The interaction between ADAM22 and 14-3-3β

ADAM family consists of a number of transmembrane proteins that contain a disintegrin and metalloprotease domain. ADAMs are involved in a highly diverse set of biological processes, including fertilization, neurogenesis, myogenesis and inflammatory response. The ADAM proteins have both cell adhesion and protease activities. Adam22 is highly expressed in human brain. The adam22-/- mice presented severe ataxia and died before weaning, but the function of ADAM22 is still unknown. 14-3-3β interacting with ADAM22 was detected by using yeast two-hybrid assay. The specificity of interaction between ADAM22 and 14-3-3β was proved by in vitro binding assay and immunoprecipitation. The major 14-3-3β binding site was located in the last 28 amino acid residues of ADAM22 cytoplasmic tail. Protein 14-3-3β is abundant and plays an important role in mediating cell diffusion, migration and cell cycle control. The interaction of ADAM22 and 14-3-3β suggests that the ADAM22 may play a crucial role in neural function and development.     

The interaction between ADAM22 and 14-3-3β

ADAM family consists of a number of transmembrane proteins that contain a disintegrin and metalloprotease domain. ADAMs are involved in a highly diverse set of biological processes, including fertilization, neurogenesis, myogenesis and inflammatory response. The ADAM proteins have both cell adhesion and protease activities.Adam22 is highly expressed in human brain. Theadam22-/- mice presented severe ataxia and died before weaning, but the function of ADAM22 is still unknown. 14-3-3 β interacting with ADAM22 was detected by using yeast two-hybrid assay. The specificity of interaction between ADAM22 and 14-3-3β was proved byin vitro binding assay and immunoprecipitation. The major 14-3-3β binding site was located in the last 28 amino acid residues of ADAM22 cytoplasmic tail. Protein 14-3-3β is abundant and plays an important role in mediating cell diffusion, migration and cell cycle control. The interaction of ADAM22 and 14-3-3β suggests that the ADAM22 may play a crucial role in neural function and development.     

High resolution phosphorus NMR spectroscopy of transfer ribonucleic acids

Summary A new activator of phosphofructokinase, which is bound to the enzyme and released during its purification, has been discovered. Its structure has been determined as -D Fructose-2,6-P₂ by chemical synthesis, analysis of various degradation products and NMR. D-Fructose-2,6-P₂ is the most potent activator of phosphofructokinase and relieves inhibition of the enzyme by ATP and citrate. It lowers the K_m for fructose-6-P from 6 mM to 0.1 mM.Fructose-6-P,2-kinase catalyzes the synthesis of fructose-2,6-P₂ from fructose-6-P and ATP, and the enzyme has been partially purified. The degradation of fructose-2,6-P₂ is catalyzed by fructose-2,6-bisphosphatase. Thus a metabolic cycle could occur between fructose-6-P and fructose-2,6-P₂, which are catalyzed by these two opposing enzymes. The activities of these enzymes can be controlled by phosphorylation. Fructose-6-P,2-kinase is inactivated by phosphorylation catalyzed by either cAMP dependent protein kinase or phosphorylase kinase. The inactive, phospho-fructose-6-P,2-kinase is activated by dephosphorylation catalyzed by phosphorylase phosphatase. On the other hand, fructose-2,6-bisphosphatase is activated by phosphorylation catalyzed by cAMP dependent protein kinase.Investigation into the hormonal regulation of phosphofructokinase reveals that glucagon stimulates phosphorylation of phosphofructokinase which results in decreased affinity for fructose-2,6-P₂, and decreases the fructose-2,6-P₂ levels. This decreased level in fructose-2,6-P₂ appears to be due to the decreased synthesis by inactivation of fructose-2,6-P₂,2-kinase and increased degradation as a result of activation of fructose-2,6-bisphosphatase. Such a reciprocal change in these two enzymes has been demonstrated in the hepatocytes treated by glucagon and epinephrine. The implications of these observations in respect to possible coordinated controls of glycolysis and glycogen metabolism are discussed.     

