Detection of surgical glove integrity.

Surgical glove integrity is essential for universal precautions; glove safety is verified by the water load test (WLT). Concerns regarding glove injury have prompted newer testing methodologies, including electrical conductance testing (ECT); however, the sensitivities of these tests are not known. We compared the sensitivity of WLT and ECT in detecting glove needle-stick injury in two commonly used brands of surgical gloves. Punctures were made with hollow-bore and solid surgical needles of various configurations. The WLT failed to detect glove holes from the smallest-caliber needles and only detected the injury in 60 per cent for the largest caliber. The ECT provided a graded index of glove injury in all holes made by both solid surgical needles and hollow-bore needles. The WLT is a poor test for clinical defects in latex surgical gloves; the ECT is significantly more sensitive and provides a gauge of the cross-sectional area of the defect. Interbrand differences in self-sealing properties of surgical gloves were evidenced and may be clinically relevant after glove perforation.

Molecular structures of proteins involved in vesicle coat formation.

This review includes 16 structures of vesicle coat components and accessory proteins and a description of their roles in vesicle budding or coat disassembly.

Molecular structures of proteins involved in vesicle fusion.

We present a summary of the structures of 13 proteins involved in the docking and fusion of intracellular transport vesicles to their target membranes.

Clathrin self-assembly is mediated by a tandemly repeated superhelix.

Clathrin is a triskelion-shaped cytoplasmic protein that polymerizes into a polyhedral lattice on intracellular membranes to form protein-coated membrane vesicles. Lattice formation induces the sorting of membrane proteins during endocytosis and organelle biogenesis by interacting with membrane-associated adaptor molecules. The clathrin triskelion is a trimer of heavy-chain subunits (1,675 residues), each binding a single light-chain subunit, in the hub domain (residues 1,074-1,675). Light chains negatively modulate polymerization so that intracellular clathrin assembly is adaptor-dependent. Here we report the atomic structure, to 2.6 A resolution, of hub residues 1,210-1,516 involved in mediating spontaneous clathrin heavy-chain polymerization and light-chain association. The hub fragment folds into an elongated coil of alpha-helices, and alignment analyses reveal a 145-residue motif that is repeated seven times along the filamentous leg and appears in other proteins involved in vacuolar protein sorting. The resulting model provides a three-dimensional framework for understanding clathrin heavy-chain self-assembly, light-chain binding and trimerization.

Crystal structure of neurotrophin-3 homodimer shows distinct regions are used to bind its receptors.

Neurotrophin-3 (NT-3) is a cystine knot growth factor that promotes the survival, proliferation, and differentiation of developing neurons and is a potential therapeutic for neurodegenerative diseases. To clarify the structural basis of receptor specificity and the role of neurotrophin dimerization in receptor activation, the structure of the NT-3 homodimer was determined using X-ray crystallography. The orthorhombic crystals diffract to 2.4 A, with dimer symmetry occurring about a crystallographic 2-fold axis. The overall structure of NT-3 resembles that of the other neurotrophins, NGF and BDNF; each protomer forms a twisted four-stranded beta sheet, with three intertwined disulfide bonds. There are notable differences, however, between NT-3 and NGF in the surface loops and in three functionally important regions, shown in previous mutagenesis studies to be critical for binding. One such difference implies that NT-3's binding affinity and specificity depend on a novel hydrogen bond between Gln 83, a residue important for binding specificity with TrkC, and Arg 103, a residue crucial for binding affinity with TrkC. NT-3's extensive dimer interface buries much of the otherwise solvent-accessible hydrophobic surface area and suggests that the dimeric state is stabilized through the formation of this hydrophobic core. A comparison of the dimer interface between the NT-3 homodimer and the BDNF/NT-3 heterodimer reveals similar patterns of hydrogen bonds and nonpolar contacts, which reinforces the notion that the evolutionarily conserved neurotrophin interface resulted from the need for receptor dimerization in signal initiation.

Distinct phosphorylation signals converge at the catalytic center in glycogen phosphorylases.

