Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.10.3.1 (tyrosinase)
 9,065 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The influence of single amino acid replacements by alanine on the binding affinity and biological activity of alpha-MSH in B16 murine melanoma cells has been studied systematically. alpha-MSH analogues were synthesized by solid-phase peptide synthesis and their binding affinities to the melanocortin receptor expressed by B16 mouse melanoma cells were determined using a radioreceptor assay. Biological activity of the analogues was determined by measuring tyrosinase stimulation. Relative activity and affinity data were generally in agreement with earlier results using terminal deletion fragments of alpha-MSH, but the alanine scan revealed important new insights into the role of individual residues. The three terminal amino acids at either end were not necessary for binding or activity, with amino acids 4-9 forming a core sequence required for receptor binding and triggering of the biological response. It was observed that replacement of the glutamic acid residue in position 5 was possible without loss of affinity or activity, whereas replacement of Met4 resulted in a 100-fold loss of binding affinity and biological activity. Each residue within the conserved melanocortin sequence His-Phe-Arg-Trp was shown to be essential with Phe7, Arg8, and Trp9 being the most sensitive to replacement by alanine. Generally, there was a rank correlation between binding affinity and tyrosinase stimulation within the group of analogues studied. Tyrosinase activity was less affected by alanine substitution than binding affinity, which suggests that full receptor binding is not required for maximum biological response.
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PMID:Synthesis and biological evaluation of alpha-MSH analogues substituted with alanine. 785 84

Although the administration of melanocyte-stimulating hormone (MSH) peptides results in skin darkening in man, cultured human melanocytes have been reported to be unresponsive to these peptides. This may be a consequence of the conditions under which the cells were maintained in vitro, particularly the use of phorbol esters and cholera toxin as melanocyte mitogens. By culturing the cells in the absence of these additives, we demonstrate that alpha-MSH and its synthetic analogue Nle4DPhe7 alpha-MSH (NDP-MSH) induce dose-related increases in melanin content and tyrosinase activity and affect cell morphology in the majority of human melanocyte cultures. In addition, NDP-MSH induces increases in tyrosinase mRNA and tyrosinase-related protein-1 (TRP-1) mRNA. The dose-response curves for the MSH peptides are sigmoidal and the two peptides are equipotent in their effects on human melanocytes. Adrenocorticotropic hormone (ACTH) also affects morphology and stimulates melanogenesis and tyrosinase activity in human melanocytes. However, the dose-response curves for ACTH are biphasic, and the melanocytes respond to lower concentrations of ACTH than MSH peptides, similar to those normally present in human plasma. These findings may be important in understanding the role of these pro-opiomelanocortin peptides in human skin pigmentation.
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PMID:Cultured human melanocytes respond to MSH peptides and ACTH. 785 66

The significance of melanotropic hormones as physiologic regulators of cutaneous pigmentation in humans is still controversial. Until recently, no direct effect for melanotropins could be demonstrated on human melanocytes. Here we present conclusive evidence that alpha-melanotropin (alpha-melanocyte-stimulating hormone, alpha-MSH) and the related hormone corticotropin (adrenocorticotropic hormone, ACTH) stimulate the proliferation and melanogenesis of human melanocytes maintained in culture in a growth medium lacking any AMP inducer. The minimal effective dose of either hormone is 0.1 nM. In time-course experiments, the increase in cell number and tyrosinase activity became evident after one treatment of the melanocytes with 100 nM alpha-MSH for 48 hr. The mitogenic effect gradually increased to 50-270% above control, depending on the individual melanocyte strain, with continuous treatment with 100 nM alpha-MSH for 8 days, whereas the melanogenic effect became maximal (70-450% increase above control) after 4 days of treatment. Western blot analysis of tyrosinase and the tyrosinase-related proteins TRP-1 and TRP-2 revealed that alpha-MSH increased the expression of those three melanogenic proteins. This was not accompanied by any change in their mRNA levels after brief (1.5-24 hr) or prolonged (6 days) treatment with 100 nM alpha-MSH, suggesting that the increased expression of these melanogenic proteins was due to posttranscriptional events. These results demonstrate both mitogenic and melanogenic effects of alpha-MSH and ACTH on human melanocytes. That both hormones are effective at subnanomolar concentrations, combined with the presence of melanotropin receptors on human melanocytes, strongly suggests that these melanotropins play a physiologic role in regulating human cutaneous pigmentation.
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PMID:Mitogenic and melanogenic stimulation of normal human melanocytes by melanotropic peptides. 787 59

