Calcium-activated neutral protease and its endogenous inhibitor Activation at the cell membrane and biological function

The structures of calcium-activated neutral protease (CANP) and its endogenous inhibitor elucidated recently have revealed novel features with respect to their structure-function relationship and enzyme activity regulation. The protease is regarded as a proenzyme which can be activated at the cell membrane in the presence of Ca²⁺ and phospholipid, and presumably regulates the functions of proteins, especially membrane-associated proteins, by limited proteolysis. Protein kinase C is hydrolysed and activated by CANP at the cell membrane to a cofactor-independent form. These results are reviewed and the possible involvement of CANP in signal transduction is discussed.     

Communication between two globular domains of calmodulin in the presence of mastoparan or caldesmon fragment. Ca2+ binding and 1H NMR

Ca2+ binding to calmodulin was measured in the presence of mastoparan or caldesmon fragment. Mastoparan and caldesmon fragment were used as model compounds of enzymes and cytoskeleton proteins, respectively, working as the target of calmodulin. Although the Ca2+ bindings of the two globular domains of calmodulin occur independently in the absence of the target peptide (or proteins), mastoparan and caldesmon fragment increased the affinity of Ca2+ and, at the same time, produced the positive cooperative Ca2+ bindings between the two domains. The result of Ca2+ binding was compared with 1H NMR spectra of calmodulin in the presence of equimolar concentration of mastoparan. It is known that a conformation change of the C-terminal half-region (C-domain) occurs by the Ca2+ binding to C-domain. A further change in conformation of C-domain was demonstrated by the Ca2+ binding to the N-terminal half-region (N-domain) in the presence of mastoparan. It indicates that the two domains of calmodulin get into communication with each other in the associated state with the target, and we concluded that the Ca2+ binding to the N-domain is responsive to the development of calmodulin function.     

Regional assignment of the gene for diphtheria toxin sensitivity using subchromosomal fragments in microcell hybrids

Human x mouse microcell hybrids resistant to G418 were constructed between mouse hepatoma cells and human x mouse whole cell hybrids containing only intact human chromosome 5 and 22 with an integrated neo ^r-gene. Among these, microcell hybrid BG15 produced four subclones, BG15-4, BG15-6, BG15-7 and BG15-9, which contained variously sized complements of human chromosome 5. BG15-6 contained an intact human chromosome 5, BG15-7 a deleted human chromosome 5 (5pter-q22) and BG15-4 and BG15-9 a translocation between parts of human chromosome 5 (pter-qter? and pter-q23, respectively) and a mouse chromosome. Southern DNA blot analysis showed that the human dihydrofolate reductase (DHFR) gene was present in all four subclones, whereas the human homolog of the v-fms gene was present in BG15-4 and 15-6, but absent from BG15-7 and 15-9. BG15-4, 15-6 and 15-9 were sensitive to diphtheria toxin, and only BG15-7 was resistant to the toxin. We used these microcell hybrids to restrict further the regional location of the gene for diphtheria toxin sensitivity to the q23 region of human chromosome 5.     

Regional assignment of five genes on human chromosome 19

A human-mouse hybrid segregant HM76Dd40-6 with new characteristics was derived from the hybrid cell line HM76Dd containing human chromosome 19 as the only human chromosome. Three virus sensitivities located on human chromosome 19 (PVS, E11S and RDRC) were lost in HM76Dd40-6, while six other genes (C3, LDLR, EF2, GPI, PEPD and MANB) were retained. Cytogenetic analysis and in situ hybridization using human or mouse repeated sequences as probes showed that the region q13.1-qter of human chromosome 19 had been replaced by a fragment of mouse chromosome. Our results permit further regional assignment for the following five genes on human chromosome 19: GPI in the region cen-q12, MANB in p13.2-q12, E11S and RDRC in q13.1-qter, and EF2 in pter-q12.     