BACKGROUND: Glycogen phosphorylases (GPs) catalyze the conversion of the storage form of carbohydrate (glycogen) to the readily usable form (glucose-1-phosphate) to provide cellular energy. Members of this enzyme family have evolved diverse regulatory mechanisms that control a conserved catalytic function. The mammalian and yeast GPs are expressed as inactive forms requiring phosphorylation for activation. Phosphorylation of yeast GP occurs at a distinct site from that of mammalian GP. This work addresses the structural basis by which distinct activation signals relay to the conserved catalytic site in yeast and mammalian GPs. Such knowledge may help understand the principles by which diverse biological regulation evolves. RESULTS: We have compared the crystal structures of the unphosphorylated and phosphorylated forms of yeast GP and propose a relay which links phosphorylation to enzyme activation. Structural components along the activation relay becomes more conserved within the GP family downstream along the relay, towards the catalytic center. Despite distinct upstream activation signals, a response element downstream of the relay leading to the catalytic center is conserved in all GPs. The response element consists of ten hydrophobic residues dispersed over two subunits of the homodimer. Phosphorylation induces hydrophobic condensation of these residues via structural rearrangement, which triggers conformation change of the active site GATE loop, leading to enzyme activation. CONCLUSIONS: Members of the GP family with diverse activation mechanisms have evolved from a constitutively active ancestral enzyme which has the TOWER hydrophobic response element in the active position. Diverse regulation evolved as a result of evolutionary constraint on the downstream response element in the active state, coupled with flexibility and variability in elements of the upstream relays.

A protein phosphorylation switch at the conserved allosteric site in GP.

A phosphorylation-initiated mechanism of local protein refolding activates yeast glycogen phosphorylase (GP). Refolding of the phosphorylated amino-terminus was shown to create a hydrophobic cluster that wedges into the subunit interface of the enzyme to trigger activation. The phosphorylated threonine is buried in the allosteric site. The mechanism implicates glucose 6-phosphate, the allosteric inhibitor, in facilitating dephosphorylation by dislodging the buried covalent phosphate through binding competition. Thus, protein phosphorylation-dephosphorylation may also be controlled through regulation of the accessibility of the phosphorylation site to kinases and phosphatases. In mammalian glycogen phosphorylase, phosphorylation occurs at a distinct locus. The corresponding allosteric site binds a ligand activator, adenosine monophosphate, which triggers activation by a mechanism analogous to that of phosphorylation in the yeast enzyme.

The evolution of an allosteric site in phosphorylase.

BACKGROUND: Glycogen phosphorylases consist of a conserved catalytic core onto which different regulatory sites are added. By comparing the structures of isozymes, we hope to understand the structural principles of allosteric regulation in this family of enzymes. Here, we focus on the differences in the glucose 6-phosphate (Glc-6-P) binding sites of two isozymes. RESULTS: We have refined the structure of Glc-6-P inhibited yeast phosphorylase b to 2.6 A and compared it with known structures of muscle phosphorylase. Glc-6-P binds in a novel way, interacting with a distinct set of secondary elements. Structural links connecting the Glc-6-P binding sites and catalytic sites are conserved, although the specific contacts are not. CONCLUSIONS: Our comparison reveals that the Glc-6-P binding site was modified over the course of evolution from yeast to vertebrates to become a bi-functional switch. The additional ability of muscle phosphorylase to be activated by AMP required the recruitment of structural elements into the binding site and sequence changes to create a binding subsite for adenine, whilst maintaining links to the catalytic site.

Mechanism of regulation in yeast glycogen phosphorylase.