Structural Characterization of the Ectodomain of a Disintegrin and Metalloproteinase-22 (ADAM22), a Neural Adhesion Receptor Instead of Metalloproteinase: INSIGHTS ON ADAM FUNCTION?

ADAMs (a disintegrin and metalloproteinases) are a family of multidomain transmembrane glycoproteins with diverse roles in physiology and diseases, with several members being drug targets for cancer and inflammation therapies. The spatial organization of the ADAM extracellular segment and its influence on the function of ADAMs have been unclear. Although most members of the ADAM family are active zinc metalloproteinases, 8 of 21 ADAMs lack functional metalloproteinase domains and are implicated in protein-protein interactions instead of membrane protein ectodomain shedding. One of such non-proteinase ADAMs, ADAM22, acts as a receptor on the surface of the postsynaptic neuron to regulate synaptic signal transmission. The crystal structure of the full ectodomain of mature human ADAM22 shows that it is a compact four-leaf clover with the metalloproteinase-like domain held in the concave face of a rigid module formed by the disintegrin, cysteine-rich, and epidermal growth factor-like domains. The loss of metalloproteinase activity is ensured by the absence of critical catalytic residues, the filling of the substrate groove, and the steric hindrance by the cysteine-rich domain. The structure, combined with calorimetric experiments, suggests distinct roles of three putative calcium ions bound to ADAM22, with one in the metalloproteinase-like domain being regulatory and two in the disintegrin domain being structural. The metalloproteinase-like domain contacts the rest of ADAM22 with discontinuous, hydrophilic, and poorly complemented interactions, suggesting the possibility of modular movement of ADAM22 and other ADAMs. The ADAM22 structure provides a framework for understanding how different ADAMs exert their adhesive function and shedding activities.The ADAM² family includes over 20 multidomain type I transmembrane glycoproteins that have diverse functions in cell adhesion/signaling and ectodomain shedding of cell-surface receptors or ligands (1, 2). They are broadly implicated in various physiological processes including sperm-egg interactions, development and function of the nervous system (e.g. cell-fate determination, axon guidance, and myelination), immune responses, and embryogenesis (2, 3). Dysregulation of the ADAM family is linked to a wide variety of pathological states including cancer, cardiovascular disease, asthma, Alzheimer disease, and inflammation (3–5). Several ADAMs have been pursued as therapeutic targets (6, 7).ADAMs, together with their phylogenic relatives, the P- III class snake venom metalloproteinases (SVMPs) and ADAMTSs (ADAM with thrombospondin type-1 motif), constitute a subgroup of the metzincin clan of zinc proteinases (8, 9). The extracellular segments of ADAMs contain a prodomain that gets cleaved off during secretion, a metalloproteinase-like domain, a disintegrin domain, and a cysteine-rich domain, which are shared by SVMPs and ADAMTSs, and a unique epidermal growth factor-like domain preceding the transmembrane segment. All ADAMs contain metalloproteinase-like domains, but in humans, only 13 of the 21 members in the family possess the complete zinc binding environment (the HEXGHXXGXXHD sequence motif and the Met turn) in the domain (10). Although these proteolytically active ADAMs can shed cell-surface proteins from the plasma membrane, the other ADAMs are suggested to be non-enzymatic cell adhesion molecules (11, 12). Several ADAMs have been reported to interact with integrins, and the disintegrin-like domains of ADAMs have been suggested for this interaction (13). Despite these suggestions, structural proof that the ADAMs without canonical zinc-binding motif lack enzymatic activities has been absent, and it remains unclear how these molecules are structurally configured to support protein-protein interaction instead of ectodomain shedding.ADAM22 (also named MDC2), one of such postulated non-catalytic ADAMs, was recently identified to serve as the postsynaptic receptor for the secreted neurotransmission modulator LGI-1 at neural synapses (14). The study supports that some ADAMs can function as adhesion molecules rather than metalloproteinases. ADAM22 is predominantly expressed in the nervous systems (15, 16). The Adam22^−/− mice suffered from hypomyelination of peripheral nerves, leading to ataxia, and died before weaning (17). At the synapse, LGI-1 and ADAM22 form a tertiary complex with postsynaptic density-95 (PSD-95), a major scaffolding protein localized to the postsynaptic density of brain synapses, which is associated with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor and other signaling proteins (14). In this complex, the extracellular domain of ADAM22 interacts with LGI-1, whereas its cytoplasmic PDZ-binding motif recruits PSD-95. The link of ADAM22 and LGI-1 to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor established their roles in glutamate neurotransmission, consistent with genetic data that all these molecules are associated with epilepsy (17–19). Recently, it was further demonstrated that LGI-1 and LGI-4 bind to ADAM22, ADAM23, and ADAM11 (20).Although ADAMs are functionally important as sheddases or adhesion receptors, the structural information about the ADAM family is limited to only isolated domains, such as the metalloproteinase domains of ADAM17 and ADAM33 and the incomplete disintegrin cysteine-rich domains of ADAM10 (21–23). Their relatives, SVMPs from the snake venom, including VAP-1, VAP-2, and RVV-X (24–26), have revealed a “C”-shaped molecular architecture. These SVMP structures and partial ADAM structures, along with those of the ADAMTS family proteins (27–29), shed light on the general mechanisms of substrate recognition and cleavage by the proteinase-type ADAMs. However, there is little structural information on those non-catalytic ADAMs, which serve as adhesion receptors. In addition, despite a low resolution electron microscopic (EM) analysis of the soluble form of pro-ADAM12, which suggested that the prodomain represents one of the leaves of the four-leaf clover-shaped ADAM12 (30), the structure of a complete ADAM ectodomain, being catalytic or non-catalytic, has been lacking. Here we report the crystal structure of the entire ectodomain of mature ADAM22.     

