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Biomedical subjects

H E Conrad

Publications and source records attributed to H E Conrad.

At least 55 records · Page 3Linked to original sources
PubMed1980

The acid lability of the glycosidic bonds of L-iduronic acid residues in glycosaminoglycans.

Heparan sulphate, heparin and dermatan sulphate were hydrolysed in 0.5M-H2SO4 at 100 degrees C. At intervals portions of the hydrolysate were removed and treated with HNO2 at pH 4.0 to cleave the glycosidic bonds of the N-unsubstituted hexosamine residues and to convert both free and combined hexosamines into anhydrohexoses. These hydrolysis/deamination mixtures were reduced with NaB3H4 and analysed by radiochromatography for alpha-L-iduronosylanhydrohexose, beta-D-glucuronosylanhydrohexose, and the free uronic acids and anhydrohexose. These data gave a kinetic profile of the cleavage of the alpha-L-iduronosyl and the beta-D-glucuronosyl bonds in these glycosaminoglycans. The beta-D-glucuronosyl bonds showed the expected resistance to acid hydrolysis, but the alpha-L-iduronosyl bonds were found to be as labile to acid as some neutral sugar glycosides. This unusual lability of alpha-D-iduronosyl-anhydromannitol and beta-D-glucuronosylanhydromannitol. The procedures used to follow the kinetics of glycosaminoglycan hydrolysis can also be sued to obtain quantitative analyses of L-iduronic acid, D-glucuronic acid and hexosamine in these polymers.

Chemical Phenomena↗
PubMed1980

Multiple kinetic forms of beta-glucuronidase.

Partially purified chick embryo liver beta-glucuronidase and highly purified beta-glucuronidase from human placenta and rat preputial gland exhibit multiple kinetic forms which appear to exist in an equilibrium which can be shifted by varying the assay conditions. All three enzymes exist in a low Km form, which predominates at pH 3 and is stabilized by bovine serum albumin, and a high Km form, which predominates at pH 5.5 to 6.0 in the absence of serum albumin. At intermediate pH values both forms are present. Addition of 0.2 M NaCl shifts the equilibrium toward the high Km form. Both forms of these enzymes are active on 4-methyl umbelliferyl-beta-D-glucuronide and on the hexasaccharides of chondroitin-6-SO4, chondroitin, and hyaluronic acid, with the low Km forms showing 2- to 20-fold more activity on the oligosaccharide substrates than the high Km forms.

Animals↗
PubMed1980

Secretion of chondroitin SO4 by monolayer cultures of chick embryo chondrocytes.

Monolayer cultures of chick embryo tibial chondrocytes incorporate 35SO42- into chondroitin SO4 which is rapidly secreted from the cells into two extracellular pools. Part of the extracellular chondroitin SO4 is recovered in a soluble form in the culture medium, and the remainder is associated with the cell matrix from which it is released by isotonic trypsinization. At 38 degrees C labeled chondroitin SO4 appears in the cell matrix fraction within 5 min after addition of 35SO42- and in the culture medium fraction 15 min after 35SO42- is added. The intracellular pool of labeled chondroitin SO4 reaches a steady state level of 150 to 200 pmol of bound SO4 per 10(6) cells in 60 min, while the cell matrix and medium fractions increase at rates of 3 and 1 nmol of bound SO4 per h per 10(6) cells, respectively. After 4 h of labeling, less than 20% of the newly synthesized cell-associated chondroitin SO4 is in the intracellular fraction. By labeling cells for 15 min at 25 degrees C 80% of the cell-associated chondroitin 35SO4 is obtained in the intracellular fraction. This material is chased without lag into both the cell matrix fraction and the medium fraction. A mixture of NaF and NaCN, both at 30 mM, lowers the cellular ATP level to 15% of normal and blocks secretion of the intracellular chondroitin SO4 into both extracellular fractions. Colchicine at 10(-6) M gives a partial inhibition of both synthesis and secretion of chondroitinSO4. Sucrose density gradient sedimentation analysis of the intracellular chondroitin SO4 and the two extracellular fractions shows that all three fractions contain both a heavy and light proteoglycan fraction. The intracellular light proteoglycan fraction is secreted preferentially into the culture medium where it represents 30% of the total culture medium pool. The ratio of 6-sulfated GalNAc to 4-sulfated GalNAc in the heavy proteochondroitin SO4 fraction is approximately twice that found for the light fraction.

