Effect of chemical modifications of tryptophan residues on the folding of reduced hen egg-white lysozyme

The effects of chemical modifications of Trp62 and Trp108 on the folding of hen egg-white lysozyme from the reduced form were investigated by means of the sulfhydryl-disulfide interchange reaction at pH 8 and 40 degrees C. The folding of reduced lysozyme was monitored by following the recovery of the original activity. Under the conditions employed, the apparent first-order rate constant for the folding of reduced lysozyme was not changed by the modifications of both Trp62 and Trp108 and the folding was completed within 30 min. However, the extent of the correct folding was changed by the modification of Trp62 but not by that of Trp108. Native and oxindolealanine108 lysozymes recovered 80 and 81% of their original activities after 30-min refolding, respectively, but Trp62-modified lysozymes recovered their activities to a lesser extent than native and oxindolealanine108 lysozymes. The recovered activities of Trp62-modified lysozymes after 30-min refolding were 63% for oxindolealanine62 lysozyme, 65% for delta 1-carboxamidomethylthiotryptophan62 lysozyme, and 52% for delta 1-carboxymethylthiotryptophan62 lysozyme. These results suggest that Trp62 is important for preventing the misfolding of reduced lysozyme, but that neither Trp62 nor Trp108 is involved in the rate-determining step (the slowest step) in the folding pathway. A decrease in the hydrophobic nature of Trp62 seems to increase the misfolding and thus to decrease the extent of the correct folding of reduced lysozyme. A mechanism for the involvement of Trp62 in the folding pathway of reduced lysozyme is proposed.     

Localization of arachidonate 12-lipoxygenase in parenchymal cells of porcine anterior pituitary

12-Lipoxygenases oxygenate arachidonic acid producing its 12S-hydroperoxy derivative and are well known as platelet and leukocyte enzymes. When a peroxidase-linked immunoassay of the enzyme according to the avidin-biotin method was applied to the cytosol fractions from various parts of porcine brain, a considerable amount of the enzyme was found in the anterior pituitary. The enzyme level (about 200 ng/mg cytosol protein) corresponded to about 6% of the enzyme content in porcine peripheral leukocytes. Posterior and intermediate lobes showed about one-tenth of the enzyme level of anterior pituitary. Other parts of porcine brain contained the 12-lipoxygenase in amounts below 7 ng/mg cytosol protein. The cytosol fraction (0.7 mg of protein) of anterior pituitary produced 12S-hydroxy-5,8,10,14-eicosatetraenoic acid from 25 microM arachidonic acid in about 34% conversion at 24 degrees C for 5 min, giving a specific enzyme activity about 3 nmol/min/mg protein. Furthermore, various octadecapolyenoic acids were oxygenated almost as fast as the arachidonate 12-oxygenation. When anterior pituitary was investigated immunohistochemically with anti-12-lipoxygenase antibody, most of the immunostained cells were certain parenchymal cells with granules, which were not blood cells. These biochemical and immunohistochemical results provide a good reason for considering that 12-lipoxygenase does play an important role in pituitary function.     

Purification and characterization of a novel GTP-binding protein with a Mr value of 24,000 from rat liver

About 15% of the total GTP-binding proteins (G proteins) of rat liver homogenate was found in the microsomes-Golgi complex fraction. From this fraction, we purified to near homogeneity and characterized a G protein with a Mr value of 24,000 (24K G). 24K G specifically bound guanosine 5'-(3-Q-thio) triphosphate (GTP gamma S), GTP and GDP with a Kd value for GTP gamma S of about 30 nM. 24K G bound maximally about 0.7 mol of GTP gamma S/mol of protein. 24K G hydrolyzed GTP to liberate Pi with a turnover number of about 0.008 min-1. 24K G was not copurified with the beta gamma subunit of heterotrimeric G proteins. The partial amino acid sequences of 24K G revealed that this protein was a novel small G protein.     