Sexual Selection and the Molecular Evolution of ADAM Proteins

Rapid evolution has been identified for many reproductive genes and recent studies have combined phylogenetic tests and information on species mating systems to test sexual selection. Here we examined the molecular evolution of the ADAM gene family, a diverse group of 35 proteins capable of adhesion to and cleavage of other proteins, using sequence data from 25 mammalian genes. Out of the 25 genes analyzed, all those expressed in male reproductive tissue showed evidence of positive selection. Positively selected amino acids within the protein adhesion domain were only found in sperm surface ADAM proteins (ADAMs 1, 2, 3, 4, and 32) suggesting selection driven by male × female interactions. We tested heterogeneity in rates of evolution of the adhesion domain of ADAM proteins by using sequence data from Hominidae and macaques. The use of the branch and branch-site models (PAML) showed evidence of higher d _N/d _S and/or positive selection linked to branches experiencing high postmating selective pressures (chimpanzee and macaque) for Adams 2, 18, and 23. Moreover, we found consistent higher proportion of nonsynonymous relative to synonymous and noncoding sequence substitutions in chimpanzee and/or macaque only for Adams 2, 18, and 23. Our results suggest that lineage-specific sexual selection bouts might have driven the evolution of the adhesion sperm protein surface domains of ADAMs 2 and 18 in primates. Adams 2 and 18 are localized in chromosome 8 of primates and adjacent to each other, so their evolution might have also been influenced by their common genome localization.     

