Ribonucleotide reductase isolated from human cells: Heterogeneity among the sources

doi:10.1016/0006-2952(78)90134-X

Biochemical Pharmacology

Volume 27, Issue 19, 1978, Pages 2297-2300

https://doi.org/10.1016/0006-2952(78)90134-X Get rights and content

Abstract

The amount of ammonium sulfate required to precipitate the ribonucleotide reductase activities derived from histocytic lymphoma cells obtained from patients or from KB or Connaughton cultured human cells is different than for reductase activity derived from Molt-4F and HeLa-S3 cells. The reductase activity of Molt-4F cells sedimented in a sucrose density gradient at a faster rate than the enzyme activity from KB cells; CDP and ADP reductase activity from either source co-sedimented. Enzymes from both cell lines sedimented at a faster rate in the presence of CDP and ATP than in the absence of these compounds, with CDP and ADP reductase still co-sedimenting. In addition, the enzymes obtained from the two sources have different stabilities at 37°. Hydroxyurea, 1-formylisoquinoline thiosemicarbazone (IQ-1), 1,10-phenanthroline and potassium chloride inhibit the enzyme activity derived from either Molt-4F or KB cells. However, a difference in the ratio of CDP to ADP reductase activity in Molt-4F and KB cells and a difference between the sensitivities of the CDP and the ADP reductase activities from the same cell line to various concentrations of inhibitors was observed.

