ALBERT

Notes: The problem of observing tyrosine emission in proteins containing tryptophan is related to both the absorption and emission properties of these two amino acids. First, the absorption of tryptophan is several times larger than tyrosine at all wavelengths. In the two compounds, acetyl tryptophan-amide and acetyl tyrosinamide, the ratio of the molar extinction coefficients of the latter to the former is maximal at 274 mμ and 232 mμ (Figure 11). At 270 mμ, the wavelength used for excitation in most experiments, the ratio is 0.235. The 270 mμ wavelength was chosen instead of 274 mμ in order to eliminate more of the scatter peak of the exciting radiation. The second problem is that the quantum yield of tyrosine is smaller than that of tryptophan in many proteins, 20 polypeptides21,22, 28 and small peptides.20 Due to these two factors, a very high fraction of the total emission is due to tryptophan. Of major importance in observing the tyrosine band is the wavelength peak of tryptophan emission. The smaller the latter, the greater the overlap and the less likely that the tyrosine band will be resolved as a shoulder or separate peak. In the absence of its resolution, the contribution of tyrosine to an emission band may be shown by exciting at two wavelength, where the ratio of absorption of tryptophan to tyrosine is high and low. In this way, the tyrosyl emission in bovine serum albumin34 and ovalbumin35 has been detected. Another method is to conjugate a dye which quenches tryptophan but not tyrosine emission by energy transfer. This result has been accomplished with the dimethylamino naphthalene sulfonyl conjugate of human serum albumin. It has shown that phosphate preferentially quenches tyrosine emission.20,36 Thus, if tyrosyl residues are accessible to phosphate their emission may be verified. It may also be possible to selectively eliminate tryptophan emission by chemical modification, as with N-bromosuccinimide. In chemical changes the product may, however, quench tyrosyl or tryptophanyl fluorescence by energy transfer or other mechanisms.There is considerable physical and chemical data on the two macromolecules, i.e., nuclease4 and bovine growth hormone,32,33 which reveal them to be highly organized structures and consequently proteins. In the case of the two hormones, PTH and ACTH analog, very little relevant data exists concerning their structure. It is instructive to compare the fluorescence behavior of the two proteins with that of the two hormones, i.e., PTH and ACTH analog.Acidification of the nuclease or bovine growth hormone produces major changes in the quantum yields of both chromophores. Very small, inconsequential changes were observed with PTH and ACTH analog. In accord with their behavior in acid, the temperature dependence of the former two proteins was very abnormal, whereas in the latter two hormones it was similar to that of the amino acid models. Moreover, the tryptophanyl emission peaks of PTH and ACTH analog occurred near 350 mμ and, therefore, at longer wavelengths than that of the nuclease and bovine growth hormone. Guanidine or dioxane in the case of the nuclease, or urea with bovine growth hormone, strongly modified their fluorescence spectra. In contrast, guanidine (5M) had very little effect on the fluorescence spectra of PTH or ACTH analog at neutral pH. We have observed that guanidine (5M) reduces the fluorescence of either N-acetyl tryptophanamide or N-acetyl tyrosinamide by only a few percent. It is evident that the tyrosyl and tryptophanyl residues in the two proteins are involved in interactions which have profound effects on their emissive properties. In the case of PTH and ACTH analog the two chromophores appear to be free and exposed to the solvent. From their fluorescence behavior under a variety of conditions, it appears likely that these two hormones have very little, if any, tertiary structure. One would expect that tyrosyl and tryptophanyl residues, because of their relatively low polarity, would be interior residues if the molecule had a globular form. It is, of course, dangerous to extrapolate from the properties of only two types of residues to that of the whole molecule. However, in the globular proteins whose structures have been determined by x-ray analysis, most amino acids have fixed coordinates.37,38 It seems unlikely, therefore, that two bulky residues that can form strong hydrophobic interactions will be free and the remainder fixed.The only data which might indicate that PTH has structure are the differences in tryptophan quenching in water compared to guanidine due to energy transfer to ionized tyrosine. This result can be explained if PTH is more extended in guanidine than in water. It is known that concentrated guanidine solutions are better solvents than water for proteins.39 Consequently, proteins or polypeptides would be expected to have a greater effective volume or root mean square end-to-end distance in guanidine.40There is chemical data which indicates that the tryptophan and the two methionine groups in PTH are free. The latter are readily oxidized, and tryptophan reacts with both N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide.3If either PTH or ACTH analog possesses structure, it would have to be formed from a less structured molecule that existed at the time of extraction in the former or synthesis in the latter. The methods of extraction and purification would disorganize any ordered structure that PTH may have in situ. It is extracted in 90% phenol and then distributed between n-butanol and 20% acetic acid in counter current distribution.1,2 In aqueous solutions at neutral pH it might, however, recover its structure. Its remarkable stability survives brief periods of boiling in 0.1M HCl or hot 80% acetic acid, treatment with guanidine, urea and trichloracetic acid.