The nucleotide sequence of the mouse immunoglobulin epsilon gene: comparison with the human epsilon gene sequence

We have determined the nucleotide sequence of the immunoglobulin epsilon gene cloned from newborn mouse DNA. The epsilon gene sequence allows prediction of the amino acid sequence of the constant region of the epsilon chain and comparison of it with sequences of the human epsilon and other mouse immunoglobulin genes. The epsilon gene was shown to be under the weakest selection pressure at the protein level among the immunoglobulin genes although the divergence at the synonymous position is similar. Our results suggest that the epsilon gene may be dispensable, which is in accord with the fact that IgE has only obscure roles in the immune defense system but has an undesirable role as a mediator of hypersensitivity. The sequence data suggest that the human and murine epsilon genes were derived from different ancestors duplicated a long time ago. The amino acid sequence of the epsilon chain is more homologous to those of the gamma chains than the other mouse heavy chains. Two membrane exons, separated by an 80-base intron, were identified 1.7 kb 3' to the CH4 domain of the epsilon gene and shown to conserve a hydrophobic portion similar to those of other heavy chain genes. RNA blot hybridization showed that the epsilon membrane exons are transcribed into two species of mRNA in an IgE hybridoma.     

Long terminal repeat-like elements flank a human immunoglobulin epsilon pseudogene that lacks introns

There are at least three immunoglobulin epsilon genes (C epsilon 1, C epsilon 2, and C epsilon 3) in the human genome. The nucleotide sequences of the expressed epsilon gene (C epsilon 1) and one (C epsilon 3) of the two epsilon pseudogenes were compared. The results show that the C epsilon 3 gene lacks the three intervening sequences entirely and has a 31-base A-rich sequence 16 bases 3' to the putative poly(A) addition signal, indicating that the C epsilon 3 gene is a processed gene. The C epsilon 3 gene sequence is homologous to the five separate DNA segments of the C epsilon 1 gene; namely, a segment in the 5'-flanking region (100 bases) and four exons, which are interrupted by a spacer region or intervening sequences. Long terminal repeat (LTR)-like sequences which contain TATAAA and AATAAA sequences as well as terminal inverted repeats are present in both 5'- and 3'-flanking regions. The 5' and 3' LTR-like sequences do not, however, constitute a direct repeat, unlike transposable elements of eukaryotes and retroviruses. The 3' LTR-like sequence is repetitive in the human genome, but is not homologous to the Alu family DNA. Models for the evolutionary origin of the processed gene flanked by the LTR-like sequences are discussed. The C epsilon 3 gene has a new open frame which codes potentially for an unknown protein of 292 amino acid residues.     

Immunohistochemical studies on the serotonergic innervation of the pia mater

Summary The direct innervation of the pial blood vessels by serotonin neurons has been demonstrated with a modified peroxidase-antiperoxidase technique in the mammalian central nervous system. The pia mater covering the ventrolateral surface of the medulla oblongata is innervated by numerous varicose serotonin fibers originating from the serotonin neurons of the lower brainstem. Scattered serotonin fibers were observed in the pia mater in every part of the brain and spinal cord.     

Effect of calcium and magnesium ions on the interaction of corticosterone with the cytosol receptor(s) in the rat brain

The effects of calcium and magnesium ions on the corticosterone binding to rat brain cytosol receptor protein(s) were investigated. The increasing amounts of CaCl2 or MgCl2 up to 5.0 mM were added, the specific [3H] corticosterone binding increased 1.3-fold and 1.5 respectively. The addition of MnCl2 and KCl did not affect this binding. The binding of corticosterone with rat brain cytosol receptor(s) were decreased by increasing amounts of EDTA and complete inhibition was observed at concentration equal to and greater than 2.5 mM. Inhibition of this binding by EDTA was less than by EGTA. Either theophylline or dibutyryl cyclic AMP had no effect on this binding.     

Effect of aliphatic alcohols on the conformation of poly (l-ornithine hydrobromide) as studied by square-pulse and reversing-pulse electric birefringence

The electric birefringence and circular dichroism spectra of poly(l-ornithine hydrobromide) have been measured in ethanol/water, 2-propanol/water and tertiary butyl alcohol/water mixtures of various compositions. This charged polypeptide underwent a transition from the coil conformation to the helical conformation at high alcohol content in every case tested. Anomalous birefringence signals, indicative of a field-induced helix-to-coil transition. were observed at high electric fields only in the case of ethanol/water mixtures. The reversing-pulse electric birefringence of this polypeptide has been studied in ethanol/water mixtures and in neutral aqueous solution. Upon rapid reversal of the pulse field, no transient could be observed. This confirms that the electric-field orientation of poly(l-ornithine hydrobromide) results predominantly from the contribution of the counterion-induced dipole moment, regardless of its molecular conformations. It is very probable that the backbone permanent dipole moment of the helical conformation is largely suppressed by the counterion-induced dipole moment in the ionized form.