordinarily high levels of preproET-1 specific mRNA and secreted ET-1 into serum-free culture medium, while the transformants of Raji cells expressed high levels of ET-1 mRNA, but secreted a negligible amount of ET-1. Immunocytochemical studies of intracellular ET-1 content revealed that there were some defects in the translation or processing of preproET-1 in the B cell line transformants. In addition, the ratio of ET-1 to ET-1 precursor (big ET-1) was much higher in the t-HUE2 transformants than in normal endothelial cells, suggesting that t-HUE2 transformants (for example t-HUE2-1) possess high levels of endothelin converting enzyme (ECE). The establishment of stable transformants producing high levels of ET-1 in serum-free medium will be useful for the study of cell-type-specific translation and processing to mature ET-1, and of the regulatory factors of ECE.     

The B subunit of cholera toxin enhances DNA synthesis in rat hepatocytes induced by insulin and epidermal growth factor.

The B subunit of cholera toxin, which binds to ganglioside GM1, enhanced DNA synthesis in rat hepatocytes in primary culture induced by insulin and/or epidermal growth factor. The effect was dose-dependent, and whole cholera toxin, activating adenylate cyclase, showed a higher effect than the B subunit alone. The B subunit acted additively with other agents that also increase cyclic AMP levels. A competitive antagonist of cyclic AMP could not suppress the effect of the B subunit completely. These data suggest that the effect is independent of the cyclic AMP signal pathway, and that GM1 plays a role in hepatocyte proliferation.