Animals↗
PubMed1979

Hybrid glycosaminoglycans synthesized by monolayers of chick embryo arterial fibroblasts.

Monolayer cultures of arterial fibroblasts from 13-day chick embryonic aorta incorporated 35SO42- into glycosaminoglycans containing both glucuronic and iduronic acids. Bacterial chondroitinase ABC converted more than 98% of the 35SO4-labeled polymer to mono- or disaccharides, including (1) N-acetyl-D-galactosamine 4-sulfate, (2) delta 4,5-glucuronic acid 2- or 3-sulfate leads to N-acetylgalactosamine 6-sulfate, and (3) the unsaturated disaccharides normally obtained from chondroitin 4-sulfate and chondroitin 6-sulfate sequences. Chondroitinase AC converted only 77% of the 35SO4-labeled polymer to the same mono- and disaccharides and yielded, in addition, the following oligosaccharide products: (1) delta 4,5-glucuronic acid leads to N-acetylgalactosamine 4- or 6-sulfate leads to iduronic acid leads to N-acetylgalactosamine 6- or 4-sulfate; (2) N-acetylgalactosamine 4-sulfate leads to iduronic acid 2- or 3-sulfate leads to N-acetylgalactosamine 6-sulfate; (3) delta 4,5-glucuronic acid leads to N-acetylgalactosamine 4-sulfate leads to (iduronic acid leads to N-acetylgalactosamine 4-sulfate)2; (4) delta 4,5-glucuronic acid leads to N-acetylgalactosamine 4- or 6-sulfate leads to (iduronic acid leads to N-acetylgalactosamine 6- or 4-sulfate)2; (5) higher oligosaccharides containing iduronic acid and N-acetylgalactosamine 4-sulfate.

Animals↗
PubMed1979

Chick embryo liver beta-glucuronidase. Comparison of activity on natural and artificial substrates.

The beta-glucuronidase in homogenates of 12-day chick embryo livers catalyzed the release of glucuronic acid from 4-methylumbelliferyl-beta-D-glucuronide and from the nonreducing terminals of the hexasaccharides of chondroitin-6-SO4 and chondroitin-4-SO4 at rates of 143, 114, and 108 nmol of glucuronic acid/h/mg of protein, respectively, when assayed at pH 3.5 in 0.05 M sodium acetate buffer. During a 60-fold purification of the enzyme, the ratios of the activities on these substrates did not change. When 4-methylumbelliferyl-beta-D-glucuronide was used as substrate the enzyme was active at pH values from 3.0 to 5.5, with maximal activity between pH values 4.0 and 4.5. Concentrations of NaCl from 0.15 to 0.3 M inhibited the activity at low pH values but activated the enzyme between pH 4.0 and 5.5. The enzyme was active on the chondroitin-6-SO4 hexasaccharide from pH 3.0 to 5.5, with a broad optimum between 3.0 and 4.5. NaCl inhibited the activity on the oligosaccharide substrate at all pH values. Eadie-Scatchard plots of rates of 4-methylumbelliferyl-beta-D-glucuronide hydrolysis at substrate concentrations ranging from 2 to 1000 microM showed multiple kinetic forms of the enzyme, a form with a Km of approximately 11 microM, and a second form with a Km of approximately 225 microM. The pH optimum of the low Km form was 3.5 to 4.0; that of the high Km form was pH 4.5. NaCl inhibited the activity of the low Km form, but activated the high Km form of the enzyme. Chondroitin SO4 oligosaccharides competed with 4-methylumbelliferyl-beta-D-glucuronide for the low Km form of the enzyme but had little effect on the hydrolysis of 4-methylumbelliferyl-beta-D-glucuronide by the high Km form of the enzyme. The activities of the beta-glucuronidase on tetra-, hexa-, octa-, and decasaccharides of chondroitin-6-SO4 and chondroitin-4-SO4, measured using a new assay procedure which can detect the formation of 1 nmol of product, were similar, although rates were somewhat lower for the higher oligosaccharides. With the exception of the chondroitin-4-SO4 tetrasaccharide, all of the oligosaccharide substrates saturated the enzyme at concentrations of 20 to 30 microM, indicating Km values of less than 10 to 15 microM for the oligosaccharides. Highly purified beta-glcuronidases from human placenta and from rat preputial gland also showed multiple kinetic forms when assayed using 4-methylumbelliferyl-beta-D-glucuronide as substrate.