Biosynthesis of bacterial glycogen: characterization of adenosine diphosphate glucose synthetases from Enterobacter hafniae and Aeromonas hydrophila

Enterobacter hafniae and Aeromonas hydrophila ADPglucose synthetases were purified approximately 39-and 61-fold, respectively, over the crude extract. Both enzymes were heat stable at 60°C in the presence of inorganic phosphate. The molecular weights of both enzymes were approximately 200,000 which are similar to other enteric ADPglucose synthetases studied. Based on kinetic results obtained from the partially purified enzymes, the E. hafniae enzyme is activated twofold by phospho-enolpyruvate while the A. hydrophila enzyme is activated twofold by fructose 6-P and 1.5-fold by fructose 1,6 bis-phosphate. The E. hafniae enzyme activity is strongly inhibited by AMP and ADP and the inhibition can be partially reversed by P-enolpyruvate. ADP is the most effective inhibitor of the A. hydrophila enzyme and its inhibiton can be partially overcome by the presence of the activators fructose 6-P and fructose 1,6-P₂. These kinetic results show that the allosteric properties of the E. hafniae enzyme are distinctly different from the ADPglucose synthetases of those previously studied from bacteria of the genus Enterobacter. Although the A. hydrophila enzyme is activated by fructose 1,6-P₂, its allosteric properties are quite different than those observed for ADPglucose synthetase of the Enterobacteriaceae.Abbreviations Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid - glucose 1-P glucose 1-phosphate - Bicine N,N-bis(2 hydroxyethyl)glycine - fructose 6-P fructose 6-phosphate - Mes 2(N-morpholino)-ethane sulfonic acid - fructose 1,6-P₂ fructose 1,6 bis-phosphate - DTE dithioerythritol; pyridoxal-P, pyridoxal-phosphate - fructose 1-P fructose 1-phosphate - P-enolpyruvate phospho-enolpyruvate - 1,6 hexanediol bis-P 1,6 hexanediol bis-phosphate; glucose 6-P, glucose 6-phosphate - dihydroxyacetone-P dihydroxyacetone phosphate - 1-glycerol-3-P 1-glycerol-3-phosphate - erythrose 4-P erythrose 4-phosphate - 2-P-glycerate 2-phosphoglycerate - sedoheptulose 1,7-P₂ sedoheptulose 1,7 bis-phosphate - 3-P-glycerate 3-phosphoglycerate - mannose-6-P mannose-6-phosphate     

Evaluation of the contribution of different ADAMs to tumor necrosis factor alpha (TNFalpha) shedding and of the function of the TNFalpha ectodomain in ensuring selective stimulated shedding by the TNFalpha convertase (TACE/ADAM17)

Tumor necrosis factor-alpha (TNFalpha), a potent pro-inflammatory cytokine, is released from cells by proteolytic cleavage of a membrane-anchored precursor. The TNF-alpha converting enzyme (TACE; a disintegrin and metalloprotease17; ADAM17) is known to have a key role in the ectodomain shedding of TNFalpha in several cell types. However, because purified ADAMs 9, 10, and 19 can also cleave a peptide corresponding to the TNFalpha cleavage site in vitro, these enzymes are considered to be candidate TNFalpha sheddases as well. In this study we used cells lacking ADAMs 9, 10, 17 (TACE), or 19 to address the relative contribution of these ADAMs to TNFalpha shedding in cell-based assays. Our results corroborate that ADAM17, but not ADAM9, -10, or -19, is critical for phorbol ester- and pervanadate-stimulated release of TNFalpha in mouse embryonic fibroblasts. However, overexpression of ADAM19 increased the constitutive release of TNFalpha, whereas overexpression of ADAM9 or ADAM10 did not. This suggests that ADAM19 may contribute to TNFalpha shedding, especially in cells or tissues where it is highly expressed. Furthermore, we used mutagenesis of TNFalpha to explore which domains are important for its stimulated processing by ADAM17. We found that the cleavage site of TNFalpha is necessary and sufficient for cleavage by ADAM17. In addition, the ectodomain of TNFalpha makes an unexpected contribution to the selective cleavage of TNFalpha by ADAM17: it prevents one or more other enzymes from cleaving TNFalpha following PMA stimulation. Thus, selective stimulated processing of TNFalpha by ADAM17 in cells depends on the presence of an appropriate cleavage site as well as the inhibitory role of the TNF ectodomain toward other enzymes that can process this site.     

