Quantitative measurements of the proton-motive force and its relation to steady state lactose accumulation in Escherichia coli.

1. Developing tail tendons from rats (19-day foetal to 126 days post partum) were examined by electron microscopy after staining for proteoglycan with a cationic copper phthalocyanin dye. Cuprolinic Blue, in a "critical electrolyte concentration" method. Hydroxyproline was measured on papain digests of tendons, from which glycosaminoglycuronans were isolated, characterized and quantified. 2. Mean collagen fibril diameters increased more than 10-fold with age according to a sigmoid curve, the rapid growth phase 2 being during 30-90 days after conception. Fibril periodicities were considerably smaller (50-55 nm) in phases 1 and 2 than in phase 3 (greater than 62 nm). 3. Dermatan sulphate is the main glycosaminoglycuronan in mature tendon. Chondroitin sulphate and hyaluronate preponderate in foetal tissue. 4. Proteoglycan was seen around but not inside collagen fibrils. Proteoglycan and collagen were quantified from electron micrographs. Their ratios behaved similarly to uronic acid/hydroxyproline and hyaluronate/hydroxyproline ratios, which decreased rapidly around birth, and then levelled off to a low plateau coincident with the onset of rapid growth in collagen fibril diameter. 5. Dermatan sulphate/hydroxyproline ratios suggest that the proteoglycan orthogonal array around the fibril is largely dermatan sulphate. In the foetus hyaluronate and chondroitin sulphate exceed that expected to be bound to collagen. 6. An inhibiting action of chondroitin sulphate-rich proteoglycan on fibril diameter growth is suggested. 7. The distributions of hyaluronate, chondroitin sulphate and dermatan sulphate are discussed in the light of secondary structures suggested to be present in hyaluronate and chondroitin sulphate, but not in dermatan sulphate.     

Structural studies on proteoglycan catabolism in rabbit articular cartilage explant cultures

Mature rabbit articular cartilage cultures have been used to study the catabolism of aggregating proteoglycan monomers in normal cartilage. During the first 4 days of culture, about 40% of monomers are degraded and lose the ability to bind to hyaluronate. The non-aggregating products (NAgg-PG) have been isolated and compared structurally and immunologically to aggregating monomers (Agg-PG) purified from fresh tissue. The results show that: (1) NAgg-PG are smaller, more heterogeneous in size and have a lower protein/glycosaminoglycan ratio than Agg-PG. (2) NAgg-PG and Agg-PG have a very similar chondroitin sulfate/keratan sulfate ratio. (3) NAgg-PG have 25-50% lower disulfide content than Agg-PG. (4) NAgg-PG have only about 20% of the reactivity of Agg-PG towards a monoclonal antibody (12-20/1-C-6) specific for the hyaluronate binding region of the core protein. These results provide further evidence that proteoglycan catabolism in cartilage explants involves proteolysis of core protein resulting in separation of the hyaluronate binding region from the glycosaminoglycan-rich regions.     

Solution conformation of glycosaminoglycans: assignment of the 300-MHz 1H-magnetic resonance spectra of chondroitin 4-sulphate, chondroitin 6-sulphate and hyaluronate, and investigation of an alkali-induced conformation change.

Complete assignments are given for the 1H nuclear magnetic resonance (NMR) spectra at 300 MHz of chondroitin 4-sulphate, chondroitin 6-sulphate and hyaluronate in deuterium oxide solution, supported by spin decoupling and computer simulation. Coupling constants and chemical shifts are as expected from spectra of the model glycosides, methyl beta-D-glucopyranosiduronate, methyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and methyl 2-acetamido-2-deoxy-beta-D-galactopyranoside, when allowance is made for systematic influences on chemical shifts of interglycosidic linkages and sulphate substitution. As reported elsewhere, addition of alkali causes the hyaluronate spectrum to sharpen considerably. This is taken to indicate that segmental motion is enhanced by disruption of some system of inter-residue bonding on ionisation of hydroxy groups. Concomitant changes in chemical shifts are seen mainly for H-2 of the glucuronate residue, and the CH3 and H-2 of the acetamidodeoxyglucose residue. Similar effects are not seen for chondroitin sulphates, either in line widths or chemical shifts. Comparison of the spectra of hyaluronate, chondroitin sulphates, and the model glycosides, indicates that proton chemical shifts are sensitive to the conformation differences between the polysaccharides in alkaline solution, but do not detect the differences in neutral solution that are known from NMR relaxation to be present. The altered configuration and/or substitution pattern of the acetamidodeoxyhexose residue in hyaluronate compared with chondroitin sulphates appears to have a critical influence on overall conformation in both alkaline and neutral solution.     

