Cellular prion protein in human plasma–derived extracellular vesicles promotes neurite outgrowth via the NMDA receptor–LRP1 receptor system

Exosomes and other extracellular vesicles (EVs) participate in cell–cell communication. Herein, we isolated EVs from human plasma and demonstrated that these EVs activate cell signaling and promote neurite outgrowth in PC-12 cells. Analysis of human plasma EVs purified by sequential ultracentrifugation using tandem mass spectrometry indicated the presence of multiple plasma proteins, including α₂-macroglobulin, which is reported to regulate PC-12 cell physiology. We therefore further purified EVs by molecular exclusion or phosphatidylserine affinity chromatography, which reduced plasma protein contamination. EVs subjected to these additional purification methods exhibited unchanged activity in PC-12 cells, even though α₂-macroglobulin was reduced to undetectable levels. Nonpathogenic cellular prion protein (PrP^C) was carried by human plasma EVs and essential for the effects of EVs on PC-12 cells, as EV-induced cell signaling and neurite outgrowth were blocked by the PrP^C-specific antibody, POM2. In addition, inhibitors of the N-methyl-d-aspartate (NMDA) receptor (NMDA-R) and low-density lipoprotein receptor–related protein-1 (LRP1) blocked the effects of plasma EVs on PC-12 cells, as did silencing of Lrp1 or the gene encoding the GluN1 NMDA-R subunit (Grin1). These results implicate the NMDA-R–LRP1 complex as the receptor system responsible for mediating the effects of EV-associated PrP^C. Finally, EVs harvested from rat astrocytes carried PrP^C and replicated the effects of human plasma EVs on PC-12 cell signaling. We conclude that interaction of EV-associated PrP^C with the NMDA-R–LRP1 complex in target cells represents a novel mechanism by which EVs may participate in intercellular communication in the nervous system.     

α3β1 integrin promotes cell survival via multiple interactions between 14-3-3 isoforms and proapoptotic proteins

Laminin-5 and α3β1 integrin promote keratinocyte survival; however, the downstream signaling pathways for laminin-5/α3β1 integrin-mediated cell survival had not been fully established. We report the unexpected finding of multiple interactions between 14-3-3 isoforms and proapoptotic proteins in the survival signaling pathway. Ln5-P4 motif within human laminin-5 α3 chain promotes cell survival and anti-apoptosis by inactivating Bad and YAP. This effect is achieved through the formation of 14-3-3ζ/p-Bad and 14-3-3σ/p-YAP complexes, which is initiated by α3β1 integrin and FAK/PI3K/Akt signaling. These complexes result in cytoplasmic sequestration of Bad and YAP and their subsequent inactivation. An increase in Akt1 activity in cells induces 14-3-3ζ and σ, p-Bad, and p-YAP, promoting cell survival, whereas decreasing Akt activity suppresses the same proteins and inhibits cell survival. Suppression of 14-3-3ζ with RNA-interference inhibits cell viability and promotes apoptosis. These results reveal a new mechanism of cell survival whereby the formation of 14-3-3ζ/p-Bad and 14-3-3σ/p-YAP complexes is initiated by laminin-5 stimulation via the α3β1 integrin and FAK/PI3K/Akt signaling pathways, thereby resulting in cell survival and anti-apoptosis.     

A Target Repurposing Approach Identifies N-myristoyltransferase as a New Candidate Drug Target in Filarial Nematodes

Myristoylation is a lipid modification involving the addition of a 14-carbon unsaturated fatty acid, myristic acid, to the N-terminal glycine of a subset of proteins, a modification that promotes their binding to cell membranes for varied biological functions. The process is catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT), an enzyme which has been validated as a drug target in human cancers, and for infectious diseases caused by fungi, viruses and protozoan parasites. We purified Caenorhabditis elegans and Brugia malayi NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and peptide substrates. Biochemical and structural analyses both revealed that the nematode enzymes are canonical NMTs, sharing a high degree of conservation with protozoan NMT enzymes. Inhibitory compounds that target NMT in protozoan species inhibited the nematode NMTs with IC₅₀ values of 2.5–10 nM, and were active against B. malayi microfilariae and adult worms at 12.5 µM and 50 µM respectively, and C. elegans (25 µM) in culture. RNA interference and gene deletion in C. elegans further showed that NMT is essential for nematode viability. The effects observed are likely due to disruption of the function of several downstream target proteins. Potential substrates of NMT in B. malayi are predicted using bioinformatic analysis. Our genetic and chemical studies highlight the importance of myristoylation in the synthesis of functional proteins in nematodes and have shown for the first time that NMT is required for viability in parasitic nematodes. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against nematode diseases including filariasis.     

