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PubMed2004

Effects of cavity-forming mutations on the internal dynamics of azurin.

The effects of two single-point cavity-forming mutations, F110S and I7S, on the internal dynamics of azurin from Pseudomonas aeruginosa were probed by the phosphorescence emission of Trp-48, deeply buried in the compact hydrophobic core of the macromolecule. Changes in flexibility of the protein matrix around the chromophore were monitored by the intrinsic phosphorescence lifetime (tau(0)) whereas more general effects on structural fluctuations were deduced from the phosphorescence acrylamide quenching rate constant (k(q)), which measures the diffusion of the solute through the protein fold. The results show a spectacular, 4-5 orders of magnitude, increase of k(q) emphasizing that large amplitude structural fluctuations permitting acrylamide migration to the protein core have been drastically enhanced in each azurin mutant. The large, 12-15 kcal/mol, decrease in the activation enthalpy associated to k(q) suggests that the rate enhancement is caused, rather than through a generalized increase of protein flexibility, by the elimination of an inner barrier to the diffusion process. According to tau(0) the chromophore environment is more fluid with I7S but strikingly more rigid with F110S, demonstrating that when internal cavities are formed local effects on the mobility at the mutation site are unpredictable. Both tau(0) and k(q) reveal a structure tightening role of bound Cd(2+) that correlates with the increase in stability from apo- to holo-azurin. While these alterations in internal dynamics of azurin do not seem to play a role on electron transfer through the central region, the enhanced migration of acrylamide emphasizes that cavities may be critical for the rapid diffusion of substrates to buried, solvent inaccessible sites of enzymes.

Acrylamide↗
PubMed1998

ATP sensitive tryptophans of hsp90.

The nature of the interaction between the nucleotide ATP and hsp90 was investigated by observing fluorescence quenching of the four tryptophan residues in hsp90 as a function of quencher type and temperature. ATP and acrylamide quench the fluorescence from tryptophan free in solution principally by static and collisional mechanisms, respectively. Acrylamide quenching of tryptophan fluorescence in hsp90 is also principally collisional and identifies two classes of residues, one readily accessible to quenching the other less accessible. ATP quenching of tryptophan fluorescence in hsp90 is more complex exhibiting no overall preferred mechanism. However, ATP competitively inhibits acrylamide quenching of the readily accessible class of tryptophan residues by static quenching with the quenching constant providing an upper limit for the ATP dissociation constant. The ATP-free tryptophan dissociation constant is more than a factor of three larger than that for ATP-hsp90 suggesting that the ATP-hsp90 interaction is specific. The static quenching of tryptophan fluorescence in hsp90 by ATP implies that the nucleotide binds in close proximity to one or more of the tryptophan residues.

Acrylamides↗
PubMedDate not supplied

Temperature sensitization of liposomes using copolymers of N-isopropylacrylamide.

To obtain liposomes which release the contents in response to ambient temperature, liposomes modified with copolymers of N-isopropylacrylamide with varying lower critical solution temperatures have been designed. Poly(N-isopropylacrylamide-co-acrylamide)s with various compositions were synthesized by free-radical copolymerization. The lower critical solution temperature of the polymer increased with increasing acrylamide content in the polymer. Poly(N-isopropylacrylamide-co-acrylamide-co-N, N-didodecylacrylamide)s were also prepared via the same method as the thermosensitive polymers having anchor groups to the liposome membrane. Calcein-loaded dioleoylphosphatidylethanolamine/egg yolk phosphatidylcholine (6:4, w/w) liposomes were coated with these polymers by incubating the liposomes with aqueous solutions of the polymers. The liposomes hardly released the contents below the lower critical solution temperature of the polymer, but the release was greatly enhanced above that temperature. The liposomes were also made from a mixture of the same lipids and the polymer. The liposome revealed a more drastic release property than the liposomes prepared by the incubation with the polymer solution, because the polymer chains were bound on both surfaces of the membrane. The close relationship between lower critical solution temperatures of the polymers and temperature regions where enhancement of the release from the polymer-fixed liposomes demonstrates that the release was triggered by alteration of the polymers from a hydrophilic state to a hydrophobic state occurring at their lower critical solution temperatures.