Changes in CD of hyaluronates and chondroitins upon periodate oxidation

Changes in conformation of hyaluronate and chondroitin sulfates following periodate oxidation were studied by CD. We monitored the progressive oxidation of these polymers by periodic acid at 4°C in pH 5.6 buffer. The negative CD band of hyaluronate at 208 nm decreased in intensity upon oxidation and changed its sign after 16 h of oxidation. In contrast, the 208-nm CD band of chondroitin sulfates decreased, but showed no change in sign even after 48 h of oxidation. A specific difference in solution conformation between hyaluronate and chondroitins may be responsible for the difference in oxidation-induced dichroic behavior. The results are discussed in terms of available x-ray diffraction analyses of these polymers.     

Secondary structure of hyaluronate in solution. A 1H-n.m.r. investigation at 300 and 500 MHz in [2H6]dimethyl sulphoxide solution.

The 1H-n.m.r. spectra of solutions in [2H6]dimethyl sulphoxide of the sodium salts of tetra-, hexa- and octa-saccharides prepared from hyaluronate by testicular-hyaluronidase digestion were examined at 300 and 500 MHz. The signals from hydroxy groups at positions 2 and 3 in the glucuronic acid moiety were assigned. Their chemical shifts and associated temperature-dependencies, as well as their coupling constants, depended on whether or not the uronic acid was at the non-reducing end. Deviations from the 'normal' pattern of hydroxy-group proton n.m.r. behaviour were attributable to participation in hydrogen bonds, either to the acetamido carbonyl oxygen atom or the pyranose ring oxygen atom of neighbouring N-acetylhexosamine moieties. A secondary structure, containing four different hydrogen bonds per trisaccharide unit of glucuronsyl-hexosaminyl-glucuronic acid, was demonstrated. This is the first complete and detailed secondary structure to be established for hyaluronate in any solvent. Hyaluronate is compared with chondroitin sulphate, dermatan sulphate, heparan sulphate and keratan sulphate in their potential to form secondary structures with features in common. The significance of the details of the structure to its overall stability, and the probability of their persistence into aqueous environments, are discussed. The presence of all or most of the secondary structure in glycosaminoglycuronans is correlated with a space-filling function in the tissue, and with a high carbohydrate content in the parent proteoglycan in the case of the chondroitin sulphates.     

Crystallization and some properties of chondroitinase from Arthrobacter aurescens.

A strain of Arthrobacter aurescens which secretes a large amount of chondroitinase into a culture broth, was isolated from soil. The chondroitinase was purified 380-fold over culture broth in 24% yield and crystallized. Some properties of the purified enzyme were studied and described: thermal stability (below 45 degrees), pH stability (pH 4.9 to 7.4), optimum temperature (50 degrees), and optimum pH (pH 6.0). Chrondroitin sulfate A and C, chondroitin, and hyaluronic acid were split by the enzyme but dermatan sulfate could not be. The initial rates of enzymic degradation of chondroitin sulfate C, chondroitin, and hyaluronic acid were 1.1, 1.95, and 3.2, respectively, compared to that of chondroitin sulfate A. When the enzyme was allowed to act on chondroitin sulfate A and C, the reducing power and the ultraviolet absorption at 232 nm increased proportionally to the decrease in viscosity of the substrate solution. Finally these substrates were degraded to the extent of 100% to disaccharides. By the enzyme action the main products from chondroitin sulfate A and C were deta 4,5-unsaturated disaccharides, which were identified as 2-acetamido-2-deoxy-3-O-(Beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose and 2-acet-amido-2-deoxy-3-O-(Beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose by paper chromatography, ultraviolet absorption spectroscophy, and infrared spectroscopy. Thus it is suggested that the chondroitinase is a chondroitin sulfate A and C lyase, one of the hyaluronate lyases (EC 4.2.99.1).     