Biosynthesis,Characterization, and Efficacy in Retinal Degenerative Diseases of Lens Epithelium-derived Growth Factor Fragment (LEDGF1–326), a Novel Therapeutic Protein

For vision-threatening retinitis pigmentosa and dry age-related macular degeneration, there are no United States Food and Drug Administration (FDA)-approved treatments. We identified, biosynthesized, purified, and characterized lens epithelium-derived growth factor fragment (LEDGF_1–326) as a novel protein therapeutic. LEDGF_1–326 was produced at about 20 mg/liter of culture when expressed in the Escherichia coli system, with about 95% purity and aggregate-free homogeneous population with a mean hydrodynamic diameter of 9 ± 1 nm. The free energy of unfolding of LEDGF_1–326 was 3.3 ± 0.5 kcal mol⁻¹, and melting temperature was 44.8 ± 0.2 °C. LEDGF_1–326 increased human retinal pigment epithelial cell viability from 48.3 ± 5.6 to 119.3 ± 21.1% in the presence of P23H mutant rhodopsin-mediated aggregation stress. LEDGF_1–326 also increased retinal pigment epithelial cell FluoSphere uptake to 140 ± 10%. Eight weeks after single intravitreal injection in Royal College of Surgeons (RCS) rats, LEDGF_1–326 increased the b-wave amplitude significantly from 9.4 ± 4.6 to 57.6 ± 8.8 μV for scotopic electroretinogram and from 10.9 ± 5.6 to 45.8 ± 15.2 μV for photopic electroretinogram. LEDGF_1–326 significantly increased the retinal outer nuclear layer thickness from 6.34 ± 1.6 to 11.7 ± 0.7 μm. LEDGF_1–326 is a potential new therapeutic agent for treating retinal degenerative diseases.     

A Highlights from MBoC Selection: Nucleotide- and Protein-Dependent Functions of Actg1

Cytoplasmic β- and γ-actin proteins are 99% identical but support unique organismal functions. The cytoplasmic actin nucleotide sequences Actb and Actg1, respectively, are more divergent but still 89% similar. Actb^–/– mice are embryonic lethal and Actb^–/– cells fail to proliferate, but editing the Actb gene to express γ-actin (Actb^c–g) resulted in none of the overt phenotypes of the knockout revealing protein-independent functions for Actb. To determine if Actg1 has a protein-independent function, we crossed Actb^c–g and Actg1^–/– mice to generate the bG/0 line, where the only cytoplasmic actin expressed is γ-actin from Actb^c–g. The bG/0 mice were viable but showed a survival defect despite expressing γ-actin protein at levels no different from bG/gG with normal survival. A unique myopathy phenotype was also observed in bG/0 mice. We conclude that impaired survival and myopathy in bG/0 mice are due to loss of Actg1 nucleotide-dependent function(s). On the other hand, the bG/0 genotype rescued functions impaired by Actg1^–/–, including cell proliferation and auditory function, suggesting a role for γ-actin protein in both fibroblasts and hearing. Together, these results identify nucleotide-dependent functions for Actg1 while implicating γ-actin protein in more cell-/tissue-specific functions.     