Multiple non-catalytic ADAMs are novel integrin α4 ligands

The ADAM (a disintegrin and metalloprotease) protein family uniquely exhibits both catalytic and adhesive properties. In the well-defined process of ectodomain shedding, ADAMs transform latent, cell-bound substrates into soluble, biologically active derivatives to regulate a spectrum of normal and pathological processes. In contrast, the integrin ligand properties of ADAMs are not fully understood. Emerging models posit that ADAM–integrin interactions regulate shedding activity by localizing or sequestering the ADAM sheddase. Interestingly, 8 of the 21 human ADAMs are predicted to be catalytically inactive. Unlike their catalytically active counterparts, integrin recognition of these “dead” enzymes has not been largely reported. The present study delineates the integrin ligand properties of a group of non-catalytic ADAMs. Here we report that human ADAM11, ADAM23, and ADAM29 selectively support integrin α4-dependent cell adhesion. This is the first demonstration that the disintegrin-like domains of multiple catalytically inactive ADAMs are ligands for a select subset of integrin receptors that also recognize catalytically active ADAMs.     

Expression patterns of ADAMs in the developing chicken lens

In the present study the expression patterns of ADAM (a disintegrin and metalloprotease) genes in the chicken developing lens were analyzed. Using in situ hybridization, we found that seven members of the ADAM family including ADAM9, ADAM10, ADAM12, ADAM13, ADAM17, ADAM22, and ADAM23 are expressed in the developing embryonic lens. From embryonic incubation day (E) 2 to E3, most of the ADAMs investigated here are expressed in the lens placode and lens vesicle. From E5 to E7, all seven ADAMs, but predominantly ADAM9 and ADAM10, are throughly expressed in the central epithelium, as well as in the proliferating lens epithelium and the equatorial lens epithelium. From E9 to E14, expression of ADAM9, ADAM10, and ADAM17 decreases moderately in these regions. ADAM12 and ADAM13 are weakly expressed in the central epithelium and the lens epithelium, and are not detectable from E14 onward. ADAM22 and ADAM23 are expressed in the central epithelium, the lens epithelium and the equatorial lens epithelium at E5 and decrease gradually afterwards in the same regions. At E16, only weak ADAM9, ADAM10 and ADAM17 signals are found in the anterior lens epithelium. The changing spatiotemporal expression of the seven ADAMs suggests a regulatory role for these molecules during chicken lens development.     

Metabolic engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for 1-butanol biosynthesis through the inverted aerobic fatty acid β-oxidation pathway

The basic reactions of the clostridial 1-butanol biosynthesis pathway can be regarded to be the inverted reactions of the fatty acid β-oxidation pathway. A pathway for the biosynthesis of fuels and chemicals was recently engineered by combining enzymes from both aerobic and anaerobic fatty acid β-oxidation as well as enzymes from other metabolic pathways. In the current study, we demonstrate the inversion of the entire aerobic fatty acid β-oxidation cycle for 1-butanol biosynthesis. The constructed markerless and plasmidless Escherichia coli strain BOX-3 (MG1655 lacI ^Q attB-P _trc-ideal-4-SD_φ10-adhE(Glu568Lys) attB-P _trc-ideal-4-SD_φ10-atoB attB-P _trc-ideal-4-SD_φ10-fadB attB-P _trc-ideal-4-SD_φ10-fadE) synthesises 0.3–1 mg 1-butanol/l in the presence of the specific inducer. No 1-butanol production was detected in the absence of the inducer.     