The 13C-NMR spectra of hyaluronate and chondroitin sulphates. Further evidence on an alkali-induced conformation change

Complete assignments are given for the 13C NMR spectra of hyaluronate and chondroitin in deuterium oxide solution at 50.32 MHz. The assignments published earlier for chondroitin 4-sulphate and chondroitin 6-sulphate were largely confirmed but were found to need some revision in detail. Our conclusions for hyaluronate and the chondroitin sulphates were confirmed by off-resonance experiments based on the proton NMR assignments. The spectra for hyaluronate show line narrowing and chemical shift changes from neutral to alkaline solution which are consistent with, and clearer than, the effects reported earlier for the proton spectra. As before no such changes occur for the chondroitin sulphates. The suggested interpretation is in terms of a conformation change for hyaluronate which originates mainly in altered interaction energies across the 1 leads to 3 linkage with the results that motional freedom is enhanced above that of the parent hyaluronate and even above the chondroitin sulphates. This, and the evidence from a temperature effect, suggests that an additional potential energy minimum is made favourable in alkali so that the overall amplitudes of the bond oscillations are increased.     

Light-scattering study on the influence of link-glycoproteins and lysozyme on the hyaluronate molecular conformation in solution

The influence of link-glycoproteins and mammalian lysozyme on the configuration and size of the hyaluronate molecule in highly diluted solutions under physiological electrolytic and pH conditions was investigated by light-scattering techniques and confirmed by column chromatography, isopycnic flotation, and boundary centrifugation. It was consistently found that link-glycoproteins induce an increase in the basic structural dimensions of the hyaluronate molecule in solution. It was also found that this increase was reversed or prevented under the action of mammalian lysozyme. Changes in configuration of the hyaluronate molecule could be related to its aggregating capacity when the hyaluronate interacts with proteoglycan subunits. It is postulated that link-glycoproteins induce structural changes in the hyaluronate molecule that might improve its aggregating capacity while mammalian lysozyme prevents or regulates such improvement.     

The interactions of cell division protein FtsZ with guanine nucleotides

Prokaryotic cell division protein FtsZ, an assembling GTPase, directs the formation of the septosome between daughter cells. FtsZ is an attractive target for the development of new antibiotics. Assembly dynamics of FtsZ is regulated by the binding, hydrolysis, and exchange of GTP. We have determined the energetics of nucleotide binding to model apoFtsZ from Methanococcus jannaschii and studied the kinetics of 2'/3'-O-(N-methylanthraniloyl) (mant)-nucleotide binding and dissociation from FtsZ polymers, employing calorimetric, fluorescence, and stopped-flow methods. FtsZ binds GTP and GDP with K(b) values ranging from 20 to 300 microm(-1) under various conditions. GTP.Mg(2+) and GDP.Mg(2+) bind with slightly reduced affinity. Bound GTP and the coordinated Mg(2+) ion play a minor structural role in FtsZ monomers, but Mg(2+)-assisted GTP hydrolysis triggers polymer disassembly. Mant-GTP binds and dissociates quickly from FtsZ monomers, with approximately 10-fold lower affinity than GTP. Mant-GTP displacement measured by fluorescence anisotropy provides a method to test the binding of any competing molecules to the FtsZ nucleotide site. Mant-GTP is very slowly hydrolyzed and remains exchangeable in FtsZ polymers, but it becomes kinetically stabilized, with a 30-fold slower k(+) and approximately 500-fold slower k(-) than in monomers. The mant-GTP dissociation rate from FtsZ polymers is comparable with the GTP hydrolysis turnover and with the reported subunit turnover in Escherichia coli FtsZ polymers. Although FtsZ polymers can exchange nucleotide, unlike its eukaryotic structural homologue tubulin, GDP dissociation may be slow enough for polymer disassembly to take place first, resulting in FtsZ polymers cycling with GTP hydrolysis similarly to microtubules.     

Secondary structures of hyaluronate and chondroitin sulphates. A 1H n.m.r. study of NH signals in dimethyl sulphoxide solution.

1H n.m.r. spectra in [2H6]dimethyl sulphoxide of dodecyltrimethylammonium salts of chondroitin sulphates and hyaluronate, or sodium salts of oligomers from hyaluronate, showed unambiguous NH signals. The acetamido NH occurs in two different environments: environment I ('normal') in simple sugars, and environment II (hydrogen-bonded NH) appearing in tri- or tetrasaccharides, indicating a secondary structure in hyaluronate (and some chondroitin sulphates) involving a hydrogen-bonded acetamido NH.     