The mechanism of yeast glycogen phosphorylase activation by covalent phosphorylation involves structural elements distinct from the mammalian homologs. To understand the role of the amino-terminal 39-residue extension in the phosphorylation control mechanism, mutants with 22 and 42 amino-terminal residues removed were expressed in Escherichia coli, and their properties were compared with the wild-type (WT) enzyme. The unphosphorylated WT enzyme had a specific activity of 0.1 unit/mg and was not activated significantly by the substrate, glucose 1-phosphate. Phosphorylation by protein kinase resulted in a 1300-fold activation. Glucose 6-phosphate inhibited the unphosphorylated enzyme more effectively than the phosphorylated form, and inhibition of the latter was cooperative. Glucose was a poor inhibitor for both the unphosphorylated and phosphorylated WT enzyme with Ki > 300 mM. The rate of phosphorylation by protein kinase depended on substrates and interactions of the amino terminus. Maltoheptaose increased the rate of phosphorylation of the WT enzyme by yeast phosphorylase kinase 5-fold. The 22-residue deletion mutant (Nd22) had overall kinetic properties similar to the WT enzyme, except that Nd22 was a better substrate for the protein kinase and the rate of phosphorylation was unaffected by maltoheptaose. The 42-residue deletion mutant (Nd42), which lacks the phosphorylation site, was measurably active, although much less active than phosphorylated WT. Sedimentation equilibrium analysis indicated that the WT, Nd22, and Nd42 exist as tetramer, partially dissociated tetramer, and dimer, respectively. Phosphorylation of the WT and Nd22 converted both to dimer. The results indicated that the amino terminus affects quaternary structure and mediates activity regulation through conformational transition.

Chimeric muscle and brain glycogen phosphorylases define protein domains governing isozyme-specific responses to allosteric activation.

Muscle and brain glycogen phosphorylases differ in their responses to activation by phosphorylation and AMP. The muscle isozyme is potently activated by either phosphorylation or AMP. In contrast, the brain isozyme is poorly activated by phosphorylation and its phosphorylated a form is more sensitive to AMP activation when enzyme activity is measured in substrate concentrations and temperatures encountered in the brain. The nonphosphorylated b form of the brain isozyme also differs from the muscle isozyme b form in its stronger affinity and lack of cooperativity for AMP. To identify the structural determinants involved, six enzyme forms, including four chimeric enzymes containing exchanges in amino acid residues 1-88, 89-499, and 500-842 (C terminus), were constructed from rabbit muscle and human brain phosphorylase cDNAs, expressed in Escherichia coli, and purified. Kinetic analysis of the b forms indicated that the brain isozyme amino acid 1-88 and 89-499 regions each contribute in an additive fashion to the formation of an AMP site with higher intrinsic affinity but weakened cooperativity, while the same regions of the muscle isozyme each contribute to greater allosteric coupling but weaker AMP affinity. Kinetic analysis of the a forms indicated that the amino acid 89-499 region correlated with the reduced response of the brain isozyme to activation by phosphorylation and the resultant increased sensitivity of the a form to activation by saturating levels of AMP. This isozyme-specific response also correlated with the glycogen affinity of the a forms. Enzymes containing the brain isozyme amino acid 89-499 region exhibited markedly reduced glycogen affinities in the absence of AMP compared to enzymes containing the corresponding muscle isozyme region. Additionally, AMP led to greater increases in glycogen affinity of the former set of enzymes. In contrast, phosphate affinities of all a forms were similar in the absence of AMP and increased approximately the same extent in AMP. The potential importance of a number of isozyme-specific substitutions in these sequence regions is discussed.

Cooperative binding is not required for activation of muscle phosphorylase.

Muscle and liver glycogen phosphorylase isozymes differ in their responsiveness to the activating ligand AMP. The muscle enzyme, which supplies glucose in response to strenuous activity, binds AMP cooperatively, and its enzymatic activity becomes greatly enhanced. The liver isozyme regulates the level of blood glucose, and AMP is not the primary activator. In muscle glycogen phosphorylase, the residue proline 48 links two secondary structural elements that bind AMP. This amino acid residue is replaced with a threonine in the liver isozyme; unlike the muscle enzyme, liver binds AMP noncooperatively, and the enzymatic activity is not greatly increased. We have substituted proline 48 in the muscle enzyme with threonine, alanine, and glycine and characterized the recombinant enzymes kinetically and structurally to determine if proline at this position is critical for cooperative AMP binding and activation. Importantly, all of the engineered enzymes were fully activated by phosphorylation, indicating that enzymatic activity was not compromised. Only the mutant enzyme with alanine at position 48 responds like the wild-type enzyme to the presence of AMP, indicating that proline is not absolutely required for full cooperative activation. The substitution of either threonine or glycine at this position, however, creates enzymes that no longer bind AMP cooperatively. The enzyme with threonine at position 48 further mimics the liver enzyme, in that the maximal enzymatic activity is also reduced. Significantly, the glycine substitution caused the enzyme to be fully activated by AMP, although binding was not cooperative. The hyperactivation of the glycine mutant by AMP suggests that the total free energy of activation has decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and crystallization of glycogen phosphorylase from Saccharomyces cerevisiae.