Sonic Hedgehog signaling impairs ionizing radiation-induced checkpoint activation and induces genomic instability

The Sonic Hedgehog (Shh) pathway plays important roles in embryogenesis, stem cell maintenance, tissue repair, and tumorigenesis. Haploinsufficiency of Patched-1, a gene that encodes a repressor of the Shh pathway, dysregulates the Shh pathway and increases genomic instability and the development of spontaneous and ionizing radiation (IR)–induced tumors by an unknown mechanism. Here we show that Ptc1^+/− mice have a defect in the IR-induced activation of the ATR–Chk1 checkpoint signaling pathway. Likewise, transient expression of Gli1, a downstream target of Shh signaling, disrupts Chk1 activation in human cells by preventing the interaction of Chk1 with Claspin, a Chk1 adaptor protein that is required for Chk1 activation. These results suggest that inappropriate Shh pathway activation promotes tumorigenesis by disabling a key signaling pathway that helps maintain genomic stability and inhibits tumorigenesis.     

Regulator of G protein signaling 2 inhibits Gαq-dependent uveal melanoma cell growth

Activating mutations in Gα_q/11 are a major driver of uveal melanoma (UM), the most common intraocular cancer in adults. While progress has recently been made in targeting Gα_q/11 for UM therapy, the crucial role for these proteins in normal physiology and their high structural similarity with many other important GTPase proteins renders this approach challenging. The aim of the current study was to validate whether a key regulator of Gq signaling, regulator of G protein signaling 2 (RGS2), can inhibit Gα_q-mediated UM cell growth. We used two UM cell lines, 92.1 and Mel-202, which both contain the most common activating mutation Gα_q^Q209L and developed stable cell lines with doxycycline-inducible RGS2 protein expression. Using cell viability assays, we showed that RGS2 could inhibit cell growth in both of these UM cell lines. We also found that this effect was independent of the canonical GTPase-activating protein activity of RGS2 but was dependent on the association between RGS2 and Gα_q. Furthermore, RGS2 induction resulted in only partial reduction in cell growth as compared to siRNA-mediated Gαq knockdown, perhaps because RGS2 was only able to reduce mitogen-activated protein kinase signaling downstream of phospholipase Cβ, while leaving activation of the Hippo signaling mediators yes-associated protein 1/TAZ, the other major pathway downstream of Gα_q, unaffected. Taken together, our data indicate that RGS2 can inhibit UM cancer cell growth by associating with Gα_q^Q209L as a partial effector antagonist.     

Distinct functionality of dishevelled isoforms on Ca2+/calmodulin-dependent protein kinase 2 (CamKII) in Xenopus gastrulation

Wnt ligands trigger the activation of a variety of β-catenin–dependent and β-catenin–independent intracellular signaling cascades. Despite the variations in intracellular signaling, Wnt pathways share the effector proteins frizzled, dishevelled, and β-arrestin. It is unclear how the specific activation of individual branches and the integration of multiple signals are achieved. We hypothesized that the composition of dishevelled–β-arrestin protein complexes contributes to signal specificity and identified CamKII as an interaction partner of the dishevelled–β-arrestin protein complex by quantitative functional proteomics. Specifically, we found that CamKII isoforms interact differentially with the three vertebrate dishevelled proteins. Dvl1 is required for the activation of CamKII and PKC in the Wnt/Ca²⁺ pathway. However, CamKII interacts with Dvl2 but not with Dvl1, and Dvl2 is necessary to mediate CamKII function downstream of Dvl1 in convergent extension movements in Xenopus gastrulation. Our findings indicate that the different Dvl proteins and the composition of dishevelled–β-arrestin protein complexes contribute to the specific activation of individual branches of Wnt signaling.     

Hypoxia Enhances Proliferation of Human Adipose-Derived Stem Cells via HIF-1ɑ Activation