Characterization and macromolecular association of proteoglycans produced by pig arterial smooth muscle cells in culture

1. Medium and cell-layer proteoglycans from pig aorta smooth muscle cells in culture were compared. In both compartments, the main proteoglycans contained chondroitin sulfate-dermatan sulfate chains of 40 kDalton. 2. However, cell-layer proteoglycans differed from those of the medium by the presence of: (a) some small-size proteoglycans; (b) a greater amount of heparan sulfate; (c) chondroitin sulfate-dermatan sulfate enriched in iduronate and in 4 sulfate- (instead of 6 sulfate-) residues. 3. During dissociation-reassociation assays of arterial proteoglycans with exogenous hyaluronate or "aggregate" proteoglycans, the in vitro formation of complexes appeared to involve inter-associations between proteoglycans molecules, in addition to aggregation with hyaluronate.     

The binding of chondroitin 6-sulfate to plasma low density lipoprotein

The interaction in vitro of several sulfated glycosaminoglycans with low density lipoproteins (LDL) has been studied. Chondroitin 6-sulfate and heparin were the only ones to produce turbidity when added to LDL in presence of Ca2+. However, when these two glycosaminoglycans were applied to LDL-affinity columns in presence of Ca2+, only chondroitin 6-sulfate was retained. Partially desulfated chondroitin 6-sulfate was not retained on LDL-affinity column, indicating the relevance of sulfate groups in the binding of LDL. Since chondroitin 4-sulfate and heparin, with a sulfate content respectively equal to and greater than that of chondroitin 6-sulfate, are not retained on LDL-affinity columns, the factors relevant to the binding of LDL are probably the conformation of the glycan in solution and the orientation of its sulfate groups.     

The metabolic heterogeneity of rabbit ear cartilage chondroitin sulphate

The only glycosaminoglycans that can be isolated from the ear cartilage of 2-month-old rabbits are chondroitin 4-sulphate and chondroitin 6-sulphate. These chondroitin sulphates exhibit molecular-weight polydispersity when isolated from tissue by papain digestion. The chondroitin sulphate is metabolically heterogeneous in that radioactive precursors [(14)C]glucose or [(35)S]sulphate are preferentially incorporated into the higher-molecular-weight polymers both in vivo and in vitro. No transfer of radioactivity from the high-molecular-weight chondroitin sulphate to the low-molecular-weight chondroitin sulphate was seen during 15 days in vivo. It is suggested that there are at least two pools of proteoglycan in the tissue. One of these pools is metabolically active whereas the other is not.     

Hydraulic conductivity of chondroitin sulfate proteoglycan solutions

The hydraulic conductivity of solutions of Swarm rat chondrosarcoma proteoglycan subunit and of chondroitin 4- and 6-sulfate up to concentrations of 80 mg ml-1 have been measured under physiological conditions using sedimentation velocity and membrane ultrafiltration techniques. This study establishes the very high flow resistance of the proteoglycan and that this resistance is due to its constituent chondroitin sulfate chains. We have also demonstrated little difference in the hydraulic conductivity of chondroitin 4-sulfate as compared to chondroitin 6-sulfate. Studies of hydraulic conductivity of chondroitin sulfate and proteoglycan subunit over a range of salt concentrations demonstrate that the chondroitin sulfates exhibit only a small degree of electrolyte dissipation indicating that their constituent charge groups do not significantly contribute to flow resistance at high mechanical pressures. It appears that the shape and conformation of the polysaccharide backbone and its glycosidic linkages are the factors that primarily govern flow resistance. This is also consistent with the fact that hydraulic conductivity of the proteoglycans and chondroitin sulfates is considerably lower than that of its more charged counterpart heparin but has similar values to hyaluronate. Qualitative agreement between sedimentation analysis and ultrafiltration measurements is also established although the latter technique suffers from not knowing over what distance, adjacent to the membrane, ultrafiltration takes place. It is predicted that the proteoglycans will significantly contribute to flow resistance of cartilagenous tissues which confirms the Maroudas correlation that high proteoglycan concentration in cartilage yields high flow resistance. Further, we establish through a comparison of hydraulic conductivity measurements on hyaluronate, desulfated chondroitin sulfate, chondroitin sulfate, and proteoglycan subunit and osmotic pressure measurements of hyaluronate and proteoglycan that the sulfate groups of the chondroitin sulfate chain play only a small role in the net movement of water relative to the proteoglycan.     

The association of bovine beta-casein. The importance of the C-terminal region.

