gamma-Carboxyglutamic acid (Gla)-domainless blood coagulation factor IXa species: preparation and properties.

To investigate the function of the gamma-carboxyglutamic acid (Gla) residues of factor IXa in the activation of factor X, a new species of bovine factor IXa, designated "factor IXa beta'," and its corresponding Gla-domainless form, designated "Gla-domainless factor IXa beta'," were prepared under controlled conditions and characterized. First, bovine factor IXa alpha was converted by alpha-chymotrypsin in the presence of calcium ions to factor IXa beta' (Mr 47,000). Compared with factor IXa beta, factor IXa beta' had essentially identical activities towards a synthetic substrate, benzoyl-L-arginine ethylester (BAEE), towards an active site titrant, p-nitrophenyl-p'-guanidinobenzoate, and towards protein substrate, namely, factor X. Next, the Gla-rich region (residues 1-41) of the light chain was removed from factor IXa beta' by additional selective cleavage by alpha-chymotrypsin in the absence of calcium ions. Gla-domainless factor IXa beta' was purified to homogeneity on a column of DEAE-Sepharose CL-6B. The heavy chain was not altered by either chymotryptic digestion. Functional comparisons of the three activated forms, namely, factor IXa alpha, factor IXa beta', and Gla-domainless factor IXa beta', with factor IXa beta revealed that all four activated forms of factor IX had one active-site residue per molecule and essentially identical specific esterase activity towards BAEE. However, the clotting activity of Gla-domainless factor IXa beta' was less than 0.5% of that of factor IXa beta'.(ABSTRACT TRUNCATED AT 250 WORDS)     

Actin polymerization promoted by a heptapeptide, an analog of the actin-binding S site on myosin head

Polymerization of G-actin to F-actin was indicated by an increase in light-scattering intensity after the addition of a heptapeptide (Ile-Arg-Ile-Cys(MT)-Arg-Lys-Gly-OEt), an analog of the actin-binding S-site on S-1 heavy chain. The half-maximal concentration of the heptapeptide which induced an increase in the light-scattering intensity at 25 degrees C was about 110 microM, which was in the range of the dissociation constant of this peptide with F-actin. The polymerization of G-actin to F-actin by binding of the heptapeptide was further demonstrated by ultracentrifugal separation, Pi liberation, and electron microscopy. The polymerization of G-actin was induced only by the heptapeptide, but not by fragments of the heptapeptide. The well known acceleration of polymerization of G-actin by the myosin head may be due to the binding of G-actin with the S-site on the myosin head.     

Anti-pituitary antibodies in patients with hypopituitarism and their families: longitudinal observation.

In an attempt to investigate the clinical significance of anti-pituitary antibodies in patients with hypopituitarism, anti-pituitary antibody in plasma was examined in 10 such patients (7 cases of isolated ACTH deficiency, 1 of partial hypopituitarism, and 2 of Sheehan's syndrome), on two or three occasions with an interval of more than 6 months (longitudinal study). In a total of 16 relatives of these 4 patients (2 cases of Sheehan's syndrome, one in each of partial hypopituitarism and isolated ACTH deficiency) and one patient not involved in the longitudinal study, anti-pituitary antibodies were also examined (family study). Anti-pituitary antibodies reacting with rat pituitary cytoplasmic antigens (pituitary cell antibodies: PCA) and pituitary cell surface antibodies (PCSA) reacting with GH3 cells and/or AtT-20 cells were measured with indirect immunofluorescence. The longitudinal study revealed the disappearance of antibodies in 3 patients, 2 PCA positive and one both PCA and PCSA positive. In 3 patients, altered antibody titers or a newly appearing antibody were found during the follow-up period. In 4 patients, the pituitary antibodies were negative during the follow-up periods. Of 16 family members studied, positive PCA was found in 3 members (2 in the families of patients with PCA positive Sheehan's syndrome, and 1 in the family of the patients with PCA positive partial hypopituitarism). Positive PCSA was found in 4 members (one in each of families of patients with partial hypopituitarism and isolated ACTH deficiency and of two cases of Sheehan's syndrome), and weakly positive PCSA was found in one family member of a patients with PCA positive Sheehan's syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complete amino acid sequence of endo-beta-N-acetylglucosaminidase from Flavobacterium sp