Acrylamides↗
PubMed1980

Correlation between activity and dynamics of the protein matrix of phosphorylase b.

Quenching of the tryptophan fluorescence of phosphorylase b was studied by using iodide and acrylamide. Steady-state measurements indicated that all indole side chains were accessible to the nonionic quencher, although only 3 out of the total of 12 residues could be quenched by I-. From Stern--Volmer plots and the fluorescence lifetime data, it was concluded that the quenching was mainly of dynamic character. The value of the collisional quenching rate constant was found to be an order of magnitude less than that obtained in the case of fully exposed tryptophans. The relatively high activation energy, 30.9 kJ/mol, of the diffusion-controlled process and the value of the activation entropy suggest that the diffusion takes place in a fluctuating, structured medium. In spite of the application of sensitive fluorescent techniques, no gross conformational changes were found in the presence of acrylamide. However, the catalytic rate of the glycogen synthesis was decreased with the residual activity of the enzyme, proportional to the concentration of the probe. Binding of activator (AMP) and substrates (glucose 1-phosphate and glycogen) was found to be unaffected by acrylamide in concentrations applied (0--0.8 M). In a similar manner, activation enthalpy did not change in the presence of the quencher either. The complete reversibility of both activity inhibition and fluorescence quenching ruled out the irreversible denaturation of the enzyme or the covalent modification of any of the functional groups. We concluded that a model, suggesting the cross-correlation of activity and fluctuation, was consistent with the experimental findings.

Acrylamides↗
PubMed2000

DNA sequence dependent and independent conformational changes in multipartite operator recognition by lambda-repressor.

Binding of regulatory proteins to multipartite DNA binding sites often occurs with protein-protein interaction, resulting in cooperative binding. The operators of bacteriophage lambda have several pairs of repressor binding sites (O(R)1-O(R)2, O(R)2-O(R)3, O(L)1-O(L)2, and O(L)2-O(L)3) separated by a variable number of base pairs, and thus, bacteriophage lambda is a model system for studying multipartite operator recognition by DNA-binding proteins. Near-UV circular dichroism spectra show that the DNA is distorted in O(R)1-O(R)2 and O(L)2-O(L)3 but much less so in O(R)2-O(R)3. Upon titration of lambda-repressor with single-operator sites O(R)1, O(R)2, and O(R)3, it was observed that the tryptophan fluorescence quenches to different degrees, suggesting different conformations of the protein in the three DNA-protein complexes. Acrylamide quenching of tryptophan fluorescence of lambda-repressor bound to these single operators also shows different Stern-Volmer constants, supporting the above conclusions. Titration of lambda-repressor with oligonucleotides containing pairs of operator sites also causes different degrees of fluorescence quenching. In particular, fluorescence quenching induced by O(R)1-O(R)2 binding is less than the quenching induced by either of the single operators alone, suggesting additional conformational changes upon establishment of protein-protein contact. Stern-Volmer constants obtained from acrylamide quenching of tryptophan fluorescence of lambda-repressor bound cooperatively to pairs of operator sites are different from those of the single-operator-site-bound repressors. For example, O(R)2-O(R)3-bound repressor has significantly higher acrylamide quenchable components than either of the O(R)2- or O(R)3-bound proteins, again suggesting additional conformational changes upon establishment of protein-protein contact. We conclude that the strategy of recognition of multipartite operator by lambda-repressor is complex and varied, involving conformational changes in both DNA and protein that are determined by the separation of the binding sites as well as the nucleic acid sequence.

Acrylamide↗
PubMed1999

Role of the cytoskeleton in adhesion stabilization of human colorectal carcinoma cells to extracellular matrix components under dynamic conditions of laminar flow.

