From phosphorylase to phosphorylase kinase.
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Animals↗
Biomedical subjects
L N Johnson
Publications and source records attributed to L N Johnson.
Oligosaccharide substrate binding in Escherichia coli maltodextrin phosphorylase.
The crystal structure of E. coli maltodextrin phosphorylase co-crystallized with an oligosaccharide has been solved at 3.0 A resolution, providing the first structure of an oligosaccharide bound at the catalytic site of an alpha-glucan phosphorylase. An induced fit mechanism brings together two domains across the catalytic site tunnel. A stacking interaction between the glucosyl residue and the aromatic group of a tyrosine residue at a sub-site remote (8 A) from the catalytic site provides a key element in substrate recognition; mutation of this residue to Ala decreases the Kcat/Km by 10(4). Extrapolation of the results to substrate binding across the site of attack by phosphorolysis indicates a likely alteration in the glycosidic torsion angles from their preferred values, an alteration that appears to be important for the catalytic mechanism.
Crystallization↗
Protein kinase inhibition by staurosporine revealed in details of the molecular interaction with CDK2.
Staurosporine exhibits nanomolar IC50 values against a wide range of protein kinases. The structure of a CDK2 staurosporine complex explains the tight binding of this inhibitor, and suggests features to be exploited in the design of specific inhibitors of CDKs.
Amino Acid Sequence↗
Ternary complex crystal structures of glycogen phosphorylase with the transition state analogue nojirimycin tetrazole and phosphate in the T and R states.
Catalysis by glycogen phosphorylase involves a mechanism in which binding of one substrate tightens the binding of the other substrate to produce a productive ternary enzyme-substrate complex. In this work the molecular basis for this synergism is probed in crystallographic studies on ternary complexes in which the glucosyl component is substituted by the putative transition state analogue nojirimycin tetrazole, a compound which has been established previously as a transition state analogue inhibitor for a number of glycosidases. Kinetic studies with glycogen phosphorylase showed that nojirimycin tetrazole is a competitive inhibitor with respect to glucose 1-phosphate and uncompetitive with respect to phosphate. Ki values for the phosphorylase-AMP-glycogen complex and the phosphorylase-AMP-glycogen-phosphate complexes are 700 microM and 53 microM, respectively, indicating that by itself norjirimycin tetrazole has poor affinity for glycogen phosphorylase but that phosphate substantially improves the binding of norjirimycin tetrazole. X-ray crystallographic binding studies to 2.4 A resolution with T state phosphorylase b crystals showed that nojirimycin tetrazole binds at the catalytic site and promotes the binding of phosphate through direct interactions. Phosphate binding is accompanied by conformational changes that bring a crucial arginine (Arg569) into the catalytic site. The positions of the phosphate oxygens were definitively established in X-ray crystallographic binding experiments at 100 K to 1.7 A resolution using synchrotron radiation. X-ray crystallographic binding studies at 2.5 A resolution with R state glycogen phosphorylase crystals showed that the protein atoms and water molecules in contact with the nojirimycin tetrazole and the phosphate are similar to those in the T state. In both T and R states the phosphate ion is within hydrogen-bonding distance of the cofactor pyridoxal 5'-phosphate group and in ionic contact with the N-1 atom of the tetrazole ring. The results are consistent with previous time-resolved structural studies on complexes with heptenitol and phosphate. The structural and kinetic results suggest that nojirimycin tetrazole in combination with phosphate exhibits properties consistent with a transition state analogue and demonstrate how one promotes the binding of the other.
1-Deoxynojirimycin↗
Active and inactive protein kinases: structural basis for regulation.
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Amino Acid Sequence↗
The relative afferent pupillary defect and a novel method of fusion recovery with the Worth 4-dot test.