Bovine beta-casein exists in the monomer form in solution (pH 6.5, 0.1 M NaCl, 0.5% w/v) at low temperatures, but associates to form polymers at higher temperatures. Gel filtration chromatography at 36 degrees showed that the polymer is large with a hydrodynamic size greater than that of a globular protein with a mol wt of 1.34 times 10(6). Removal of two C-terminal amino acids per molecule decreased the proportion of polymer in the solution, although the chromatographic behavior of the modified beta-casein monomers and polymers was retained. Removal of a 20 amino acid peptide from the C terminus of the beta-casein completely destroyed its ability to form polymers and removed the 8-anilino-1-naphthalenesulfonate binding site. However, deletion of segments of the protein from the N terminus did not decrease the ability of the modified beta-casein to associate, nor did it affect the 8-anilino-1-naphthalenesulfonate binding site greatly. It seems likely that all, or some, of the 20 amino acids at the C terminus are responsible for the associative behavior of beta-casein, possibly by the direct participation of their side chains in hydrophobic bond formation. However, removal of the C-terminal peptides may have disrupted the spatial structure of the native protein so that it could no longer associate normally.     

FtsZ fiber bundling is triggered by a conformational change in bound GTP

Polymer formation by the essential FtsZ protein plays a crucial role in the cytokinesis of most prokaryotes. Lateral associations between these FtsZ polymers to form bundles or sheets are widely predicted to be extremely important for FtsZ function in vivo. We have carried out a study in vitro of FtsZ polymer formation and bundling using linear dichroism (LD) to assess structural properties of the polymers. We demonstrate proof-of-principle experiments to show that LD can be used as a technique to follow FtsZ polymerization, and we present the LD spectra of FtsZ polymers. Our subsequent examination of FtsZ polymer bundling induced by calcium reveals a substantial increase in the LD signal indicative of increased polymer length and rigidity. We also detect a specific conformational change in the guanine moiety associated with bundling, whereas the conformation and configuration of the FtsZ monomers within the polymer remain largely unchanged. We demonstrate that other divalent cations can induce this conformational change in FtsZ-bound GTP coincident with polymer bundling. Therefore, we present "flipping" of the guanine moiety in FtsZ-bound GTP as a mechanism that explains the link between reduced GTPase activity, increased polymer stability, and polymer bundling.     

Detection of secondary structure in glycosaminoglycans via the H n.m.r. signal of the acetamido NH group.

Two simple methods for dissolving salts of acid glycosaminoglycans with inorganic cations (e.g. Li+ and Na+) in dry dimethyl sulphoxide are described. Complete n.m.r. spectra of, e.g., Na+ and Li+ salts of chondroitin sulphate and keratan sulphate were obtained on these solutions. In [2H6]dimethyl sulphoxide the NH resonance of 2-acetamido-2-deoxy hexosides is in the range 7.2-8.0 delta, but is downfield (8.3-9.3 delta) when the NH is H-bonded to -CO2-. Heparan sulphate shows two NH resonances, of which one (at 8.3 delta) is probably indicative of H-bonding. Space-filling models show that a very close approach of NH to -CO2- across the alpha-glucosaminidic bond is possible, and a solution configuration for heparan sulphate is proposed. The n.m.r. results are entirely compatible with interpretations of periodate-oxidation kinetics, based on H-bonded secondary structures present in hyaluronate and chondroitin sulphates, but not in dermatan (or keratan) sulphate.     

Bioenergetics and Life's Origins

Bioenergetics is central to our understanding of living systems, yet has attracted relatively little attention in origins of life research. This article focuses on energy resources available to drive primitive metabolism and the synthesis of polymers that could be incorporated into molecular systems having properties associated with the living state. The compartmented systems are referred to as protocells, each different from all the rest and representing a kind of natural experiment. The origin of life was marked when a rare few protocells happened to have the ability to capture energy from the environment to initiate catalyzed heterotrophic growth directed by heritable genetic information in the polymers. This article examines potential sources of energy available to protocells, and mechanisms by which the energy could be used to drive polymer synthesis.Previous research on life''s origins has for the most part focused on the chemistry and energy sources required to produce the small molecules of life—amino acids, nucleobases, and amphiphiles—and to a lesser extent on condensation reactions by which the monomers can be linked into biologically relevant polymers. In modern living cells, polymers are synthesized from activated monomers such as the nucleoside triphosphates used by DNA and RNA polymerases, and the tRNA-amino acyl conjugates that supply ribosomes with activated amino acids. Activated monomers are essential because polymerization reactions occur in an aqueous medium and are therefore energetically uphill in the absence of activation.A central problem therefore concerns mechanisms by which prebiotic monomers could have been activated to assemble into polymers. Most biopolymers of life are synthesized when the equivalent of a water molecule is removed to form the ester bonds of nucleic acids, glycoside bonds of polysaccharides, and peptide bonds in proteins. In life today, the removal of water is performed upstream of the actual bond formation. This process involves the energetically downhill transfer of electrons, which is coupled to either substrate-level oxidation or generation of a proton gradient that in turn is the energy source for the synthesis of anhydride pyrophosphate bonds in ATP. The energy stored in the pyrophosphate bond is then distributed throughout the cell to drive most other energy‐dependent reactions. This is a complex and highly evolved process, so here we consider simpler ways in which energy could have been captured from the environment and made available for primitive versions of metabolism and polymer synthesis. Because the atmosphere of the primitive Earth did not contain appreciable oxygen, this review of primitive bioenergetics is limited to anaerobic sources of energy.     