Adhesion stabilization of malignant cells in the microcirculation is necessary for successful metastasis formation. The adhesion of colon carcinoma cells to microcirculation extracellular matrix (ECM) components is mediated, in part, by integrins that can be intracellularly linked to cytoskeletal proteins. Thus the functional status of at least certain integrins can be regulated by complex interactions with cytosolic, cytoskeletal and membrane-bound proteins. Wall shear stress caused by fluid flow also influences cellular functions, such as cell morphology, cytoskeletal arrangements and cell signaling. Using a parallel plate laminar flow chamber dynamic adhesion of human HT-29 colon carcinoma cells to collagen was investigated and compared with cell adhesion under static conditions. Cells were pretreated with cytochalasin D, nocodazole, colchicine or acrylamide to disrupt actin filaments, microtubules or intermediate filaments. Disruption of actin filaments completely inhibited all types of adhesive interactions. In contrast, impairment of tubulin polymerization or disruption of intermediate filaments resulted in different effects on static and dynamic adhesion. Treatment with acrylamide did not interfere with dynamic cell adhesion, whereas under static conditions it partially reduced adhesion rates. Under dynamic conditions increased initial adhesive interactions between HT-29 cells and collagen were found after disruption of microtubules, and the adherent cells demonstrated extensive crawling on collagen surfaces. In contrast, under static adhesion disrupting microtubules did not affect cell adhesion rates. Cytochalasin D and acrylamide were found to inhibit Tyr-phosphorylation of FAK and paxillin, whereas microtubule disrupting agents at low but not high concentrations increased phosphorylation of these focal adhesion proteins. Our results revealed that cytoskeletal components appear to be involved in adhesion stabilization of HT-29 cells to ECM components, and hydrodynamic shear forces modulate this involvement. Tyr-phosphorylation of focal adhesion proteins, such as paxillin and FAK, appears to be a part of this cytoskeleton-mediated process.

Acrylamide↗
PubMed1999

Fluorescence and circular dichroism spectroscopic studies on bovine lactoperoxidase.

Intrinsic steady-state fluorescence of lactoperoxidase (LPO) and its ligand-bound complexes has been characterized as a structural probe of its structure in solution. On excitation at 295 nm, a broad emission maximum is observed around 338 nm for LPO and for its ligand-bound complexes. The quantum yield is 0.0185 +/- 0.0005 for LPO and indicates tryptophan-->heme energy transfer. Tryptophan residues are located away from heme and are approximately equally distributed among hydrophobic and hydrophilic environments. From Förster resonance energy transfer equations, the "average" distance between tryptophans and heme within the enzyme is computed to be 25.1 +/- 0.2 A. These fluorescence properties are consistent with the recent theoretical three-dimensional model for LPO and reveal that Trp337 and Trp404 dominate the intrinsic fluorescence, and together contribute approximately 64% of the observed intensity. The effects of the denaturing agents guanidine hydrochloride and urea on the intrinsic fluorescence of LPO and CD of the backbone amide chromophores have been examined. The considerably red shifted emission maximum at 356 nm indicates that tryptophans, buried in the hydrophobic environment, are exposed to the solvent on denaturation. A simple two-state transition between the native and denatured forms of the protein has been used to explain the results. [Denaturant]1/2 approximately 5.5 M, determined from both these experiments, indicates that LPO is relatively stable toward the denaturing agents. Quenching studies using. I-, Cs+ and polar neutral acrylamide are consistent with this picture. Acrylamide can penetrate the protein matrix. It is an efficient quencher and the quenching process is essentially homogeneous with all the tryptophans being accessible. Cs+ ion is a very inefficient quencher but the iodide ion shows the quenching process to be predominantly heterogeneous with widely differing tryptophan accessibility. The Stern-Volmer constants deduced are Ksv = 8.4 +/- 1.4 M-1 and Ksv = 4.05 +/- 0.65 M-1 for acrylamide and iodide quenching, respectively. The fractional accessibility, fa, deduced is fa = 0.52 +/- 0.03 for iodide quenching.

Acrylamide↗
PubMed2004

Rhodococcus pyridinovorans MW3, a bacterium producing a nitrile hydratase.