The complete amino acid sequence of endo-beta-N-acetylglucosaminidase from Flavobacterium sp. has been determined by analysis of peptides after cleavage with lysyl endopeptidase, pepsin and chymotrypsin. The protein consists of a single polypeptide chain consisting of 267 amino acid residues and a molecular mass of 27972 Da. The sequence of Flavobacterium endo-beta-N-acetylglucosaminidase is very close to that of the Streptomyces enzyme (endo-H), having 60% similarity and very similar hydropathy profiles. Similarities were also found between Flavobacterium endo-beta-N-acetylglucosaminidase and chitinases from Bacillus circulans, Serratia marcescens and Phaseolus vulgaris.     

Augmentation of retinoic acid-induced granulocytic differentiation in HL-60 leukemia cells by serine/threonine protein phosphatase inhibitors.

To evaluate the involvement of protein phosphatases (PP) in differentiation of human myelogenous leukemia HL-60 cells, we made use of potent inhibitors of PP1 and PP2A, calyculin-A (CAL-A) and okadaic acid (OKA). CAL-A and OKA could augment all-trans retinoic acid (ATRA)-induced granulocytic differentiation, whereas the differentiation toward macrophage lineage by 12-o-tetradecanoylphorbol acetate (TPA) was unchanged in the presence of CAL-A. CAL-A augmented the phosphorylation of 18K, 23K and 30K proteins induced by ATRA. The PP1 and PP2A were identified and were present mainly in the cytosol of HL-60 cells. These results suggest that either PP1 or PP2A or both may be involved in regulating granulocytic differentiation of HL-60 cells.     

Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction

For determination of the physiological role and mechanism of vacuolar proteolysis in the yeast Saccharomyces cerevisiae, mutant cells lacking proteinase A, B, and carboxypeptidase Y were transferred from a nutrient medium to a synthetic medium devoid of various nutrients and morphological changes of their vacuoles were investigated. After incubation for 1 h in nutrient-deficient media, a few spherical bodies appeared in the vacuoles and moved actively by Brownian movement. These bodies gradually increased in number and after 3 h they filled the vacuoles almost completely. During their accumulation, the volume of the vacuolar compartment also increased. Electron microscopic examination showed that these bodies were surrounded by a unit membrane which appeared thinner than any other intracellular membrane. The contents of the bodies were morphologically indistinguishable from the cytosol; these bodies contained cytoplasmic ribosomes, RER, mitochondria, lipid granules and glycogen granules, and the density of the cytoplasmic ribosomes in the bodies was almost the same as that of ribosomes in the cytosol. The diameter of the bodies ranged from 400 to 900 nm. Vacuoles that had accumulated these bodies were prepared by a modification of the method of Ohsumi and Anraku (Ohsumi, Y., and Y. Anraku. 1981. J. Biol. Chem. 256:2079-2082). The isolated vacuoles contained ribosomes and showed latent activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase. These results suggest that these bodies sequestered the cytosol in the vacuoles. We named these spherical bodies "autophagic bodies." Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle. Genetic analysis revealed that the accumulation of autophagic bodies in the vacuoles was the result of lack of the PRB1 product proteinase B, and disruption of the PRB1 gene confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.     

Secretory regulation of 19-hydroxyandrostenedione in normal man.