Expression patterns of the ADAMs in early developing chicken cochlea

Members of the ADAM (a disintegrin and metalloprotease) family are type I transmembrane proteins involved in biological processes of proteolysis, cell adhesion, cell–matrix interaction, as well as in the intracellular signaling transduction. In the present study, expression patterns of seven members of the ADAM family were investigated at the early stages of the developing cochlea by in situ hybridization. The results show that each individual ADAM is expressed and regulated in the early developing cochlea. ADAM9, ADAM10, ADAM17, and ADAM23 are initially and widely expressed in the otic vesicle at embryonic day 2.5 (E2.5) and in the differential elements of the cochlear duct at E9, while ADAM12 is expressed in acoustic ganglion cells at E7. ADAM22 is detectable in cochlear ganglion cells as early as from E4 and in the basilar papilla from E7. Therefore, the present study extends our previous results and suggests that ADAMs also play a role in the early cochlear development.     

The isolated N-terminal domains of TIMP-1 and TIMP-3 are insufficient for ADAM10 inhibition

ADAM (a disintegrin and metalloproteinase) 10 is a key member of the ADAM family of disintegrin and metalloproteinases which process membrane-associated proteins to soluble forms in a process known as 'shedding'. Among the major targets of ADAM10 are Notch, EphrinA2 and CD44. In many cell-based studies of shedding, the activity of ADAM10 appears to overlap with that of ADAM17, which has a similar active-site topology relative to the other proteolytically active ADAMs. The tissue inhibitors of metalloproteinases, TIMPs, have proved useful in the study of ADAM function, since TIMP-1 inhibits ADAM10, but not ADAM17; however, both enzymes are inhibited by TIMP-3. In the present study, we show that, in comparison with ADAM17 and the MMPs (matrix metalloproteinases), the N-terminal domains of TIMPs alone are insufficient for the inhibition of ADAM10. This knowledge could form the basis for the design of directed inhibitors against different metalloproteinases.     

Selective modulation of integrin-mediated cell migration by distinct ADAM family members

A disintegrin and a metalloprotease (ADAM) family members have been implicated in many biological processes. Although it is recognized that recombinant ADAM disintegrin domains can interact with integrins, little is known about ADAM-integrin interactions in cellular context. Here, we tested whether ADAMs can selectively regulate integrin-mediated cell migration. ADAMs were expressed in Chinese hamster ovary cells that express defined integrins (alpha4beta1, alpha5beta1, or both), and cell migration on full-length fibronectin or on its alpha4beta1 or alpha5beta1 binding fragments was studied. We found that ADAMs inhibit integrin-mediated cell migration in patterns dictated by the integrin binding profiles of their isolated disintegrin domains. ADAM12 inhibited cell migration mediated by the alpha4beta1 but not the alpha5beta1 integrin. ADAM17 had the reciprocal effect; it inhibited alpha5beta1- but not alpha4beta1-mediated cell migration. ADAM19 and ADAM33 inhibited migration mediated by both alpha4beta1 and alpha5beta1 integrins. A point mutation in the ADAM12 disintegrin loop partially reduced the inhibitory effect of ADAM12 on cell migration on the alpha4beta1 binding fragment of fibronectin, whereas mutations that block metalloprotease activity had no effect. Our results indicate that distinct ADAMs can modulate cell migration mediated by specific integrins in a pattern dictated, at least in part, by their disintegrin domains.     

Quantitative and dynamic expression profile of premature and active forms of the regional ADAM proteins during chicken brain development

The ADAM (A Disintegrin and Metalloprotease) family of transmembrane proteins plays important roles in embryogenesis and tissue formation based on their multiple functional domains. In the present study, for the first time, the expression patterns of the premature and the active forms of six members of the ADAM proteins — ADAM9, ADAM10, ADAM12, ADAM17, ADAM22 and ADAM23 — in distinct parts of the developing chicken brain were investigated by quantitative Western blot analysis from embryonic incubation day (E) 10 to E20. The results show that the premature and the active forms of various ADAM proteins are spatiotemporally regulated in different parts of the brain during development, suggesting that the ADAMs play a very important role during embryonic development.     