Rhodococcus pyridinovorans MW3 was isolated from an arable land of manioc from the Congo for its ability to transform acrylonitrile to acrylamide. This strain contains a cobalt nitrile hydratase (NHase) showing high sequence homology with NHases so far described. The specific NHase activity was 97 U mg(-1) dry wt. NHase production by R. pyridinovorans MW3 was urea and Co-dependent. The NHase was active for acrylamide up to 60% (w/v) indicating its potential for acrylamide production.

Acrylamide↗
PubMed1996

Regioselective immobilization of short oligonucleotides to acrylic copolymer gels.

Four types of polyacrylamide or polydimethyl-acrylamide gels for regioselective (by immobilization at the 3' end) of short oligonucleotides have been designed for use in manufacturing oligonucleotide microchips. Two of these supports contain amino or aldehyde groups in the gel, allowing coupling with oligonucleotides bearing aldehyde or amino groups, respectively, in the presence of a reducing agent. The aldehyde gel support showed a higher immobilization efficiency relative to the amino gel. Of all reducing agents tested, the best results were obtained with a pyridine-borane complex. The other supports are based on an acrylamide gel activated with glutaraldehyde or a hydroxyalkyl-functionalized gel treated with mesyl chloride. The use of dimethylacrylamide instead of acrylamide allows subsequent gel modifications in organic solvents. All the immobilization methods are easy and simple to perform, give high and reproducible yields, allow long durations of storage of the activated support, and provide high stability of attachment and low non-specific binding. Although these gel supports have been developed for preparing oligonucleotide microchips, they may be used for other purposes as well.

Acrylamides↗
PubMed1999

Synthesis of polyacrylamides N-substituted with PNA-like oligonucleotide mimics for molecular diagnostic applications.

Two types of oligonucleotide mimics relative to peptide nucleic acids (PNAs) were tested as probes in nucleic acid hybridisation assays based on polyacrylamide technology. One type of mimic oligomers represented a chimera constructed of PNA and phosphono-PNA (pPNA) monomers, and the other one contained pPNA residues alternating with PNA-like monomers on the base of trans -4-hydroxy-L-proline (HypNA). A chemistry providing efficient and specific covalent attachment of these DNA mimics to acrylamide polymers using a convenient approach based on the co-polymerisation of acrylamide and some reactive acrylic acid derivatives with oligomers bearing 5'- or 3'-terminal acrylamide groups has been developed. A comparative study of polyacrylamide conjugates with oligonucleotides and mimic oligomers demonstrated the suitability and high potential of PNA-pPNA and HypNA-pPNA chimeras as sequence-specific probes in capture and detection of target nucleic acid fragments to serve current forms of DNA arrays.

Acrylamide↗
PubMed1989

The interaction of Abrus precatorius agglutinin with saccharides as analyzed by fluorescence spectroscopy.

The binding of saccharides to Abrus precatorius agglutinin (APA) was analyzed by fluorescence spectroscopy. Upon binding of specific saccharides, the fluorescence emission maximum of APA (338 nm) shifted to shorter wavelength by 5 nm, owing to the change in the environment of tryptophan. By analyzing the change in the fluorescence intensity at 338 nm as a function of concentration of saccharides, the association constants for binding of saccharides to APA were determined. The results suggest that in the saccharide binding site on each B-chain of APA, there may be a site which interacts with the saccharide residue linked to galactopyranoside at the non-reducing end, in addition to the site which recognizes the galactopyranosyl residue. Fluorescence quenching data indicate that 8 out of 24 tryptophans in APA are located at or near the surface of the protein molecule and are available for quenching with both KI and acrylamide, and 10 tryptophans are involved in the environment to which acrylamide has access but KI does not. Binding of lactose to APA reduced by 4 the number of tryptophan residues accessible to quenchers. Based on the results, it is suggested that the tryptophan residues at the saccharide binding site on each B-chain of APA are present on the surface of the APA molecule, and they are shielded from quenching by KI and acrylamide upon binding with specific saccharides.