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The saturation binding curves were plotted using GraphPad software (San Diego, CA, USA), comparing the best fit for single-binding-site models (Kd, Bmax and IC50 values were obtained from nonlinear regression analysis). Inhibition constants (Ki) were calculated from the IC50 values using the Cheng–Prusoff equation [11]. Food intake results are expressed as the means ± S.E.M. Data were evaluated by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test using GraphPad software (Graph-Pad Software, San Diego, CA, USA).
Obesity is an escalating epidemic, but an effective non-invasive therapy is still scarce. For obesity treatment, anorexigenic neuropeptides are promising tools, but their delivery from the periphery to the brain is complicated by their peptide character. In order to overcome this unfavorable fact, we have applied the lipidization of neuropeptide prolactin-releasing peptide (PrRP), whose strong anorexigenic effect was demonstrated. A palmitoylated analog of human PrRP (h palm-PrRP31) was injected in free-fed Wistar rats by three routes: subcutaneous (s.c.), intraperitoneal (i.p) (both 5 mg/kg) and intravenous (i.v.) (from 0.01 to 0.5 mg/kg). We found a circulating compound in the blood after all three applications with the highest concentration after i.v. administration. This corresponds to the effect on food intake, which was also strongest after i.v. injection. Moreover, this is in agreement with the fact that the expression of c-Fos in specific brain regions involved in food intake regulation was also highest after intravenous application. Pharmacokinetic data are further supported by results obtained from dynamic light scattering and CD spectroscopy. Human palm-PrRP31 analog showed a strong tendency to micellize, and formation of aggregates suggested lower availability after i.p. or s.c. application. We have demonstrated that palm-PrRP influenced food intake even in free fed rats. Not surprisingly, the maximal effect was achieved after the intravenous application even though two orders of magnitude lower dose was used compared to both two other applications. We believe that palm-PrRP could have a potential as an antiobesity drug when its s.c. application would be improved.
Neuropeptide FF analog RF9 is not an antagonist of NPFF receptor and decreases food intake in mice after its central and peripheral administration
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Saturation and competitive binding curves were plotted using Graph-Pad Software (San Diego, CA, USA), comparing the best fit for single binding site models (Kd, Bmax and IC50 values were obtained from a non-linear regression analysis). Inhibition constants (Ki) were calculated from IC50 values using the Cheng–Prusoff equation (Chang and Cheng, 1978). The Kd of 125I-PrRP31 was determined previously (Maixnerová et al., 2011).
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New analogs of the CART peptide with anorexigenic potency: The importance of individual disulfide bridges
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Data are presented as the means ± S.E.M. Competitive binding curves were plotted using Graph-Pad Software (San Diego, CA, USA) using the best fit for single-binding-site models (IC50 values were obtained from a non-linear regression analysis). Inhibition constants (Ki) were calculated from the IC50 using the Cheng–Prusoff equation [6]. The Kd from saturation experiments was 0.48 nM for non-differentiated cells and 1.90 nM for differentiated cells, as described previously [25].
The CART (cocaine- and amphetamine-regulated transcript) peptide is an anorexigenic neuropeptide that acts in the hypothalamus. The receptor and the mechanism of action of this peptide are still unknown. In our previous study, we showed that the CART peptide binds specifically to PC12 rat pheochromocytoma cells in both the native and differentiated into neuronal phenotype. Two biologically active forms, CART(55–102) and CART(61–102), with equal biological activity, contain three disulfide bridges. To clarify the importance of each of these disulfide bridges in maintaining the biological activity of CART(61–102), an Ala scan at particular S–S bridges forming cysteines was performed, and analogs with only one or two disulfide bridges were synthesized. In this study, a stabilized CART(61–102) analog with norleucine instead of methionine at position 67 was also prepared and was found to bind to PC12 cells with an anorexigenic potency similar to that of CART(61–102). The binding study revealed that out of all analogs tested, [Ala^68,86]CART(61–102), which contains two disulfide bridges (positions 74–94 and 88–101), preserved a high affinity to both native PC12 cells and those that had been differentiated into neurons. In food intake and behavioral tests with mice after intracerebroventricular administration, this analog showed strong and long-lasting anorexigenic potency. Therefore, the disulfide bridge between cysteines 68 and 86 in CART(61–102) can be omitted without a loss of biological activity, but the preservation of two other disulfide bridges and the full-length peptide are essential for biological activity.
Biological properties of prolactin-releasing peptide analogs with a modified aromatic ring of a C-terminal phenylalanine amide
2011, Peptides
Citation Excerpt :
The saturation and competitive binding curves were plotted using GraphPad software (San Diego, CA, USA) and compared the best fit for single-binding-site models (IC50 values were obtained from nonlinear regression analysis). Inhibition constants (Ki) were calculated from the IC50 values using the Cheng–Prusoff equation [4]. In prolactin release experiments, dose-response curves and half-maximal effective concentrations (EC50) were obtained from the nonlinear regression analyses using GraphPad software.
Prolactin-releasing peptide (PrRP)-induced secretion of prolactin is not currently considered a primary function of PrRP, but the development of late-onset obesity in both PrRP and PrRP receptor knock-out mice indicates the unique anorexigenic properties of PrRP. In our recent study, we showed comparable potencies of peptides PrRP31 and PrRP20 in binding, intracellular signaling and prolactin release in pituitary RC-4B/C cells, and anorexigenic effect after central administration in fasted mice. In the present study, eight analogs of PrRP20 with C-terminal Phe amide modified with a bulky side chain or a halogenated aromatic ring revealed high binding potency, activation of mitogen-activated protein kinase/extracellular-regulated kinase (MAPK/ERK1/2) and cAMP response element-binding protein (CREB) and prolactin release in RC-4B/C cells. In particular, [PheNO₂³¹]PrRP20, [1-Nal³¹]PrRP20, [2-Nal³¹]PrRP20 and [Tyr³¹]PrRP20 showed not only in vitro effects comparable or higher than those of PrRP20, but also a very significant and long-lasting anorexigenic effect after central administration in fasted mice. The design of potent and long-lasting PrRP analogs with selective anorexigenic properties promises to contribute to the study of food intake disorders.
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2011, Peptides
Citation Excerpt :
Data are presented as means ± S.E.M. Saturation and competitive binding curves were plotted using Graph-Pad Software (San Diego, CA, USA), comparing the best fit for single binding site models (Kd, Bmax and IC50 values were obtained from non-linear regression analysis). Inhibition constants (Ki) were calculated from IC50 values using the Cheng–Prusoff equation [9]. Data from cell signaling and hormone release, as well as food intake experiments were analyzed by one-way ANOVA (analysis of variance) followed by Tukey's post hoc test using Graph-Pad Software; P < 0.05 was considered statistically significant.
The recently discovered prolactin-releasing peptide (PrRP) binds to the PrRP receptor and is involved in endocrine regulation and energy metabolism. However, its main physiological role is currently unknown. Two biologically active isoforms of PrRP exist: the 31 (PrRP31) and the 20 (PrRP20) amino acid forms, which both contain a C-terminal Phe amide sequence. In the present study, the PrRP receptor was immunodetected in three rodent tumor pituitary cell lines: GH3, AtT20 and RC-4B/C cells. The saturation binding of radioiodinated PrRP31 to intact cells demonstrated a K_d in the 10⁻⁹ M range and a B_max in the range of tens of thousands binding sites per cell. For binding to RC-4B/C cells, both PrRP31 and PrRP20 competed with ¹²⁵I-PrRP31 with a similar K_i. The C-terminal analog PrRP13 showed lower binding potency compared to PrRP31 and PrRP20. All PrRP analogs increased the phosphorylation of MAPK/ERK1/2 (mitogen-activated phosphorylase/extracellular-regulated kinase) and CREB (cAMP response element-binding protein) in RC-4B/C cells. Additionally, prolactin release was induced by the PrRP analogs in a dose-dependent manner in RC-4B/C cells. Finally, food intake after intracerebroventricular administration of PrRP analogs in fasted mice was followed. Both PrRP31 and PrRP20 decreased food intake, but PrRP13 did not show significant effect. Studies on pituitary cell lines expressing the PrRP receptor are more physiologically relevant than those on cells transfected with the receptor. This cell type can be used as a model system for pharmacological studies searching for PrRP antagonists and stable effective PrRP agonists, as these drugs may have potential as anti-obesity agents.
Study of ribonucleotide reductase in cells infected with six clinical isolates of herpes simplex virus type 2 (HSV-2) with mutations in its larger subunit
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Herpes simplex virus type 2 (HSV-2) induces a novel ribonucleotide reductase (RR) composed of two subunits (140 and 38 kDa) in infected cells. Other investigators have developed a monoclonal antibody, A6, against the 140-kDa subunit of RR and have found, in about 1 % of the cases, an inability to detect this protein in cells infected with clinical isolates of HSV-2. We therefore investigated whether in such cases the clinical isolates were capable of inducing viral RR activity and whether the lack of detection of the 140-kDa protein by the monoclonal antibody was due to an alteration in the antigenic site of this protein. Six such isolates were examined and were found to induce RR activity, similar to HSV-2 (strain 333) RR, which did not require ATP for CDP reduction. Western blot analyses using A6 failed to detect the protein. However, R1, a polyclonal antibody raised against viral RR was capable of detecting this subunit. In addition, R1 was also capable of neutralizing RR activity induced by all the isolates and HSV-2 (strain 333). In conclusion, the lack of detection of the large subunit of RR was not due to the lack of induction but was due to an alteration in the antigenic site recognized by A6; this alteration did not appear to affect the properties of the induced RR activity.

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Ribonucleotide reductase isolated from human cells: Heterogeneity among the sources

Abstract

J. biol. Chem.

J. biol. Chem.

Biochem. biophys. Res. Commun.

Analyt. Biochem.

Analyt. Biochem.

Archs Biochem. Biophys.

Biochim. biophys. Acta

Eur. J. Biochem.

J. biol. Chem.