Functional classification of ADAMs based on a conserved motif for binding to integrin alpha 9beta 1: implications for sperm-egg binding and other cell interactions

ADAMs (a disintegrin and metalloproteases) are members of the metzincin superfamily of metalloproteases. Among integrins binding to disintegrin domains of ADAMs are alpha(9)beta(1) and alpha(v)beta(3), and they bind in an RGD-independent and an RGD-dependent manner, respectively. Human ADAM15 is the only ADAM with the RGD motif in the disintegrin domain. Thus, both integrin alpha(9)beta(1) and alpha(v)beta(3) recognize the ADAM15 disintegrin domain. We determined how these integrins recognize the ADAM15 disintegrin domain by mutational analysis. We found that the Arg(481) and the Asp-Leu-Pro-Glu-Phe residues (residues 488-492) were critical for alpha(9)beta(1) binding, but the RGD motif (residues 484-486) was not. In contrast, the RGD motif was critical for alpha(v)beta(3) binding, but the other residues flanking the RGD motif were not. As the RX(6)DLPEF alpha(9)beta(1) recognition motif (residues 481-492) is conserved among ADAMs, except for ADAM10 and 17, we hypothesized that alpha(9)beta(1) may recognize disintegrin domains in all ADAMs except ADAM10 and 17. Indeed we found that alpha(9)beta(1) bound avidly to the disintegrin domains of ADAM1, 2, 3, and 9 but not to the disintegrin domains of ADAM10 and 17. As several ADAMs have been implicated in sperm-oocyte interaction, we tested whether the functional classification of ADAMs, based on specificity for integrin alpha(9)beta(1), applies to sperm-egg binding. We found that the ADAM2 and 15 disintegrin domains bound to oocytes, but the ADAM17 disintegrin domain did not. Furthermore, the ADAM2 and 15 disintegrin domains effectively blocked binding of sperm to oocytes, but the ADAM17 disintegrin domain did not. These results suggest that oocytes and alpha(9)beta(1) have similar binding specificities for ADAMs and that alpha(9)beta(1), or a receptor with similar specificity, may be involved in sperm-egg interaction during fertilization. As alpha(9)beta(1) is a receptor for many ADAM disintegrins and alpha(9)beta(1) and ADAMs are widely expressed, alpha(9)beta(1)-ADAM interaction may be of a broad biological importance.     

Glycolysis and the regulation of cryoprotectant synthesis in liver of the freeze tolerant wood frog

Summary Wood frogs,Rana sylvatica, were sampled after freezing at –4°C (a short time course from 2 to 70 min after the appearance of the freezing exotherm) and thawing (20 h at 3°C after 70 min of freezing) and the regulation of liver glycolysis with respect to cryoprotectant glucose synthesis was examined. Within 5 min of the initiation of freezing, cryoprotectant concentrations in blood and liver had begun to increase. This was correlated with a rapid rise in the levels of hexose monophosphates in liver, including a 2.5 fold increase in glucose-6-P and 10 fold rise in fructose-6-P contents within the first 5 min post-exotherm. Contents of fructose-1,6-P₂, fructose-2,6-P₂, triose phosphates, P-enolpyruvate, and pyruvate did not significantly change over the course of freezing. Thawing sharply reduced the levels of hexose monophosphates in liver but raised P-enolpyruvate content by 2.3 fold. Changes in the contents of glycolytic intermediates over the freeze/thaw course are consistent with an inhibitory block of glycolysis at phosphofructokinase during freezing in order to facilitate a rapid glycogenolysis and production of cryoprotectant; during thawing, however, glycolysis appears to be inhibited at the level of pyruvate kinase.Possible regulatory control of cryoprotectant synthesis by covalent modification of liver glycolytic enzymes was examined. Glycogenolysis during freezing was facilitated by an increase in the percentage of glycogen phosphorylase in the activea (phosphorylated) form and also by an increase in the total amount (a+b) of enzyme expressed. For phosphofructokinase, kinetic changes as a result of freezing included a 40% reduction inK _m for fructose-6-P, a 60% decrease inK _a for fructose-2,6-P₂, and a 2 fold increase in I₅₀ for ATP. These changes imply a freezing-induced covalent modification of the enzyme but are not, apparently, the factors responsible for inhibition of glycolytic flux at the phosphofructokinase locus during glucose synthesis. Kinetic parameters of pyruvate kinase were not altered over the freeze/thaw course.     