Acrylamides↗
PubMed1983

The accessibility of the active site and conformation states of the beta 2 subunit of tryptophan synthase studied by fluorescence quenching.

The rate of quenching of the fluorescence of pyridoxal 5'-phosphate in the active site of the beta 2 subunit of tryptophan synthase from Escherichia coli was measured to estimate the accessibility of the coenzyme to the small molecules iodide and acrylamide. The alpha subunit and the substrate L-serine substantially reduced the quenching rate. For iodide, the order of decreasing quenching was: Schiff's base of N alpha-acetyl-lysine with pyridoxal 5'-phosphate greater than holo beta 2 subunit greater than holo alpha 2 beta 2 complex approximately equal to holo beta 2 subunit + L-serine greater than holo alpha 2 beta 2 complex + L-serine. The coenzyme in the beta 2 subunit is apparently freely accessible to both iodide and acrylamide (kappa approximately equal to 2 X 10(9) M-1 s-1), but the alpha subunit and L-serine decrease the rate by factors of 2-5. Quenching of the fluorescence of the single tryptophan residue of the beta 2 subunit revealed that the apo and holo forms exist in different states, whereas the alpha subunit stabilizes a third conformation. As the alpha subunit binds to the beta 2 subunit, the tryptophan residue, which is within 2.2 nm of the active site of the beta 2 subunit, probably rotates with respect to the plane of the ring of the coenzyme, such that fluorescence energy transfer from tryptophan to pyridoxal phosphate is greatly reduced. The alpha subunit strongly protects the active-site ligand indole propanol phosphate from quenching with acrylamide, consistent with the active site being deep in a cleft in the protein. Iodide induces dissociation of the holo alpha 2 beta 2 complex [E. W. Miles & M. Moriguchi (1977) J. Biol. Chem. 252, 6594-6599]. The effect of iodide on the fluorescence properties of holo alpha 2 beta 2 complex allows us to estimate an upper limit for the dissociation constant for the alpha 2 beta 2 complex of 10(-8) M, in the absence of iodide.

Acrylamides↗
PubMed2004

Conformational changes of beta-lactoglobulin in sodium bis(2-ethylhexyl) sulfosuccinate reverse micelles. A fluorescence and CD study.

The effect of beta-lactoglobulin encapsulation in sodium bis(2-ethylhexyl) sulfosuccinate reverse micelles on the environment of protein and on Trp was analysed at different water contents (omega0). CD data underlined the distortion of the beta-sheet and a less constrained tertiary structure as the omega0 increased, in agreement with a concomitant red shift and a decrease in the signal intensity obtained in steady-state fluorescence measurements. Fluorescence lifetimes, evaluated by biexponential analysis, were tau1 = 1.28 ns and tau2 = 3.36 ns in neutral water. In reverse micelles, decay-associated spectra indicated the occurrence of important environmental changes associated with omega0. Bimolecular fluorescence quenching by CCl4 and acrylamide was employed to analyse alterations in the accessibility of the two Trp residues in beta-lactoglobulin, induced by changes in omega0. The average bimolecular quenching constant was found not to depend on omega0, confirming the insolubility of this quencher in the aqueous interface, while increases with omega0. The drastic decrease with omega0 of kq, associated with the longest lifetime kq2(CCl4), comparatively to the increase of kq2(acrylamide), emphasizes the location of beta-lactoglobulin in the aqueous interfacial region especially at omega0> or = 10. The fact that (omega0 = 30) >> kq2(acrylamide) (water) also confirms the important conformational changes of encapsulated beta-lactoglobulin.

Acrylamide↗
PubMed1999

An investigation into the electro-physical characteristics of microbial cells during the metabolism of toxic compounds under conditions of limited O2 availability.