BackgroundAdipose tissue-derived stem cells (ASCs) have been recently isolated from human subcutaneous adipose tissue. ASCs may be useful in regenerative medicine as an alternative to bone marrow-derived stem cells. Changes in the oxygen concentration influence physiological activities, such as stem cell proliferation. However, the effects of the oxygen concentration on ASCs remain unclear. In the present study, the effects of hypoxia on ASC proliferation were examined.MethodsNormal human adipose tissue was collected from the lower abdomen, and ASCs were prepared with collagenase treatment. The ASCs were cultured in hypoxic (1%) or normoxic (20%) conditions. Cell proliferation was investigated in the presence or absence of inhibitors of various potentially important kinases. Hypoxia inducible factor (HIF)-1α expression and MAP kinase phosphorylation in the hypoxic culture were determined with western blotting. In addition, the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2 in hypoxic or normoxic conditions were determined with real-time RT-PCR. The effects of these growth factors on ASC proliferation were investigated. Chromatin immunoprecipitation (ChIP) of the HIF–1α-binding hypoxia responsive element in FGF–2 was performed. HIF–1α was knocked down by siRNA, and FGF–2 expression was investigated.ResultsASC proliferation was significantly enhanced in the hypoxic culture and was inhibited by ERK and Akt inhibitors. Hypoxia for 5–15 minutes stimulated the phosphorylation of ERK1/2 among MAP kinases and induced HIF–1α expression. The levels of VEGF and FGF–2 mRNA and protein in the ASCs were significantly enhanced in hypoxia, and FGF–2 increased ASC proliferation. The ChIP assay revealed an 8-fold increase in the binding of HIF–1α to FGF–2 in hypoxia. HIF–1α knockdown by siRNA partially inhibited the FGF–2 expression of ASCs induced by hypoxia.ConclusionASC proliferation was enhanced by hypoxia. HIF–1α activation, FGF–2 production, and the ERK1/2 and Akt pathway were involved in this regulatory mechanism.     

Low-resolution structure of the soluble domain GPAA1 (yGPAA170–247) of the glycosylphosphatidylinositol transamidase subunit GPAA1 from Saccharomyces cerevisiae

The GPI (glycosylphosphatidylinositol) transamidase complex catalyses the attachment of GPI anchors to eukaryotic proteins in the lumen of ER (endoplasmic reticulum). The Saccharomyces cerevisiae GPI transamidase complex consists of the subunits yPIG-K (Gpi8p), yPIG-S (Gpi17p), yPIG-T (Gpi16p), yPIG-U (CDC91/GAB1) and yGPAA1. We present the production of the two recombinant proteins yGPAA1^70–247 and yGPAA1^70–339 of the luminal domain of S. cerevisiae GPAA1, covering the amino acids 70–247 and 70–339 respectively. The secondary structural content of the stable and monodisperse yGPAA1^70–247 has been determined to be 28% α-helix and 27% β-sheet. SAXS (small-angle X-ray scattering) data showed that yGPAA1^70–247 has an R_g (radius of gyration) of 2.72±0.025 nm and D_max (maximum dimension) of 9.14 nm. These data enabled the determination of the two domain low-resolution solution structure of yGPAA1^70–247. The large elliptical shape of yGPAA1^70–247 is connected via a short stalk to the smaller hook-like domain of 0.8 nm in length and 3.5 nm in width. The topological arrangement of yGPAA1^70–247 will be discussed together with the recently determined low-resolution structures of yPIG-K^24–337 and yPIG-S^38–467 from S. cerevisiae in the GPI transamidase complex.     

The GPCR–β-arrestin complex allosterically activates C-Raf by binding its amino terminus

G protein–coupled receptors (GPCRs) convert external stimuli into cellular signals through heterotrimeric guanine nucleotide-binding proteins (G-proteins) and β-arrestins (βarrs). In a βarr-dependent signaling pathway, βarrs link GPCRs to various downstream signaling partners, such as the Raf–mitogen-activated protein kinase extracellular signal–regulated kinase–extracellular signal-regulated kinase cascade. Agonist-stimulated GPCR–βarr complexes have been shown to interact with C-Raf and are thought to initiate the mitogen-activated protein kinase pathway through simple tethering of these signaling partners. However, recent evidence shows that in addition to canonical scaffolding functions, βarrs can allosterically activate downstream targets, such as the nonreceptor tyrosine kinase Src. Here, we demonstrate the direct allosteric activation of C-Raf by GPCR–βarr1 complexes in vitro. Furthermore, we show that βarr1 in complex with a synthetic phosphopeptide mimicking the human V2 vasopressin receptor tail that binds and functionally activates βarrs also allosterically activates C-Raf. We reveal that the interaction between the phosphorylated GPCR C terminus and βarr1 is necessary and sufficient for C-Raf activation. Interestingly, the interaction between βarr1 and C-Raf was considerably reduced in the presence of excess activated H-Ras, a small GTPase known to activate C-Raf, suggesting that H-Ras and βarr1 bind to the same region on C-Raf. Furthermore, we found that βarr1 interacts with the Ras-binding domain of C-Raf. Taken together, these data suggest that in addition to canonical scaffolding functions, GPCR–βarr complexes directly allosterically activate C-Raf by binding to its amino terminus. This work provides novel insights into how βarrs regulate effector molecules to activate downstream signaling pathways.     