Animals↗
PubMed1979

Chondroitin SO4 catabolism in chick embryo chondrocytes.

An enzyme preparation from cultured chick embryo vertebral chondrocytes attacks chondroitin SO4 oligosaccharides from the nonreducing terminal in a recycling pathway involving the sequential action of a beta-glucuronidase, a 4- or a 6-sulfatase, and a beta-N-acetylgalactosaminidase. The sequence is blocked by saccharo-1,4-lactone, an inhibitor of the beta-glucuronidase, or by 2-acetamido-2-deoxy-D-galactonolactone, an inhibitor of the beta-N-acetylgalactosaminidase. The level of 4-sulfatase activity is low relative to the other activities and limits the rate of catabolism of hybrid oligosaccharide structures containing both 6-sulfated galactosamine residues and 4-sulfated galactosamine residues. This results in the accumulation of shortened oligosaccharides, most of which have galactosamine-4-SO4 residues at their nonreducing terminals. In the presence of the lactone inhibitors, polymeric chondroitin SO4 is broken down by the enzyme preparation to oligosaccharides which are 10 to 15 monosaccharides long, indicating that degradation of chondroitin SO4 chains is initiated by an endoglycosidase which generates oligosaccharide substrates for the recycling exoglycosidase system.

Acetylgalactosamine↗
PubMed1979

Effect of growth conditions on the content of the major groups of carbohydrates in chick embryo fibroblasts.

The levels of glycogen, hyaluronic acid, chondroitin sulfates, N-acetylneuraminic acid, all of the monosaccharide components of the glycoprotein and glycolipid fractions, and the monosaccharide pools were measured in cultured chick embryo fibroblasts. Under all growth conditions, the glycogen plus the glucose phosphate pool contained approximately 50% of total monosaccharide content of the cells. However, marked qualitative and quantitative alterations were found in the glycoprotein, glycolipid, and mucopolysaccharide fractions when growing cells reached confluence, when the growth temperature was shifted from 36 to 41 degrees, or when the cells were transformed with Rous sarcoma virus. From 65 to 95% of the total monosaccharide residues in these complex carbohydrates were found in the glycoprotein fraction, while the glycolipids contained only 5 to 10% of the residues, and the mucopolysaccharides contained 5 to 25%. Changes in the complex carbohydrates in normal cells following changes in cell density or growth temperature were so great that they obscured any transformation-dependent changes that might have occurred consistently in the virus-infected cells under different growth conditions.

Animals↗
PubMed1979

Development of the tibiotarsus in the chick embryo: biosynthetic activities of histologically distinct regions.

The dynamics of the histological changes which occur in the distal half of the tibial portion of the embryonic chick tibiotarsus from day 8 to day 18 of incubation are correlated with the capacities of histologically distinct zones to incorporate isotopic precursors into mucopolysaccharides and collagen. At the distal end of the tibia, which abuts the suture line formed by the fusion of the two tarsals with the tibia, there persists throughout embryonic development a narrow band of small, round or oval, rapidly dividing chondrocytes which synthesize chondroitin SO4 and collagen at low rates. Just proximal to this zone is a broader zone of flattened, disc-shaped chondrocytes which divide more slowly and are extremely active in chondroitin SO4 and collagen synthesis. Proximal to the zone of flattened chondrocytes is a zone of non-dividing, hypertrophied chondrocytes which are large and round and increase continually in size going from the distal to the proximal end of the zone. The biosynthetic activities of the cells in this zone fall sharply with their distance from the zone of flattened chondrocytes. Finally, there is a fourth zone, the marrow cavity, formed by a proximal to distal disintegration of the hypertrophied chondrocytes, starting at mid-diaphysis. The marrow cavity is surrounded by a shell of periosteal and intra-membranous bone which extends to the distal end of the zone of hypertrophied chondrocytes. Our data suggest that as the tibiotarsus grows in length the small, round rapidly dividing cells of the tibia differentiate first to flattened chondrocytes which synthesize matrix at a high rate and ultimately to low activity, hypertrophying chondrocytes. This sequence proceeds in a linear fashion.