OBJECTIVE: To examine the relation of the relative afferent pupillary defect and commonly used clinical tests of visual cortex binocular cell activity, namely, fusion and stereopsis. METHODS: Sixty-seven subjects with neuro-ophthalmologic disorders underwent measurement of relative afferent pupillary defect and assessment of binocular sensory status by fusion on the Worth 4-dot test and stereopsis on the Titmus stereo test. Recovery of fusion on the Worth 4-dot test and improvement of stereopsis were tested by placing increasing neutral density filters in front of the eye that did not have the afferent pupillary defect. RESULTS: Recovery of fusion on the Worth 4-dot test occurred with use of neutral density filters (Worth 4-dot neutralization test). No improvement of stereopsis was observed. A significant positive association was identified between the measured afferent pupillary defect and the measured log filter required for fusion (r = .83, P < .001 by the Spearman rank correlation coefficient). In no case was a relative afferent pupillary defect observed when fusion without use of neutral density filters was present at baseline. For subjects with relative afferent pupillary defects, the measurements of the Worth 4-dot neutralization test were equal before and after pupillary dilation. CONCLUSIONS: The relative afferent pupillary defect is a dynamic process that results from an imbalance of brightness sensitivity between the two eyes. Altering the latency ("timing") and amplitude of the light stimuli by use of neutral density filters can restore fusion on the Worth 4-dot neutralization test but does not alter stereopsis. The Worth 4-dot neutralization test may be a useful adjunct in the assessment of visual dysfunction.
Adolescent↗
Activator anion binding site in pyridoxal phosphorylase b: the binding of phosphite, phosphate, and fluorophosphate in the crystal.
It has been established that phosphate analogues can activate glycogen phosphorylase reconstituted with pyridoxal in place of the natural cofactor pyridoxal 5'-phosphate (Change YC. McCalmont T, Graves DJ. 1983. Biochemistry 22:4987-4993). Pyridoxal phosphorylase b has been studied by kinetic, ultracentrifugation, and X-ray crystallographic experiments. In solution, the catalytically active species of pyridoxal phosphorylase b adopts a conformation that is more R-state-like than that of native phosphorylase b, but an inactive dimeric species of the enzyme can be stabilized by activator phosphite in combination with the T-state inhibitor glucose. Co-crystals of pyridoxal phosphorylase b complexed with either phosphite, phosphate, or fluorophosphate, the inhibitor glucose, and the weak activator IMP were grown in space group P4(3)2(1)2, with native-like unit cell dimensions, and the structures of the complexes have been refined to give crystallographic R factors of 18.5-19.2%, for data between 8 and 2.4 A resolution. The anions bind tightly at the catalytic site in a similar but not identical position to that occupied by the cofactor 5'-phosphate group in the native enzyme (phosphorus to phosphorus atoms distance = 1.2 A). The structural results show that the structures of the pyridoxal phosphorylase b-anion-glucose-IMP complexes are overall similar to the glucose complex of native T-state phosphorylase b. Structural comparisons suggest that the bound anions, in the position observed in the crystal, might have a structural role for effective catalysis.
Animals↗
The effect of aspirin on the visual outcome of nonarteritic anterior ischemic optic neuropathy.
PURPOSE: To examine the effect of aspirin use on the visual outcome of nonarteritic anterior ischemic optic neuropathy. METHODS: This retrospective case-controlled study included 23 patients who used aspirin regularly before and during the course of nonarteritic anterior ischemic optic neuropathy and 55 control patients with nonarteritic anterior ischemic optic neuropathy. Snellen visual acuity in logMAR and mean deviation on automated perimetry were compiled from the initial and final follow-up examinations. RESULTS: The comparisons of patients who took aspirin with control patients disclosed that neither initial logMAR (P = .36) nor the follow-up logMAR (P = .95) data differed significantly. There was no significant difference between the groups for the initial (P = .60) and the follow-up mean deviation (P = .41). CONCLUSIONS: Aspirin use does not improve the visual outcome of patients with nonarteritic anterior ischemic optic neuropathy. Additional studies are needed to further elucidate this issue.
Aged↗
Effect of levodopa and carbidopa on recovery of visual function in patients with nonarteritic anterior ischemic optic neuropathy of longer than six months' duration.