The purpose of the work reported here was to experimentally clarify the interconnection between changes in the electro-physical characteristics of microbial suspensions and processes of the metabolism of certain toxic compounds (acrylamide and p-nitrophenol (PNP)) in cells containing enzyme systems of the initial metabolism of these compounds. In this work, we used cells of two strains, Acinetobacter calcoaceticum A-122 and Brevibacterium sp. 13PA, which are capable of utilising PNP and acrylamide, respectively, as the sole source of carbon. Suspensions of these cells exhibited appreciable decreases in the magnitude of the electro-optical signal when the microbial metabolism of PNP and acrylamide was inhibited under conditions of limited O2 supply. This attests to a relationship between the electro-physical characteristics of microbial suspensions and cellular metabolic reactions and also to the negligibly small effect that the non-specific interaction of substrate with the cells has on the suspensions' electro-optical properties.

Acinetobacter calcoaceticus↗
PubMed1979

Bacitracin production by whole cells immobilized in polyacrylamide gel.

Whole cells of Bacillus sp., bacitracin-producing bacteria, were immobilized in polyacrylamide gel prepared by using 5% total acrylamide (95% acrylamide monomer and 5% N, N'-methylenebis acrylamide). Bacitracin production by the immobilized whole cells was examined by using various fermentation media. In starch-bouillon medium, the initial activity of immobilized whole cells for bacitracin production was 20 to 25% that of an equivalent amount of washed cells. With successive utilizations in 1% peptone as reaction medium, activity of the immobilized whole cells increased gradually and reached a steady-state maximum having a value of 80 to 90% of the activity obtained initially with washed cells, whereas the washed cells lost most activity when utilized successively. What appeared to be growth of the bacteria in the gel was observed by electron microscopy. Therefore, the activation of immobilized whole cells during successive utilizations apparently resulted from the growth of whole cells in the gel, especially at the gel surface.

Acrylamides↗
PubMed1972

Conduction in unmyelinated fibres in experimental neuropathy.

Conduction velocity of autonomic unmyelinated fibres has been measured in the cervical sympathetic trunk of normal rats, and in rats intoxicated by acrylamide or by isoniazid. The mean maximal conduction velocity in nerves from normal rats is 2·0 m/sec. There is no significant reduction in velocity of the unmyelinated fibres in nerves from intoxicated rats, although histological studies of the sural nerve confirmed severe degeneration of myelinated fibres in the same animals. It is shown that the amplitude of the compound nerve action potential is proportional to the resistance between the recording electrodes. If this is taken into account, there is no reduction in the amplitude of the monophasic action potential of unmyelinated fibres recorded from the cervical sympathetic trunk of intoxicated rats. The amplitude of the A component of the sural nerve compound action potential is markedly reduced in rats intoxicated by acrylamide or by isoniazid, but there is no significant reduction in the amplitude of the C component in the same nerve. It is concluded that in the rat an insignificant number of unmyelinated fibres of autonomic or dorsal root origin are affected in the neuropathy produced by acrylamide or isoniazid. The relevance of these findings to human neuropathies is discussed.

Acrylamides↗
PubMed2006

[Time dependent changes in antioxidation of rat serums and sciatic nerves caused by toxicology].

