Characterization and purification of polymorphic arylalkylamine N-acetyltransferase from the American cockroach, Periplaneta americana.

We separated two forms of arylalkylamine N-acetyltransferase (AANAT) from various organs of the American cockroach, Periplaneta americana. Both forms of the enzyme had an equivalent molecular mass of 28 kDa. One form isolated from the testicular accessory glands had high enzyme activity at acidic pHs. The isoelectric point was 5-6 and the substrate specificity was wider than the other type. The other isolated form from female midguts had a higher level of enzyme activity at basic pHs. These findings suggested that P. americana contains polymorphic AANAT, as is the case in Drosophila melanogaster. These forms differed not only in pH specificity, and substrate specificity but in chromatographic behavior and kinetic properties. Most of the organs we examined contained a mixture of the two forms since two types of AANAT activity were separated in different chromatographic fractions when two pH conditions were used for activity measurement.     

In vitro translocation of protein across Escherichia coli membrane vesicles requires both the proton motive force and ATP

The energy requirement for protein translocation across membrane was studied with inverted membrane vesicles from an Escherichia coli strain that lacks all components of F1F0-ATPase. An ompF-lpp chimeric protein was used as a model secretory protein. Translocation of the chimeric protein into membrane vesicles was totally inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or valinomycin and nigericin and partially inhibited when either valinomycin or nigericin alone was added. Depletion of ATP with glucose and hexokinase resulted in the complete inhibition of the translocation process, and the inhibition was suppressed by the addition of ATP-generating systems such as phosphoenolpyruvate-pyruvate kinase or creatine phosphate-creatine kinase. These results indicate that both the proton motive force and ATP are required for the translocation process. The results further suggest that both the membrane potential and the chemical gradient of protons (delta pH), of which the proton motive force is composed, participate in the translocation process.     

Analysis of overall pathophysiological relationships between serum levels of TSH and thyroid hormones using a large laboratory database

Factors associated with the basal level of serum thyrotropin (TSH) were analyzed over a wide range of pathophysiological conditions by means of a large laboratory database on thyroid function. When data were analyzed two-dimensionally, serum TSH showed significant inverse correlations with total triiodothyronine (T3), free T3 index (FT3I), total thyroxin (TT4) and free T4 index (FT4I) in the order of increasing intensity. The three-dimensional analysis, however, revealed that 1) total hormone levels were actually unrelated to serum TSH when the levels of free hormone indices were held constant, 2) the relation between FT3I and TSH became obscure when the influence of FT4I was similarly removed. On the other hand, 3) the relation of FT4I with TSH was unaffected by the level of FT3I. These results suggest that free T4 is the main determinant of the serum TSH level. This study also implies that it is possible to use large amounts of laboratory data to elucidate the overall profile of a given patho-physiological system, whose structure is only partially revealed by conventional clinical or animal studies.     

Nucleotide sequence of Suncus murinus immunoglobulin mu gene and comparison with mouse and human mu genes

RNA polymerase from the archaebacterium Sulfolobus acidocaldarius was chemically modified with AMP o-formylphenyl ester followed by reduction with borohydride. The modified protein catalyzes the labeling of its own largest subunit when incubated with [-³³P]UTP in the presence of poly[d(A-T)]. On cleaving of the labeled protein using cyanogen bromide, hydroxylamine or amino acid-specific endoproteinases for a very brief period, the pattern and size of the radioactive fragments formed are best explained by attachment of the label between Gly⁸⁴³ and Met⁸⁹⁵ of the largest subunit. In this region there exists a highly conserved sequence which is also found in other archaebacterial, eukaryotic and prokaryotic RNA polymerases. This suggests that the binding site for the initiating substrate of RNA polymerases has been conserved during evolution.     

Separation of yeast proteasome subunits. Immunoreactivity with antibodies against ATP-dependent protease Ti from Escherichia coli

On two-dimensional gel electrophoresis, proteasomes (multicatalytic proteinase complexes) from the yeast Saccharomyces cerevisiae were separated into a characteristic set of approximately 20 components with molecular weights of 21,000 to 31,000 and isoelectric points of 3.5 to 7.5. The main components were isolated by reverse-phase high performance liquid chromatography on a TSK gel phenyl-5PW RP column and named YC1 to YC11, in order of their elution. Immuno-blot analysis showed that two components (YC1-alpha and YC1-beta) with molecular weights of 30,800 and 28,300 strongly cross-reacted with antibody against the P-component of ATP-dependent protease Ti from Escherichia coli, but no components were found to react with antibodies against the A-component of protease Ti or another ATP-dependent protease La (the Ion gene product) of Escherichia coli. These results indicate a structural relationship between eukaryotic proteasomes and bacterial ATP-dependent protease Ti.     