Analysis of mutations affecting the dissmilation of galactitol (dulcitol) in Escherichia coli K 12.

Summary Three mutations clustered at 45.5 min of the genetic map of E. coli K12 have been shown previously (Lengeler, 1975a) to affect specifically galactitol transport via an enzyme II-complex^Gat (gatA) of the PEP dependent phosphotransferase system and a soluble, NAD dependent dehydrogenase (gatD). In the present report data are given further supporting the existence of a gat operon, made up by a control gene gatC and the structural genes gatA and gatD. The enzyme II-complex^Gat is shown to catalyze the formation of galactitol-1-P and the dehydrogenase to catalyze the reversible conversion of galactitol-1-P and D-tagatose-6-P. Loss of a phosphofructokinase activity controlled by the gene pfkA prevents growth on galactitol and concomitantly the formation of D-tagatose-1,6-P₂, while the suppressing mutation pfkB-1 restores a phosphofrucokinase activity and growth on galactitol.As shown further the erratic growth behaviour of E. coli K12, B and C on galactitol is apparently due to a temperature sensitive ketose-bis-phosphate aldolase inactive at temperatures >35° C. This enzyme reacts with D-tagatose-1,6-P₂ and to a lesser extent with D-fructose-1,6-P₂ and thus is able to suppress fda mutations. It is controlled by a new gene locus kba located within 1 min of the marker argG, remoted from the gat operon and the gene fda. Galactitol dissimilation in E. coli K12 thus seems to be via galactitol-1-P-D-tagatose-6-P-D-tagatose-1,6-P₂ to dihydroxyacetone-P+glyceraldehyde-P, controlled by the genetic loci gatC A D, pfkA, pfkB-1 and kba respectively.     

Sequence-specific interaction between the disintegrin domain of mouse ADAM 3 and murine eggs: role of beta1 integrin-associated proteins CD9, CD81, and CD98

ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-alpha6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-alpha6 mAb, or by mAbs against either the alphav or beta3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other beta1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg beta1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface "tetraspan web" facilitates fertilization and that it may do so by fostering ADAM-integrin interactions.     

Studies on the Formation and the Degradation of Glucose 1,6-Bisphosphate in the Beef Liver Homogenate

Four kinds of the enzyme reactions have been reported for the synthesis of Glc-1,6-P₂. However, any activity of Glc-1-P dismutase and phosphoglucokinase was not observed in the beef liver homogenate. When the liver homogenate was incubated with Glc-1-P and Fru-1,6-P₂, a significant amount of Glc-1,6-P₂ was formed. The Glc-1,6-P₂ synthesis activity from Glc-1-P and Fru-1,6-P₂ was caused by the action of phosphoglucomutase present in the liver homogenate. The most remarkable activity for Glc-1,6-P₂ synthesis was observed when the homogenate was incubated with Glc-1-P and glycerate-1,3-P₂. The Glc-1,6-P₂ synthesis activity from Glc-1-P and glycerate-1,3-P₂ was separated from the major peak of phosphoglucomutase activity by DEAE-Sephadex chromatography. The peak of Glc-1,6-P₂ synthesis activity, however, still retained phosphoglucomutase activity.

Glc-1,6-P₂ phosphatase activity was mainly observed in the mitochondria and microsome fraction. The properties of Glc-1,6-P₂ phosphatase were differentiated from those of acid phosphatase and Glc-6-P phosphatase.