PURPOSE: We conducted a pilot clinical trial to determine the efficacy of levodopa in promoting visual recovery in eyes with nonarteritic anterior ischemic optic neuropathy of greater than six months' duration. METHODS: This prospective, randomized, double-masked, placebo-controlled clinical trial involved 20 subjects with nonarteritic anterior ischemic optic neuropathy of 30 months' mean duration. Subjects were randomly assigned to receive either low-dose levodopa and carbidopa or a placebo for three weeks. At 12 weeks after the baseline visit, the levodopa group then was provided a higher, conventional dose of levodopa and carbidopa for three more weeks. Change in visual function was monitored at four, 12, 16, and 24 weeks after the baseline visit. RESULTS: At 12 weeks after the baseline visit, the levodopa group experienced a significant (P = .016) mean difference in improvement of visual acuity of 5.9 letters from the placebo group. At 24 weeks after the baseline visit, a significant treatment effect (P = .036) for visual acuity was still evident; the levodopa group had a mean gain in improvement of 7.5 letters difference from baseline from the placebo group. Three subjects in the levodopa group experienced a doubling of the visual angle as denoted by a gain of at least 15 letters. Significant improvement was not observed for color vision (P = .82) or mean deviation of visual field loss (P = .82). CONCLUSION: The study found significant improvement of visual acuity among subjects receiving levodopa and carbidopa despite long-standing visual loss from nonarteritic anterior ischemic neuropathy. Confirmation of our results is awaited from larger population studies and with a longer follow-up time interval regarding the efficacy of levodopa in reversing visual loss in this disease.
Aged↗
Control by phosphorylation.
The two examples of phospho and dephospho proteins for which structural data were previously available (glycogen phosphorylase and isocitrate dehydrogenase) demonstrated two different mechanisms for control. In glycogen phosphorylase, activation by phosphorylation results in long-range allosteric changes. In isocitrate dehydrogenase, inhibition by phosphorylation is achieved by an electrostatic blocking mechanism with no conformational changes. During the past year, the structures of the phospho and dephospho forms of two more proteins, the cell cycle protein kinase CDK2 and yeast glycogen phosphorylase, have been determined. The new results highlight the importance of the phosphoamino acids both in the organization of local regions of protein structure through phosphate-arginine interactions and in the promotion of long-range conformational responses.
Binding Sites↗
The effect of sandblasting on the retention of orthodontic bands.
A variety of luting cements are available for use with orthodontic bands. This in vitro study was conducted to evaluate the force required to cause debanding when zinc phosphate, polycarboxylate and glass ionomer cements are used as the luting agents; and to determine whether sandblasting the inner surface of orthodontic bands affects the force required to deband. The data were obtained by debanding cemented stainless steel bands from 20 extracted third molars. Glass ionomer cement demonstrated the highest mean force value required to deband both the nonsandblasted and sandblasted orthodontic bands. Sandblasting the inner surface of the bands proved to be a significant (P < 0.001) method for increasing band retention for all three cements tested. The mean force required to deband using zinc phosphate, polycarboxylate and glass ionomer cements was approximately doubled following sandblasting.
Dental Bonding↗
Optic nerve compression by a dolichoectatic internal carotid artery: case report.
OBJECTIVE AND IMPORTANCE: Compression of the optic nerve by a dolichoectatic internal carotid artery is a rare and correctable cause of visual loss. CLINICAL PRESENTATION: A young woman presented with right eye visual loss without obvious cause and was found to have compression of the right optic nerve by the ipsilateral internal carotid artery, as revealed by magnetic resonance imaging. INTERVENTION: Pterional craniotomy and decompression of the optic nerve by unroofing the optic canal resulted in improvement of vision in the affected eye. CONCLUSION: Compression of the optic nerve by a dolichoectatic internal carotid artery may produce visual loss. The disorder is well demonstrated by magnetic resonance imaging, and timely intervention can improve vision.
Adult↗
Distinct early and late subcomponents of the photic blink reflex: response characteristics in patients with retrogeniculate lesions.