Receptor-binding domain of SARS-CoV-2 is a functional αv-integrin agonist

Among the novel mutations distinguishing SARS-CoV-2 from similar coronaviruses is a K403R substitution in the receptor-binding domain (RBD) of the viral spike (S) protein within its S1 region. This amino acid substitution occurs near the angiotensin-converting enzyme 2–binding interface and gives rise to a canonical RGD adhesion motif that is often found in native extracellular matrix proteins, including fibronectin. Here, the ability of recombinant S1-RBD to bind to cell surface integrins and trigger downstream signaling pathways was assessed and compared with RGD-containing, integrin-binding fragments of fibronectin. We determined that S1-RBD supported adhesion of fibronectin-null mouse embryonic fibroblasts as well as primary human small airway epithelial cells, while RBD-coated microparticles attached to epithelial monolayers in a cation-dependent manner. Cell adhesion to S1-RBD was RGD dependent and inhibited by blocking antibodies against α_v and β₃ but not α₅ or β₁ integrins. Similarly, we observed direct binding of S1-RBD to recombinant human α_vβ₃ and α_vβ₆ integrins, but not α₅β₁ integrins, using surface plasmon resonance. S1-RBD adhesion initiated cell spreading, focal adhesion formation, and actin stress fiber organization to a similar extent as fibronectin. Moreover, S1-RBD stimulated tyrosine phosphorylation of the adhesion mediators FAK, Src, and paxillin; triggered Akt activation; and supported cell proliferation. Thus, the RGD sequence of S1-RBD can function as an α_v-selective integrin agonist. This study provides evidence that cell surface α_v-containing integrins can respond functionally to spike protein and raises the possibility that S1-mediated dysregulation of extracellular matrix dynamics may contribute to the pathogenesis and/or post-acute sequelae of SARS-CoV-2 infection.     

A Novel GH7 Endo-β-1,4-Glucanase from Neosartorya fischeri P1 with Good Thermostability,Broad Substrate Specificity and Potential Application in the Brewing Industry

An endo-β-1,4-glucanase gene, cel7A, was cloned from the thermophilic cellulase-producing fungus Neosartorya fischeri P1 and expressed in Pichia pastoris. The 1,410-bp full-length gene encodes a polypeptide of 469 amino acids consisting of a putative signal peptide at residues 1–20, a catalytic domain of glycoside hydrolase family 7 (GH7), a short Thr/Ser-rich linker and a family 1 carbohydrate-binding module (CBM 1). The purified recombinant Cel7A had pH and temperature optima of pH 5.0 and 60°C, respectively, and showed broad pH adaptability (pH 3.0–6.0) and excellent stability at pH3.0–8.0 and 60°C. Belonging to the group of nonspecific endoglucanases, Cel7A exhibited the highest activity on barley β-glucan (2020 ± 9 U mg^–1), moderate on lichenan and CMC-Na, and weak on laminarin, locust bean galactomannan, Avicel, and filter paper. Under simulated mashing conditions, addition of Cel7A (99 μg) reduced the mash viscosity by 9.1% and filtration time by 24.6%. These favorable enzymatic properties make Cel7A as a good candidate for applications in the brewing industry.     