Complete structural analysis of globoseries glycolipids by two-dimensional nuclear magnetic resonance

Combined two-dimensional proton nuclear magnetic resonance allowed the determination of complete oligosaccharide structures of glycolipids belonging to the globo series, without any other analytical methods. Although a chemical modification by peracetylation was required for the above purpose, the derivatization permitted facile assignment of the pyranose ring proton resonances of the oligosaccharide moiety. Two-dimensional chemical-shift-correlated spectroscopy of the acetylated glycolipid enabled us to elucidate the glycosidic positions from the chemical shifts of the protons at the substituted sites. The monosaccharide species were also identified from the characteristic splitting patterns of the methine protons on individual pyranose rings. The sequence of the monosaccharides was inferred from the interresidue connectivity across glycosidic linkages shown by two-dimensional nuclear Overhauser effect spectroscopy, which also gave intraresidue interaction on the pyranose rings. The linkage sites of long oligosaccharide chains having more than five monosaccharides, such as globopentaosylceramide, were analyzed by two-dimensional J-relayed coherence transfer, which yielded 1,3 interactions along with 1,2 interactions.     

Pharmacokinetic study of exogenously administered ß-endorphin using a rapid radioreceptor assay in rats

A rapid radioreceptor assay for measuring ß-endorphin (ß-EP) in unextracted serum has been developed. The method is based upon the inhibition by ß-EP of ³H-naloxone binding to the specific receptors on rat brain membranes, prepared in a stable form of pellets. Effect of serum on the assay was minimized by adding pooled serum to the equal dilution of total serum in the assay mixture. Pharmacokinetic analysis of pharmacologically active ß-EP equivalents (ß-EP eq.) in rats was performed using this method. The serum disappearance of ß-EP eq. after iv administration followed a biexponential decline and pharmacokinetic parameters were calculated by a two-compartment open model. The half-lives of α-phase and ß-phase were 2.6 ± 0.5 min and 6.2 ± 1.6 hr (mean ± SE; n=6), respectively. The volume of the central compartment (V₁) and that of steady-state (Vd_ss) were 67 ± 16 and 480 ± 75 ml/kg (mean ± SE; n=6), respectively. The total body serum clearance (CL_tot) was 2.1 ± 0.9 ml/min/kg (mean ± SE; n=6). The serum disappearance curve of ß-EP eq. obtained in the present study was similar to that previously reported by Houghten et al. (Proc. Natl. Acad. Sci. U.S.A. $\text{77}$, 4588–4591 (1980)), in which the disapperance of total radioactivity of tritiated ß-EP in rats was examined.     

Effect of High-intensity Light Given Prior to Low-temperature Treatment on the Long-day Flowering of Pharbitis nil

Pharbitis nil, strain Violet which had been exposed to high-intensitylight (18,000 lux at 23?C) for 7 days followed by a low-temperaturetreatment (13–14?C) for 7 days initiated flower buds evenunder continuous light, but plants given these treatments inreverse order failed to bud. Three days of high-intensity lightat 23?C was most effective in promoting the flower-inducingeffect of the subsequent low-temperature period. Six days oflow temperature following the 3-day high-intensity light periodinduced near-maximum flowering response. DCMU (5?10^–6M) given during the high-intensity light period inhibited flowering,but when given during or after the low-temperature period itwas ineffective. DCMU at the same concentration given before,during or after an inductive 16-hr dark period at 26?C did notinhibit flowering. Sucrose, ATP, NADPH and some other reducingagents tested did not nullify the DCMU effect nor substitutefor the effect of high-intensity light. But, the high-intensitylight effect could be substituted, at least partly, by 5-chlorosalicylicacid, 3,4-dichlorobenzoic acid and some other benzoic acid derivatives,which are highly effective in inducing long-day flowering inthe short-day plant, Lemna paucicostata. (Received October 20, 1981; Accepted February 3, 1982)     

Crystallization and preliminary X-ray structure analysis of pigeon egg-white lysozyme.

Calcium binding lysozyme from pigeon egg-white was crystallized by the hanging drop vapor diffusion technique using ammonium sulphate as a precipitant. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), and have unit cell dimensions of a = 34.2 A, b = 34.8 A, and c = 99.4 A. One asymmetric unit contains one molecule of the pigeon lysozyme. The crystals diffract X-rays at least to 2.0 A resolution and are suitable for high resolution structure analysis. The diffraction data up to 3.0 A resolution were collected with a diffraction image processor, DIP100, using a Fuji imaging plate as an area detector. The structure was solved by the molecular replacement technique and refined to an R factor of 0.216. Least-squares fitting of the main-chains of pigeon egg-white lysozyme with those of chicken egg-white lysozyme and baboon alpha-lactalbumin showed that the main-chain folding of pigeon lysozyme is more similar to that of chicken lysozyme than that of alpha-lactalbumin. The largest differences between the pigeon and chicken lysozymes are in the surface loop regions.