To assess cortical contributions to the photic blink reflex, signal averaged electromyograms (EMG) were compared for responses to strobe flashes presented within the blind and sighted hemifields of 13 patients with occipital lobe lesions. Reflexes evoked by flashes within the scotoma were virtually identical to those evoked by flashes within the intact visual field. This suggests that both the early and late components of this reflex (R50 and R80, respectively) are mediated by subcortical structures that do not require, or benefit from, conscious visual processing. Additional findings included larger R80s at the eyelid contralateral to the lesion, regardless of stimulated hemifield. This presumably reflects the loss of a tonic descending influence of visual cortex onto the motor limb of the reflex arc. The R80 was also larger for stimuli activating the crossed (temporal hemifield) rather than the uncrossed (nasal hemifield) afferent pathway.
Adult↗
Recurrent herpes labialis as a potential risk factor for nonarteritic anterior ischemic optic neuropathy.
This case-control study was undertaken to determine if recurrent herpes labialis posed an independent risk factor for the development of nonarteritic anterior ischemic optic neuropathy among individuals with small optic nerve cup:disk ratio. The study population was comprised of 43 nonarteritic anterior ischemic optic neuropathy cases and 30 consecutive control subjects with < or = 0.1 optic nerve cup:disk ratio. The two groups were compared with respect to the history of recurrent herpes labialis, cigarette smoking status, hypertension, diabetes mellitus, cardiovascular disease, and cerebrovascular disease. The nonarteritic anterior ischemic optic neuropathy cases were comparable to controls with respect to cigarette smoking status, hypertension, diabetes mellitus, cardiovascular disease, and cerebrovascular disease status. Nonarteritic anterior ischemic optic neuropathy cases were significantly more likely to have a history of recurrent herpes labialis than controls. Herpes simplex virus as identified by recurrent herpes labialis plays a role in the development of nonarteritic anterior ischemic optic neuropathy. However, a casual relationship cannot be established.
Aged↗
Intrasellar vascular malformation mimicking a pituitary macroadenoma.
We report the case of a 61-year-old man with an unusual intrasellar vascular malformation, who presented with symptoms of hypopituitarism and whose neuroradiologic evaluation mimicked a pituitary macroadenoma. The histopathologic and radiologic findings are discussed. This rare lesion should be considered in the diagnosis of a sellar mass lesion.
Adenoma↗
The binding of 2-deoxy-D-glucose 6-phosphate to glycogen phosphorylase b: kinetic and crystallographic studies.
Kinetic and crystallographic studies have characterized the effect of 2-deoxy-glucose 6-phosphate on the catalytic and structural properties of glycogen phosphorylase b. Previous work on the binding of glucose 6-phosphate, a potent physiological inhibitor of the enzyme, to T state phosphorylase b in the crystal showed that the inhibitor binds at the allosteric site and induces substantial conformational changes that affect the subunit-subunit interface. The hydrogen-bond from the O-2 hydroxyl of glucose 6-phosphate to the main-chain oxygen of Val40' represents the only hydrogen bond from the sugar to the other subunit, and this interaction appears important for promoting a more "tensed" structure than native T state phosphorylase b. 2-Deoxy-glucose 6-phosphate acts competitively with both the activator AMP and the substrate glucose 1-phosphate, with Ki values of 0.53 mM and 1.23 mM, respectively. The binding of 2-deoxy-glucose 6-phosphate to T state glycogen phosphorylase b in the crystal, has been investigated and the complex phosphorylase b: 2-deoxy-glucose 6-phosphate has been refined to give a crystallographic R factor of 17.3%, for data between 8 A and 2.3 A. 2-Deoxy-glucose 6-phosphate binds at the allosteric site as the a anomer and adopts a different conformation compared to glucose 6-phosphate. The two conformations differ by 160 degrees in the torsion angle about the C-5-C-6 bond. The contacts from the phosphate group are essentially identical to those made by the phosphate of glucose 6-phosphate but the 2-deoxy glucosyl moiety binds in a quite different orientation compared to the glucosyl of glucose 6-phosphate. 2-Deoxy-glucose 6-phosphate can be accommodated in the allosteric site with very little change in the protein, while structural comparisons show that the phosphorylase b: 2-deoxy-glucose 6-phosphate complex structure is overall more similar to a glucose-like complex than to the Glc-6-P complex structure.
Allosteric Site↗
The crystal structure of cyclin A.