Resveratrol Inhibits β-Amyloid-Induced Neuronal Apoptosis through Regulation of SIRT1-ROCK1 Signaling Pathway

Alzheimer’s disease (AD) is characterized by the accumulation of β-amyloid peptide (Aβ) and loss of neurons. Recently, a growing body of evidences have indicated that as a herbal compound naturally derived from grapes, resveratrol modulates the pathophysiology of AD, however, with a largely unclear mechanism. Therefore, we aimed to investigate the protection of resveratrol against the neurotoxicity of β-amyloid peptide 25–35 (Aβ_25–35) and further explore its underlying mechanism in the present study. PC12 cells were injuried by Aβ_25–35, and resveratrol at different concentrations was added into the culture medium. We observed that resveratrol increased cell viability through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) colorimetric assays. Flow cytometry indicated the reduction of cell apoptosis by resveratrol. Moreover, resveratrol also stabilized the intercellular Ca²⁺ homeostasis and attenuated Aβ_25–35 neurotoxicity. Additionally, Aβ_25–35-suppressed silent information regulator 1 (SIRT1) activity was significantly reversed by resveratrol, resulting in the downregulation of Rho-associated kinase 1 (ROCK1). Our results clearly revealed that resveratrol significantly protected PC12 cells and inhibited the β-amyloid-induced cell apoptosis through the upregulation of SIRT1. Moreover, as a downstream signal molecule, ROCK1 was negatively regulated by SIRT1. Taken together, our study demonstrated that SIRT1-ROCK1 pathway played a critical role in the pathomechanism of AD.     

Angiotensin-(1-7) Attenuates Kidney Injury Due to Obstructive Nephropathy in Rats

Background

Angiotensin-(1–7) [Ang-(1–7)] counteracts many actions of the renin-angiotensin-aldosterone system. Despite its renoprotective effects, extensive controversy exists regarding the role of Ang-(1–7) in obstructive nephropathy, which is characterized by renal tubulointerstitial fibrosis and apoptosis.

Methods

To examine the effects of Ang-(1–7) in unilateral ureteral obstruction (UUO), male Sprague-Dawley rats were divided into three groups: control, UUO, and Ang-(1–7)-treated UUO rats. Ang-(1–7) was continuously infused (24 μg/[kg·h]) using osmotic pumps. We also treated NRK-52E cells in vitro with Ang II (1 μM) in the presence or absence of Ang-(1–7) (1 μM), Mas receptor antagonist A779 (1 μM), and Mas receptor siRNA (50 nM) to examine the effects of Ang-(1–7) treatment on Ang II-stimulated renal injury via Mas receptor.

Results

Angiotensin II (Ang II) and angiotensin type 1 receptor (AT₁R) protein expression was higher in UUO kidneys than in controls. Ang-(1–7) treatment also decreased proapoptotic protein expression in UUO kidneys. Ang-(1–7) also significantly ameliorated TUNEL positive cells in UUO kidneys. Additionally, Ang-(1–7) reduced profibrotic protein expression and decreased the increased tumor growth factor (TGF)-β1/Smad signaling present in UUO kidneys. In NRK-52E cells, Ang II induced the expression of TGF-β1/Smad signaling effectors and proapoptotic and fibrotic proteins, as well as cell cycle arrest, which were attenuated by Ang-(1–7) pretreatment. However, treatment with A779 and Mas receptor siRNA enhanced Ang II-induced apoptosis and fibrosis. Moreover, Ang II increased tumor necrosis factor-α converting enzyme (TACE) and decreased angiotensin-converting enzyme 2 (ACE2) expression in NRK-52E cells, while pretreatment with Ang-(1–7) or A779 significantly inhibited or enhanced these effects, respectively.

Conclusion

Ang-(1–7) prevents obstructive nephropathy by suppressing renal apoptosis and fibrosis, possibly by regulating TGF-β1/Smad signaling and cell cycle arrest via suppression of AT₁R expression. In addition, Ang-(1–7) increased and decreased ACE2 and TACE expression, respectively, which could potentially mediate a positive feedback mechanism via the Mas receptor.     