Glycogen phosphorylase from Saccharomyces cerevisiae is activated by the covalent phosphorylation of a single threonine residue in the N terminus of the protein. We have hypothesized that the structural features that effect activation must be distinct from those characterized in rabbit muscle phosphorylase because the two enzymes have unrelated phosphorylation sites located in dissimilar protein contexts. To understand this potentially novel mechanism of activation by phosphorylation, we require information at atomic resolution of the phosphorylated and unphosphorylated forms of the enzyme. To this end, we have purified, characterized and crystallized glycogen phosphorylase from S. cerevisiae. The enzyme was isolated from a phosphorylase-deficient strain harboring a multicopy plasmid containing the phosphorylase gene under the control of its own promoter. One liter of cultured cells yields 12 mg of crystallizable material. The purified protein was not phosphorylated and had an activity of 4.7 units/mg in the presence of saturating amounts of substrate. Yeast phosphorylase was crystallized in four different crystal forms, only one of which is suitable for diffraction studies at high resolution. The latter belongs to space group P4(1)2(1)2 with unit cell constants of a = 161.1 A and c = 175.5 A Based on the density of the crystals, the solvent content is 49.7%, indicating that the asymmetric unit contains the functional dimer of yeast phosphorylase.

Molecular analysis of GPH1, the gene encoding glycogen phosphorylase in Saccharomyces cerevisiae.

In yeast cells, the activity of glycogen phosphorylase is regulated by cyclic AMP-mediated phosphorylation of the enzyme. We have previously cloned the gene for glycogen phosphorylase (GPH1) in Saccharomyces cerevisiae. To assess the role of glycogen and phosphorylase-catalyzed glycogenolysis in the yeast life cycle, yeast strains lacking a functional GPH1 gene or containing multiple copies of the gene were constructed. GPH1 was found not to be an essential gene in yeast cells. Haploid cells disrupted in GPH1 lacked phosphorylase activity and attained higher levels of intracellular glycogen but otherwise were similar to wild-type cells. Diploid cells homozygous for the disruption were able to sporulate and give rise to viable ascospores. Absence of functional GPH1 did not impair cells from synthesizing and storing trehalose. Increases in phosphorylase activity of 10- to 40-fold were detected in cells carrying multiple copies of GPH1-containing 2 microns plasmid. Northern (RNA) analysis indicated that GPH1 transcription was induced at the late exponential growth phase, almost simultaneous with the onset of intracellular glycogen accumulation. Thus, the low level of glycogen in exponential cells was not primarily maintained through regulating the phosphorylation state of a constitutive amount of phosphorylase. GPH1 did not appear to be under formal glucose repression, since transcriptional induction occurred well in advance of glucose depletion from the medium.

The family of glycogen phosphorylases: structure and function.

Glycogen phosphorylase plays a central role in the mobilization of carbohydrate reserves in a wide variety of organisms and tissues. While rabbit muscle phosphorylase remains the most studied and best characterized of phosphorylases, recombinant DNA techniques have led to the recent appearance of primary sequence data for a wide variety of phosphorylase enzymes. The functional properties of rabbit muscle phosphorylases are reviewed and then compared to properties of phosphorylases from other tissues and organisms. Tissue expression patterns and the chromosomal localization of mammalian phosphorylases are described. Differences in functional properties among phosphorylases are related to new structural information. Evolutionary relationships among phosphorylases as afforded by comparative analysis of proteins and gene sequences are discussed.

Sequence analysis of the cDNA encoding human liver glycogen phosphorylase reveals tissue-specific codon usage.