BACKGROUND: Eukaryotic cell cycle progression is regulated by cyclin dependent protein kinases (CDKs) whose activity is regulated by association with cyclins and by reversible phosphorylation. Cyclins also determine the subcellular location and substrate specificity of CDKs. Cyclins exhibit diverse sequences but all share homology over a region of approximately 100 amino acids, termed the cyclin box. From the determination of the structure of cyclin A, together with results from biochemical and genetic analyses, we can identify which parts of the cyclin molecular may contribute to cyclin A structure and function. RESULTS: We have solved the crystal structure, at 2.0 A resolution, of an active recombinant fragment of bovine cyclin A, cyclin A-3, corresponding to residues 171-432 of human cyclin A. The cyclin box has an alpha-helical fold comprising five alpha helices. This fold is repeated in the C-terminal region, although this region shares negligible sequence similarity with the cyclin box. CONCLUSIONS: Analysis of residues that are conserved throughout the A, B, and E cyclins identifies two exposed clusters of residues, one of which has recently been shown to be involved in the association with human CDK2. The second cluster may identify another site of cyclin A-protein interaction. Comparison of the structure of the unbound cyclin with the structure of cyclin A complexed with CDK2 reveals that cyclin A does not undergo any significant conformational changes on complex formation. Threading analysis shows that the cyclin-box fold is consistent with the sequences of the transcription factor TFIIB and other functionally related proteins. The structural results indicate a role for the cyclin-box fold both as a template for the cyclin family and as a generalised adaptor molecule in the regulation of transcription.
Algorithms↗
Effects of C-1-substituted glucose analogue on the activation states of glycogen synthase and glycogen phosphorylase in rat hepatocytes.
A series of glucose-analogue inhibitors of glycogen phosphorylase b (GPb) has been designed, synthesized and investigated in crystallographic binding and kinetic studies. The aim is to produce a compound that may exert more effective control over glycogen metabolism than the parent glucose molecule and which could alleviate hyperglycaemia in Type-II diabetes. N-Acetyl-beta-D-glucopyranosylamine (1-GlcNAc) has a Ki for muscle GPb in crude extracts of 30 microM, 367-fold lower than that of beta-D-glucose [Board, Hadwen and Johnson (1995) Eur. J. Biochem. 228, 753-761]. In the current work, the effects of 1-GlcNAc on the activation states of GP and glycogen synthase (GS) in cell-free preparations and in isolated hepatocytes are reported. In gel-filtered extracts of liver, which lack ATP for kinase activity, 1-GlcNAc produced a rapid and time-dependent inactivation of GP with a subsequent activation of GS. Effects of 1-GlcNAc on both enzymes were stronger than those of glucose, with 0.8 mM 1-GlcNAc being equipotent with 50 mM glucose. At 1 mM, 1-GlcNAc enhanced the dephosphorylation of exogenous GPa by liver extracts (600%) and by muscle extracts (75%). This represents an approximately 500-fold improvement on glucose for the liver activity and 40-fold for the muscle activity. In whole hepatocytes, 1-GlcNAc showed an approximately 5-fold enhancement of glucose effects for GP inactivation but failed to elicit activation of GS. Glucose-induced activation of GS in whole hepatocytes was reversed by subsequent addition of 1-GlcNAc. However, when GS activation was achieved via the adenosine analogue and kinase inhibitor, 5'-iodotubercidin (ITU), subsequent addition of 1-GlcNAc allowed continued activation of GS. Phosphorylation of 1-GlcNAc in rat hepatocytes was established using radiolabelled material. The rate of phosphorylation was 1.60 nmol/min per 10(6) cells at 20 mM 1-GlcNAc but was reduced by the presence of 50 microM ITU (0.775 nmol/min per 10(6) cells). It is suggested that the phosphorylated derivative of 1-GlcNAc formed in hepatocytes is 1-GlcNAc 6-phosphate and that the presence of this species is responsible for the failure of 1-GlcNAc to activate GS. The relative importance of the reduction in concentration of GPa versus increased glucose 6-phosphate levels for activation of GS is discussed.
Acetylglucosamine↗