In malignant melanoma, melanocyte-stimulating hormone (alpha-MSH) has been found to influence the cellular metabolism of melanoma cells (c-AMP production, protein and RNA synthesis, and tyrosinase activation). In some publications elevated alpha-MSH levels have been described in melanoma patients. In the present study we used a commercially available radioimmunoassay to examine the alpha-MSH levels in patients with malignant melanoma and a control group consisting of apparently healthy volunteers (laboratory assistants) and dermatological patients without malignant tumours. The plasma alpha-MSH levels were (mean +/- SD) 12.2 +/- 12.9 for 37 melanoma patients (17 female, 20 male) and 7.9 +/- 3.5 pmol/l for 38 control persons (18 female, 20 male). The difference is significant according to the distribution-free U-test of Mann and Whitney. In 13 (35%) of the melanoma patients values were above the normal range defined by the 95.5% confidence limit. alpha-MSH cannot be classified as a typical tumour marker. Nonetheless, in our opinion alpha-MSH levels may be useful in monitoring melanoma patients with reference to prognosis and follow up during and after therapy.
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PMID:[The diagnostic significance of alpha-MSH in malignant melanoma of man]. 792 41

The influence of the terminal amino acids of alpha-MSH on its biological action in B16 murine melanoma cells has been systematically studied. Fragments of alpha-MSH lacking various sequences of terminal residues were synthesized by solid-phase peptide synthesis and their binding affinity to melanoma cells was measured using a radioreceptor assay. Biological activity was determined by measuring both tyrosinase activity and melanogenesis. The relative affinities and activities of the fragments generally followed the same pattern as found previously in other assay systems (frog and lizard bioassay and Cloudman S91 mouse melanoma), with the three amino acids at each terminal not being essential for binding and biological activity, although the C-terminal amino acids 11-13 are more important than those in the N-terminus. The differences in biological activity between the fragments can be explained by their relative binding affinities for the receptor.
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PMID:Influence of alpha-MSH terminal amino acids on binding affinity and biological activity in melanoma cells. 793 16

While ACTH is known to induce skin pigmentation in man, its effects on cultured human melanocytes have not been investigated. Using a culture system free of artificial mitogens, we report for the first time that ACTH stimulates melanogenesis in cultured human melanocytes. While ACTH, alpha-MSH and the synthetic alpha-MSH analogue Nle4DPhe7 alpha-MSH all stimulate the activity of tyrosinase, the rate limiting enzyme in melanogenesis, and all produce a 50% increase in the melanin content of the cells at a concentration of 10(-8)-10(-7) mol/l, the shapes of the dose response curves differ: those for the MSH peptides are sigmoidal while those for ACTH are biphasic. In addition, human melanocytes are able to respond to concentrations of ACTH comparable with physiological plasma levels. We suggest that ACTH may be relatively more important than alpha-MSH as a pigmentary hormone in man and could have a physiological role in skin pigmentation.
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PMID:ACTH stimulates melanogenesis in cultured human melanocytes. 813 43

To learn more of the role of calcium in the regulation of melanogenesis, we have used direct manipulation of medium calcium and pharmacological modulation of intracellular calcium to examine the consequences on unstimulated and cyclic AMP elevated tyrosinase activity and melanin synthesis and distribution in B16 melanoma cells. In unstimulated cells, calcium is clearly inhibitory to tyrosinase activity. However, in cells stimulated with cAMP-elevating agents the requirement for extracellular calcium was changed such that cells required a minimum of 0.4-0.6 mmol medium calcium for maximum tyrosinase response to these agents. Paradoxically, pharmacologically increasing intracellular calcium in cAMP-stimulated cells with ionophore inhibited tyrosinase activity, and the calcium-lowering agent TMB8 and the calcium channel blocker verapamil both stimulated tyrosinase activity. When melanin synthesis was measured in cAMP-stimulated cells, TMB8 was found to significantly increase the sensitivity and the maximum melanogenic response to alpha-MSH, suggesting the presence of at least one level of endogenous calcium inhibitory control operative in these cells. In addition, TMB8 changed the distribution of melanin between the cell and the medium such that, in the presence of alpha-MSH and TMB8, significantly more melanin was secreted into the medium. These data suggest that calcium is required for several steps in melanogenesis, having an apparently inhibitory effect on pre-tyrosinase activity in unstimulated cells, but also showing evidence of a positive role in cyclic AMP-stimulated tyrosinase activity, as well as a further possible inhibitory role in melanin movement or secretion.
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PMID:Calcium plays a complex role in the regulation of melanogenesis in murine B16 melanoma cells. 814 88