To evaluate the secretory regulation of 19-hydroxyandrostenedione (19-OH-AD), its plasma concentration was measured before and after stimulation and inhibition tests for the ACTH-adrenal axis and the renin-angiotensin system in 50 normal subjects. Basal levels of plasma 19-OH-AD did not correlate with either those of plasma renin activity (PRA) or the plasma aldosterone concentration (PAC), but positively correlated with those of plasma cortisol. Plasma 19-OH-AD was stimulated by 0.25 mg ACTH-(1-24) and was suppressed by 1 mg dexamethasone (DEX) as were plasma cortisol and PAC. On the other hand, with 2-h standing alone or iv 40 mg furosemide plus 2-h standing, plasma 19-OH-AD and cortisol did not increase but PRA and PAC did. With iv furosemide plus 2-h standing with 3 mg DEX pretreatment, plasma 19-OH-AD and cortisol did not respond either, but PRA and PAC increased. With 25 mg oral captopril following 1-h standing with 3 mg DEX pretreatment, plasma 19-OH-AD and cortisol did not change but PAC decreased. These results indicate that the secretion of 19-OH-AD is mainly under the control of the ACTH-adrenal axis rather than the renin-angiotensin system.     

Induction of micronucleated reticulocytes by potassium bromate and potassium chromate in CD-1 male mice.

Micronucleus tests of potassium bromate (KBrO3) and potassium chromate (K2CrO4) were conducted with peripheral blood reticulocytes (PB-RETs) of CD-1 male mice dose intraperitoneally. Peripheral blood cells collected from the tail were stained supravitally with acridine orange (AO) using AO-coated glass slides. Both KBrO3 and K2CrO4 induced micronuclei in PB-RETs in the same manner as in polychromatic erythrocytes of bone marrow.     

Exchange of the fluorescence-labeled 20,000-dalton light chain of smooth muscle myosin.

The 20,000-dalton light chain of smooth muscle myosin was exchanged with exogenous light chain in a solution containing 0.5 M NaCl and 10 mM EDTA at 40 degrees C. The light chain was almost completely exchanged within 30 min under the above conditions. The exchange was markedly inhibited either below 37 degrees C or in the presence of Mg2+ concentrations higher than 10 microM. The 20,000-dalton light chain was selectively labeled of a single thiol (Cys-108) with 5-[[2-[(iodoacetyl)amino]ethyl]amino-naphthalene-1-sulfonic acid (1,5-IAEDANS). The labeled light chain was exchanged stoichiometrically into myosin and was used as a probe to investigate the conformation of smooth muscle myosin. The resulting myosin hybrids showed enzymatic properties virtually identical with those of the control, untreated myosin; i.e., actin-activated ATPase activity was dependent on the 20,000-dalton light-chain phosphorylation catalyzed by myosin light chain kinase, and the 10S-6S conformational transition of myosin correlating with the changes in ATPase was also affected either by the light-chain phosphorylation or by the change in the ionic strength. Steady-state fluorescence antisotropy measurements were performed by varying the temperature. The Perrin-Weber plots were constructed in order to obtain information about the average rotational mobility of the probe and to estimate the rotational correlation time for the AEDANS-myosin head. The fluorescence probe on the 20,000-dalton light chain was found to be quite immobile as indicated by its limiting anisotropy (A0 = 0.33).(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of a K(+)-channel protein of sarcoplasmic reticulum by assaying the channel activity in the planar lipid bilayer system

A K(+)-channel protein of the sarcoplasmic reticulum (SR) was purified by assaying the channel activity in a planar lipid bilayer system. The light fraction of SR vesicles was solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and fractionated by an anion-exchange chromatography and followed by gel filtration chromatography and affinity chromatography with concanavalin A. All fractions in each steps were mixed with asolectin solubilized in CHAPS and reconstituted into vesicles by dialysis. The channel activity of each fraction was assayed after the reconstituted vesicles had been fused into a planar lipid bilayer. The final fraction which showed the K(+)-channel activity contained only 100 kDa protein in a silver-stained gel after SDS-PAGE and an anti-Ca(2+)-ATPase antibody did not recognize the protein. The characteristics of the K(+)-channel were identical to those observed in native SR vesicles when using the same method. The channel showed a single-channel conductance of 120 pS in 0.1 M KCl and marked voltage dependence. The channel did not permeate Ca2+ and Cl- and was blocked by neomycin B.