Recombinant production and biochemical characterization of a hyperthermostable α-glucan/maltodextrin phosphorylase from Pyrococcusfuriosus

Alpha-glucan phosphorylase catalyzes the reversible cleavage of α-1-4-linked glucose polymers into α-D-glucose-1-phosphate. We report the recombinant production of an α-glucan/maltodextrin phosphorylase (PF1535) from a hyperthermophilic archaeon, Pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. The apparent 98 kDa recombinant enzyme was active over a broad range of temperatures and pH, with optimal activity at 80 °C and pH 6.5–7. This archaeal protein retained its complete activity after 24 h at 80 °C in Tris-HCl buffer. Unlike other previously reported phosphorylases, the Ni-affinity column purified enzyme showed broad substrate specificity in both the synthesis and degradation of maltooligosaccharides. In the synthetic direction of the enzymatic reaction, the lowest oligosaccharide required for the chain elongation was maltose. In the degradative direction, the archaeal enzyme can produce glucose-1-phosphate from maltotriose or longer maltooligosaccharides including both glycogen and starch. The specific activity of the enzyme at 80 °C in the presence of 10 mM maltoheptaose and at 10 mg ml^–1 glycogen concentration was 52 U mg^–1 and 31 U mg^–1, respectively. The apparent Michaelis constant and maximum velocity for inorganic phosphate were 31 ± 2 mM and 0.60 ± 0.02 mM min^–1 µg^–1, respectively. An initial velocity study of the enzymatic reaction indicated a sequential bi-bi catalytic mechanism. Unlike the more widely studied mammalian glycogen phosphorylase, the Pyrococcus enzyme is active in the absence of added AMP.     

Determination of base and backbone contributions to the thermodynamics of premelting and melting transitions in B DNA

In previous papers of this series the temperature-dependent Raman spectra of poly(dA)·poly(dT) and poly(dA–dT)·poly(dA–dT) were used to characterize structurally the melting and premelting transitions in DNAs containing consecutive A·T and alternating A·T/T·A base pairs. Here, we describe procedures for obtaining thermodynamic parameters from the Raman data. The method exploits base-specific and backbone-specific Raman markers to determine separate thermodynamic contributions of A, T and deoxyribosyl-phosphate moieties to premelting and melting transitions. Key findings include the following: (i) Both poly(dA)·poly(dT) and poly(dA–dT)· poly(dA–dT) exhibit robust premelting transitions, due predominantly to backbone conformational changes. (ii) The significant van’t Hoff premelting enthalpies of poly(dA)·poly(dT) [ΔH_vH^pm = 18.0 ± 1.6 kcal·mol^–1 (kilocalories per mole cooperative unit)] and poly(dA–dT)·poly(dA–dT) (ΔH_vH^pm = 13.4 ± 2.5 kcal·mol^–1) differ by an amount (~4.6 kcal·mol^–1) estimated as the contribution from three-centered inter-base hydrogen bonding in (dA)_n·(dT)_n tracts. (iii) The overall stacking free energy of poly(dA)· poly(dT) [–6.88 kcal·mol_bp^–1 (kilocalories per mole base pair)] is greater than that of poly(dA–dT)· poly(dA–dT) (–6.31 kcal·mol_bp^–1). (iv) The difference between stacking free energies of A and T is significant in poly(dA)·poly(dT) (ΔΔG_st = 0.8 ± 0.3 kcal· mol_bp^–1), but marginal in poly(dA–dT)·poly(dA–dT) (ΔΔG_st = 0.3 ± 0.3 kcal·mol_bp^–1). (v) In poly(dA)· poly(dT), the van’t Hoff parameters for melting of A (ΔH_vH^A = 407 ± 23 kcal·mol^–1, ΔS_vH^A = 1166 ± 67 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 60.0 ± 3.2 kcal·mol^–1) are clearly distinguished from those of T (ΔH_vH^T = 185 ± 38 kcal·mol^–1, ΔS_vH^T = 516 ± 109 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 27.1 ± 5.5 kcal·mol^–1). (vi) Similar relative differences are observed in poly(dA–dT)· poly(dA–dT) (ΔH_vH^A = 333 ± 54 kcal·mol^–1, ΔS_vH^A = 961 ± 157 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 45.0 ± 7.6 kcal· mol^–1; ΔH_vH^T = 213 ± 30 kcal·mol^–1, ΔS_vH^T = 617 ± 86 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 29.3 ± 4.9 kcal·mol^–1). The methodology employed here distinguishes thermodynamic contributions of base stacking, base pairing and backbone conformational ordering in the molecular mechanism of double-helical B DNA formation.