Although melanocyte stimulating hormone (MSH) peptides are known to stimulate pigmentation in man, previous reports suggest that human melanocytes are relatively unresponsive to these peptides in vitro. This may be related to the conditions under which the melanocytes were cultured. Thus, we have re-investigated the in vitro effects of MSH peptides using human melanocytes cultured in the absence of artificial mitogens. Human melanocytes were incubated with alpha-MSH or its potent analogue Nle4Dphe7 alpha-MSH for 3 days. After 18 hours, melanocyte morphology had evolved from mainly bipolar to dendritic in approximately 66% of cultures. Nle4DPhe7 alpha-MSH produced dose-related increases in both tyrosinase activity and melanin content although the degree of response was variable and tyrosinase activity was the relatively more responsive to the peptide. Similar results were obtained with alpha-MSH, but, although the effect on melanin content was similar to that of Nle4DPhe7 alpha-MSH, the effect on tyrosinase activity was less marked. The preliminary EC50 values for the actions of the MSH peptides suggest that they may be equipotent in their actions on human melanocytes. In addition, we have demonstrated that the common melanocyte mitogens 12-O-tetradecanoyl phorbol-13-acetate (TPA) and cholera toxin affect basal melanogenesis and modulate the effects of the MSH peptides. However, not all melanocyte cultures showed melanogenic responses to the MSH peptides. Ability to respond was unrelated to basal levels of tyrosinase activity or melanin content. In at least some cultures, morphological and melanogenic responses appear to be independent of one another.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Alpha-melanocyte stimulating hormone and its analogue Nle4DPhe7 alpha-MSH affect morphology, tyrosinase activity and melanogenesis in cultured human melanocytes. 817 9

The melanocyte-stimulating hormone (alpha-MSH) used at 10(-6)-5 x 10(-8) M concentrations inhibited the growth of amelanotic cells of human malignant melanoma BRO and influenced cell morphology without any effect on melanization or tyrosinase activity. Inhibition of tumour cell growth was accompanied by marked elevation of intracellular cAMP levels but not that of cGMP. Dibutyryl-cAMP and the cAMP-dependent protein kinase A inhibitor also inhibited the cell growth. alpha-MSH increased mono-, di- and 1.4.5-myoinositol triphosphate concentrations and influenced the activities of phosphatidylinositol kinase and phosphatidylinositol-4-phosphate kinase determining phosphatidylinositol-4-phosphate kinase and phosphatidylinositol-4.5-diphosphate levels. Myoinositol phosphate concentrations changed on a second scale and levelled off by the 3rd-5th min, whereas that of cAMP increased drastically by the 30th min.
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PMID:[Melanocyte-stimulating hormone induces growth of human malignant melanoma amelanotic cells with a change in cAMP, phosphatidylinositols, and inositol phosphate concentration]. 838 47

The possible mechanisms for the reduced melanin content and poor melanogenic response to MSH was investigated in B16-F10DD differentiation deficient melanoma cells. In particular, the MSH receptor status and associated signal transduction pathway linking to tyrosinase activity in these cells was studied for evidence of any defects. F10DD cells contained high-affinity binding sites for alpha-MSH, with KD values similar to those previously reported for other variants of the B16 melanoma. SDS-PAGE analysis after radioactive ligand cross-linking showed no evidence of gross structural alterations of the receptor. The F10DD cells expressed approximately twice as many receptors as the F10 parent cell line, suggesting a possible feedback response attempting to compensate for the amelanotic condition. The functional integrity of the MSH receptors in F10DD cells was confirmed by the presence of increased levels of cAMP in response to MSH stimulation. These results, coupled with the observation that F10 and F10DD cells express similar levels of tyrosinase mRNA and protein, point to a structural defect in tyrosinase or in the post-translational control mechanisms by which the activity of this enzyme is regulated.
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PMID:MSH receptors and function in amelanotic B16 melanoma cells. 839 Aug 76


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