We have cloned the cDNA encoding glycogen phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyl-transferase, EC 2.4.1.1) from human liver. Blot-hybridization analysis using a large fragment of the cDNA to probe mRNA from rabbit brain, muscle, and liver tissues shows preferential hybridization to liver RNA. Determination of the entire nucleotide sequence of the liver message has allowed a comparison with the previously determined rabbit muscle phosphorylase sequence. Despite an amino acid identity of 80%, the two cDNAs exhibit a remarkable divergence in G+C content. In the muscle phosphorylase sequence, 86% of the nucleotides at the third codon position are either deoxyguanosine or deoxycytidine residues, while in the liver homolog the figure is only 60%, resulting in a strikingly different pattern of codon usage throughout most of the sequence. The liver phosphorylase cDNA appears to represent an evolutionary mosaic; the segment encoding the N-terminal 80 amino acids contains greater than 90% G+C at the third codon position. A survey of other published mammalian cDNA sequences reveals that the data for liver and muscle phosphorylases reflects a bias in codon usage patterns in liver and muscle coding sequences in general.

Convergent and divergent evolution of regulatory sites in eukaryotic phosphorylases.

The activity of many proteins in eukaryotic cells is regulated by reversible covalent phosphorylation. This regulatory modification is often linked to other allosteric controls within the same protein, and such overlapping regulatory mechanisms are best characterized for glycogen phosphorylase (EC 2.4.1.1). Phosphorylases from different organisms or cell types exhibit markedly contrasting regulatory features; this makes the enzyme attractive for studying the evolution of interacting molecular regulatory mechanisms. Extensive biochemical and crystallographic studies of rabbit muscle phosphorylase have led to a characterization of five regulatory regions (phosphorylation, glycogen storage, AMP, glucose and purine sites). Here we report the complete primary structure of the yeast Saccharomyces cerevisiae glycogen phosphorylase, deduced from the sequence of the cloned gene. Regions that are highly conserved between muscle and yeast enzymes include the active site, the glycogen storage site and possibly the glucose and purine inhibition sites. Partial conservation of the residues involved in AMP-binding suggests a binding site for the yeast enzyme inhibitor, glucose 6-phosphate. Other parts of the AMP site and the intersubunit contacts involved in AMP allostery are disrupted in the yeast enzyme by extreme sequence divergence. The poor alignment of amino termini and lack of homology at phosphorylation sites indicate that regulation by reversible phosphorylation evolved independently in yeast and vertebrate phosphorylases.

Complete cDNA sequence for rabbit muscle glycogen phosphorylase.

The cDNA for the nearly full-length rabbit muscle glycogen phosphorylase mRNA has been isolated and sequenced. The cDNA is rich in G and C nucleotides. This feature is especially striking at the 3rd position of codons, where 86% of the 843 amino acid codons terminate with G or C. Methionine, presumably the initiation residue, is found at position-1, suggesting that the removal of only a single methionine residue precedes the amino-terminal acetylation at serine. Eight differences between the deduced amino acid sequence and the previously determined protein sequence are discussed.

Comparative sequence analysis of rat, rabbit, and human muscle glycogen phosphorylase cDNAs.

As an initial step in the investigation of the structure, evolution and developmental regulation of the glycogen phosphorylase gene family, we have isolated partial cDNAs to rat, rabbit and human muscle phosphorylase mRNAs. Sequence comparisons of these cDNAs in regions that encode portions of the enzyme located near and encompassing the C terminus show that there is a high degree of interspecies conservation of structure in this region. Conservation of amino acid and nucleotide sequence is high, approximately 96% and 90% homology, respectively, among all three species. In addition, most of the amino acid changes that have occurred conserve the chemical nature of the amino acid side-chains affected. The changes can be easily accommodated in the rabbit muscle phosphorylase tertiary structure and appear to have little effect on the overall conformation. Interestingly the rat and human enzymes lack the carboxyl-terminal proline (residue 841) present in the rabbit enzyme and terminate at isoleucine (residue 840). The genetic basis for this difference in carboxyl termini is unknown. However, unlike the other amino acid changes, it cannot be accounted for by a single base-pair substitution. A comparison of the 3' untranslated regions in these cDNAs shows that there has been little constraint on the evolutionary divergence of most of this region (70% homology among the three species). There are, however, two repeated segments of DNA flanking the stop codons that are identical among all three species. Similar sequences are found within regions of DNA that contain a variety of transcriptional enhancers, suggesting the possibility that the repeats may be functional.