Molecular cloning of cDNA for proteasomes (multicatalytic proteinase complexes) from rat liver: primary structure of the largest component (C2)

Proteasomes (multicatalytic proteinase complexes) from rat liver are composed of at least 13 nonidentical components [Tanaka, K., Yoshimura, T., Ichihara, A., Ikai, A., Nishigai, M., Morimoto, M., Sato, M., Tanaka, N., Katsube, Y., Kameyama, K., & Takagi, T. (1988) J. Mol. Biol. 203, 985-996]. The nucleotide sequence of one major component (C2) of the proteasomes has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a mixture of synthetic deoxyribonucleotides as a probe. The sequence was composed of 1174 nucleotides including a coding region for the entire protein and noncoding regions of both the 5'- and 3'-sides. The polypeptide deduced from the open reading frame consisted of 263 amino acid residues, and its molecular weight was calculated to be 29,516. The partial amino acid sequences of several fragments (approximately 45% of the total residues), which were obtained by cleavage of C2 with lysyl endopeptidase and cyanogen bromide, were determined by automated Edman degradation and found to be in complete accordance with those deduced from the cDNA sequence. The amino acid composition of C2, determined by chemical analysis, was also consistent with that deduced from the cDNA sequence, indicating that the cloned cDNA actually encoded component C2. Computer analysis revealed little structural similarity of C2 to other proteins reported so far. Northern blot hybridization analyses showed that the mRNA encoding this novel protein C2 was expressed in all the rat tissues examined and in a variety of eukaryotic organisms such as amphibia, birds, and mammals with slight species-specific differences in size.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autocrine mechanism of growth of neonatal rat hepatocytes in primary culture

Hepatocytes from neonatal rats of 0 to 3 days old grew actively in primary culture without added serum or growth factors. In these culture conditions, growth of hepatocytes decreased progressively with increase in age of the rats from which they were isolated, and hepatocytes from rats of 2 weeks old showed scarcely any growth. Actively growing hepatocytes were found to secrete a growth factor that promoted their growth and that of Swiss 3T3 cells, but not that of adult hepatocytes. This growth factor in conditioned medium of growing hepatocytes was heat- and acid-stable, but sensitive to trypsin, and had a molecular weight of over 10,000. It did not inhibit the binding of [125I]epidermal growth factor to its receptor, and its growth promoting activity was not inhibited by monoclonal antibody against insulin-like growth factor II. Therefore, it seems to be a new growth factor. These results, together with previous findings (Nakamura, T., Nagao, M., & Ichihara, A. (1987) Exp. Cell Res. 169, 1-14) demonstrated a reciprocal relation between growth and maturation of neonatal hepatocytes during development, like that of adult cells, but indicated that unlike growth of the latter, growth of neonatal cells is induced by an autocrine mechanism.     

A high molecular weight protease in the cytosol of rat liver. II. Properties of the purified enzyme

The properties of a soluble endoprotease from rat liver were studied. The enzyme was purified in a latent form. It sedimented as a single component with a sedimentation coefficient (S(0)20,w) of 19.8 S. Measurement by quasi-elastic light scattering gave a diffusion coefficient (D(0)20,w) of 2.5 X 10(-7) cm2 X s-1 and an effective hydrodynamic radius of 85 A. The enzyme had an unusually high molecular weight, estimated as 743,000 by sedimentation equilibrium and 722,000 by sedimentation velocity and diffusion measurements and as 760,000 by a recently developed low-angle laser light scattering method. Judging from electron microscopic observation and the calculated frictional and axial ratios, the enzyme molecule is disc-shaped. Analysis of the far-ultraviolet circular dichroic spectrum showed that the enzyme contains 50% alpha-helical, 25% beta-sheet, and 15% unordered structures with 10% beta-turns. The isoelectric point of the enzyme is 5.0. These properties indicate that the purified enzyme is a homogeneous molecule. In addition, the enzyme is a simple protein since it contains no measurable amounts of nucleic acid carbohydrate or lipid.     

A unique trypsin-like protease associated with plasma membranes of rat liver

One way in which serum promotes survival of primary cultured hepatocytes is by inhibiting plasma membrane protease (Nakamura, T., Asami, O., Tanaka, K., and Ichihara, A. (1984) Exp. Cell Res. 155, 81-91). One of these proteases was solubilized from the plasma membranes of rat liver with 4% octyl glucoside and purified to a homogeneous state by affinity chromatography on bovine pancreatic trypsin inhibitor linked to Sepharose 4B. The protease had an apparent Mr = 120,000 by Sephacryl S-200 gel filtration and the Mr of its subunits was 30,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It appeared to be a glycoprotein. A high concentration of detergent was necessary to keep the protein soluble. The purified enzyme readily hydrolyzed synthetic tripeptide nitroanilides at sites adjacent to Arg or Lys residues, but did not degrade synthetic substrates of chymotrypsin, elastase, or aminopeptidase. It showed endopeptidase activity, hydrolyzing various proteins such as casein, hemoglobin, and denatured albumin. The enzyme was strongly inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, bovine pancreatic trypsin inhibitor, leupeptin, antipain, and alpha 1-antitrypsin, but not by chymostatin, elastatinal, or inhibitors of carboxyl, thiol, or metallo proteases, suggesting that it is a seryl trypsin-like protease. This protease was found in plasma membranes of rat and mouse liver and in small amounts in those of kidney, but not in those of brain, red cells, Ehrlich ascites tumor, or two Morris hepatomas, suggesting that it may be involved in differentiated functions of normal hepatocytes.