Notes: [Auszug] Figure 1 a, a plot of peak current as a function of test pulse potential, shows that the dopamine agonist apomorphine (0.1 |xM) greatly increases the Ca2+ current. Nisoldipine (1 jxM), a dihydropyridine antagonist, completely inhibited the Ca2+ current recruited by apomorphine (Fig. la). It has ...

Notes: [Auszug] Figure 1 a shows the time course for the recruitment of facilita-tion Ca2+ current by pre-pulses. Pre-pulses as brief as 1 ms produced a measurable increase in Ca2+ current. The time course for the recruitment of facilitation was well fitted by a single exponential with T = 26 ms. The ...

Notes: [Auszug] Transient and catabolite repression of the synthesis of β-galactosidase in E. coli are related phenomena, the result of a decreased intracellular concentration of cyclic AMP. The lac operon promoter is the site of action of the cyclic ...

Notes: Summary 1. Antiphosphotyrosine antibodies were used to detect phosphotyrosine-containing proteins in immunoblots of bovine chromaffin cell proteins. 2. Unstimulated cells exhibited two major phosphotyrosine-containing proteins, which hadM r 's of 121,000 and 70,000. Insulin-like growth factor I (IGF-I) had little effect on the phosphotyrosine content of these two proteins but greatly increased the phosphotyrosine content of three other proteins ofM r 185,000, 170,000, and 96,000. These proteins were found predominantly in the particulate fraction of cell homogenates. 3. The effects of the IGF-I were time and concentration dependent, with maximal increases in phosphorylation occurring after 1 min of treatment with 10 nM IGF-I. Na3VO4, an inhibitor of phosphotyrosine phosphatases, potentiated the effects of IGF-I. 4. Thus, the IGF-I receptor appears to function as an IGF-I-activated protein tyrosine kinase in chromaffin cells. The tyrosine kinase activity of the IGF-I receptor presumably mediates the effects of IGF-I on chromaffin cell function.

Notes: Summary 1. 32P-Labeled proteins from the superior cervical ganglion of the rat were separated by two-dimensional gel electrophoresis and visualized by autoradiography. 2. The most heavily labeled phosphoprotein in the ganglion had a relative molecular weight of 83,000 and apI of 4.5. Phosphorylation of this protein was increased by phorbol 12,13-dibutyrate, an activator of the Ca2+/phospholipiddependent protein kinase, protein kinase C. This protein appears to be similar or identical to a specific protein kinase C substrate that has been described in other tissues (Blackshear, P. J.,et al., J. Biol. Chem. 261:1459–1469, 1986). 3. Phosphorylation of this protein was also increased by treatment of the ganglion with phospholipase C (Bacillus cereus) but was not increased by 8-bromocyclic AMP or by nicotinic agonists. Vasopressin increased the hydrolysis of inositolcontaining phospholipids in the ganglion and also increased the labeling of the 83,000M r protein. Thus, vasopressin appears to activate protein kinase C in the ganglion. 4. Muscarine, which also increased phospholipid metabolism in the ganglion, did not increase the phosphorylation of the 83,000M r protein. Muscarine and vasopressin stimulate phospholipid metabolism in different structures within the ganglion (Horwitz, J.,et al., J. Pharmacol. Exp. Ther. 237:312–317, 1986). Muscarine may increase phospholipid metabolism in structures that do not contain significant amounts of the 83,000M r protein.

Notes: Summary Normal postnatal rat chromaffin cells and rat pheochromocytoma cells are known to show extensive Nerve Growth Factor (NGF)-induced process outgrowth in culture, and this outgrowth from the postnatal chromaffin cells is abolished by the corticosteroid dexamethasone. To determine whether adult rat chromaffin cells respond to NGF and dexamethasone, dissociated adrenal medullary cells from 3-month-old rats were cultured for 30 days in the presence or absence of these agents. Such cultures contained typical chromaffin cells, chromaffin cells with processes, and neurons. Fewer than 2 % of normal adult chromaffin cells formed processes under any of the conditions studied, and statistically significant changes in this proportion were not detectable in the presence of NGF or dexamethasone. Adrenal medullary neurons, however, were observed only in the presence of NGF, in cultures with or without dexamethasone, and thus appear to be previously unreported NGF targets which require NGF for survival or process outgrowth. Dexamethasone markedly increased total catecholamine content, total content of epinephrine, and tyrosine hydroxylase activity in cultures with or without NGF. In contrast, postnatal rat chromaffin and rat pheochromocytoma cells which have been studied in culture do not produce epinephrine under any of these conditions. It is concluded that rat adrenal chromaffin cells undergo age-related changes in both structural and functional plasticity. The in vitro characteristics of rat pheochromocytoma cells more closely resemble those of postnatal than of adult rat chromaffin cells, but may not entirely reflect the properties of the majority of chromaffin cells in either age group.