OBJECTIVE: To study the time dependent antioxidation changes of serum and sciatic nerve in rats intoxicated with acrylamide. METHODS: Male Wistar rats weighing 180 to 220 g were given acrylamide dissolved in physiological saline (40 mg/kg ip 3 days/week). The control groups received normal saline. The gait was observed and antioxidant indexes of rat serum and sciatic nerve were determined on 0, second, fourth, sixth, 10th week. RESULTS: With the extension of the intoxication period, compared with the control, the contents of glutathione in serum and sciatic nerve gradually decreased (P < 0.05; after 6 and 10 weeks to 92% and 77%; after 2, 4, 6 and 10 weeks to 92%, 82%, 67% and 66%); the levels of malondialdehyde gradually increased (P < 0.05; after 4, 6 and 10 weeks to 113%, 118% and 120%; after 4, 6 and 10 weeks to 153%, 167%, 174%); the abilities of the resistance to reactive oxygen species gradually decreased (P < 0.05; after 10 weeks to 82%; after 6 and 10 weeks to 76% and 71%); the activities of glutathione peroxidase gradually increased (P < 0.05; after 2, 4, 6 and 10 weeks to 122%, 130%, 160% and 124%; after 4, 6 and 10 weeks to 134%, 152% 164%); the activities of glutathione reductase increased at early stage (P < 0.01; after 4 and 6 weeks to 300% and 217%; after 4 weeks to 142%) and decreased later (P < 0.01; 6 and 10 weeks to 59% and 33% in sciatic nerve); the activities of superoxide dismutase increased primitively (P < 0.05; after 2 weeks to 110%; after 4 weeks to 124%) and decreased later (P < 0.05; after 10 weeks to 85% in serum). The changes of antioxidant indexes in serum and sciatic nerve according to gait score were similar. The level of MDA in serum was in high correlation (P < 0.01) with that in sciatic nerve. The regression coefficients were 0.99 and 0.96 according to the administration time and gait score respectively. CONCLUSION: The changes of the antioxidant indexes in serum and sciatic nerve of rat treated with acrylamide are time dependent. The changes in serum and sciatic nerve are similar but those in sciatic nerve are more remarkable.

Acrylamide↗
PubMed1978

Adhesion of chicken hepatocytes to polyacrylamide gels derivatized with N-acetylglucosamine.

Complex carbohydrates on the surfaces of eukaryotic cells are thought to participate in a wide variety of cell-cell interactions. A model system has therefore been developed to study these processes. In the present experiments, the ability of chicken hepatocytes to recognize and adhere to sugars covalently linked to polyacrylamide gels was investigated. The gels were snythesized by two methods. Type I gels were prepared from a co-polymer of an active ester of acrylic acid (N-succinimidyl acrylate), acrylamide, and bisacrylamide. The "activated" polyacrylamide gel was then treated with the desired ligand containing an amino group, such as 6-aminohexyl O- or S-glycoside. Type II gels were formed by treating similar ligands with acryloyl chloride, followed by co-polymerization of the resulting N-substituted acrylamide with acrylamide and N,N'-methylenebisacrylamide. These polyacrylamide derivatives offer many advantages for studies with intact cells. They are not toxic to any cell type studied, can be cast in any desired shape, are transparent and stable over a wide range of pH values, and contain no cationic and low to negligible levels of anionic charge (charged groups can be introduced if desired), and the polyacrylamide matrix is stable to common biological agents such as bacteria and enzymes. In addition, type I gels can be synthesized using a broad range of molecules containing amino groups, such as glycopeptides, proteins, etc. The hepatocytes were prepared by collagenase perfusion of intact chicken livers. The rate and extent of adhesion of the cells to the derivatized gels was determined by measuring lactate dehydrogenase in these cells. This enzyme was also used to assay viability and cell "leakiness." At 37 degrees C, 70 to 100% of the cells adhered within 60 min to gels derivatized with N-acetylglucosamine, i.e. gels derivatized with 6-aminohexyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (or the corresponding thioglycoside). By contrast, less than 5% of the cells adhered to polyacrylamide or to gels derivatized with 6-aminohexanol or the 6-aminohexyl glycosides of beta-D-glucose, beta-D-galactose, alpha-D-mannose, beta-D-maltose, beta-D-melibiose, beta-D-cellobiose, and (alpha or beta)-D-lactose. Kinetic studies with the chicken hepatocytes and N-acetylglucosamine gels showed that cell-gel binding was dependent upon Ca2+ and was decreased at low temperatures. Binding was inhibited by N-acetylglucosamine or by glycosides of this sugar, the most effective inhibitor being orosomucoid (alpha1-acid glycoprotein) pretreated with sialidase and beta-galactosidase. The cell surface receptor(s) involved in this interaction is not known, but may be related or identical to the chicken liver binding protein described by Lunney and Ashwell (Lunney, J., and Ashwell, G. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 341--343). The present results suggest that this model system should prove useful in delineating cell surface interactions with carbohydrates.

Acetylglucosamine↗