Immunochemical characterization and ultrastructural localization of chondroitin sulfates and keratan sulfate in embryonic chick bone marrow

Summary Monoclonal antibodies directed against specific carbohydrate epitopes on chondroitin 4-/dermatan sulfate, chondroitin 6-sulfate, keratan sulfate, and a monoclonal antibody directed against the hyaluronate binding region were used to characterize proteoglycans extracted from embryonic chick bone marrow. About half of the proteoglycans separate into the high density fraction on a CsCl gradient. Glycosaminoglycan-specific antibodies recognize proteoglycans from all fractions; this includes an antibody directed against keratan sulfate. Some proteoglycans, principally in the high buoyant density fraction, contain sites recognized by the antibody specific for the hyaluronate binding region. Within limits of detection, all core proteins belong to the high-molecular-weight category, with weights in excess of 212 kD. Antibodies directed against chondroitin 4-/dermatan sulfate and against keratan sulfate primarily bind to extracellular matrix material located in the extracellular spaces and to matrix elements in the pericellular regions of fibroblastic stromal cells. The antibody that recognizes chondroitin 6-sulfate binds to sites on surfaces of fibroblastic stromal cells and also to extracellular matrix material. Little or no antibody binding is detected on surfaces of granulocytic cells. These studies indicate that chondroitin sulfate and keratan sulfate chains are both present in the proteoglycan extract.     

The nature and origin of the glycosaminoglycans of the embryonic chick vitreous body

Glycosaminoglycans of the embryonic chicken vitreous were characterized and then were used as markers to establish which tissues synthesize the vitreous humor during development. The glycosaminoglycans are predominantly chondroitin sulfates by several criteria. They are resistant to streptomyces hyaluronidase, an enzyme which degrades only hyaluronate, and are digested by testicular hyaluronidase and chondroitinase AC, enzymes which degrade hyaluronate plus chondroitin 4- and 6-sulfates. On electrophoresis on cellulose acetate in 0.15 M phosphate buffer, pH 6.7, the vitreous glycosaminoglycans migrate slightly slower than authentic chondroitin sulfate, but, in 0.1 N HCl, they migrate very close to chondroitin sulfate standards. Finally, the disaccharides produced by digestion of these radioactively labeled glycosaminoglycans with chondroitinases AC and ABC were identified as Δdi-4S and Δdi-6S, as expected for chondroitin 4- and 6-sulfate. By using incorporation of radioactive precursors into chondroitin sulfates in vitro, we than determined which tissues synthesize the vitreous humor in the developing eye. Late in development, on Day 12–13, the isolated vitreous is very active in chondroitin sulfate synthesis, while the neural retina, the lens, and the pecten are less active and produce a high proportion of enzyme-resistant GAG. The eye tissues isolated from embryos labeled in ovo retain similar amounts and types of glycosaminoglycans, indicating that cells within the vitreous synthesize the vitreous humor glycosaminoglycans at this time. Earlier in development, from Days 6 to 8, the isolated vitreous incorporates very low levels of radioactivity into GAG, but the neural retina incorporates high levels of radioactivity into chondroitin sulfate. When the embryos are labeled in ovo and the same tissues are isolated following incorporation, the vitreous retains more radioactive chondroitin sulfate than does the neural retina. Thus, the vitreous humour glycosaminoglycan is initially synthesized by the neural retina and is secreted into the vitreous space.