Animals↗
PubMed1978

Turnover of cellular carbohydrates in normal and Rous sarcoma virus-transformed cells.

We have analyzed the distribution of glucosamine-labeled polymers on the cell surface, in the growth medium, and inside the cell and the net turnover of these polymers during the process of malignant transformation of chick embryo fibroblasts by Rous sarcoma virus. The distribution of label and the turnover kinetics for hyaluronic acid, total glycoproteins, and chondroitins were found to be identical in both normal and transforming cultures. We conclude that nonspecific degradative processes are probably not involved in causing transformation-related alterations in cell surface carbohydrates, althougn degradation of specific macromolecules is not excluded.

Avian Sarcoma Viruses↗
PubMed1976

Kinetics of mucopolysaccharide and glycoprotein synthesis by chick embryo chondrocytes. Effect of D-glucose concentration in the culture medium.

Late log cultures of chick embryo vertebral chondrocytes in Dulbecco's Modified Eagle's Medium with 10% fetal calf serum consume D-glucose from the culture medium at a rate of approximately 0.40 mumol per h per 10(6) cells. When the D-glucose concentration in the medium drops below 1 mumol per ml the glycogen stores are rapidly exhausted, and cell growth ceases. 35SO4(2)- is incorporated into chondroitin-6-SO4 and chondroitin-4-SO4 at linear rates of 1.2 and 0.4 nmol per h per 10(6) cells, respectively, until the D-glucose level in the medium drops below 1 mumol per ml, but there is always a slight lag in the initial appearance of chondroitin-4-SO4. Throughout the period of 35SO4 2- labeling, the amount of chondroitin-6-SO4 that is recovered in the cells exceeds the amount that is recovered in the medium, but the opposite is true for chondroitin-4-SO4. However, when cells prelabeled with 35SO4(2-) are then transferred to a label-free medium, the secretion of chondroitin sulfates proceeds at much slower rates, and the kinetics of chondroitin-6-SO4 and chondroitin-4-SO4 secretion are very similar. In this chase experiment the chondroitin sulfates are recovered quantitatively after a 24-h incubation period, indicating that these embryonic chondrocytes do not degrade the chondroitin sulfates under these culture conditions. The rate of incorporation of counts from D-[14C]glucosamine into mucopolysaccharides and glycoproteins increase with time. This nonlinear rate results from a progressive increase in the specific activity of the UDP-N-acetyl-D-[14C]hexosamine pool as the D-glucose in the culture medium is depleted. A linear relationship is demonstrated between the logarithm of the 14C counts per min per nmol of UDP-N-acetylhexosamine and the logarithm of the concentration of D-glucose in the culture medium over a range of 1 to 20 mumol of D-glucose per ml. The relative rates of appearance of counts from 35SO4(2-) and D-[14]glucosamine in chondroitin 4-SO4 and chondroitin-6-SO4 are used to calculate the specific activity of the UDP-N-acetyl-D-[14C]hexosamine pool at each stage in the labeling period. The resulting values are then used to calculate the rates of synthesis of the nonsulfated polymers, namely, chondroitin, hyaluronic acid, and glycoprotein.

Animals↗
PubMed1976

Formation of anhydrosugars in the chemical depolymerization of heparin.

In the reactions used to break heparin down to mono- and oligosaccharides, androsugars are formed at two stages. The first of these is the well-known cleavage of heparin with nitrous acid to convert the N-sulfated D-glucosamines to anhydro-D-mannose residues; this reaction has been studied in detail. It is demonstrated here that only low pH (less than 2.5) reaction conditions favor the deamination of N-sulfated D-glucosamine residues; the reaction proceeds very slowly at pH 3.5 or above. On the other hand, N-unsubstituted amino sugars are deaminated at a maximum rate at pH 4 with markedly reduced rates at pH2 or pH6. At room temperature solutions of nitrous acid lose one-fourth to one-third of their capacity to deaminate amino sugars in 1 h at all pHs. A low pH nitrous acid reagent which will convert heparin quantitatively to its deamination products in 10 min at room temperature is described, and a comparison of the effectiveness of this reagent with other commonly used nitrous acid reagents is presented. It is also shown that conditions used for acid hydrolysis of heparin convert approximately one-fourth of the L-iduronosyluronic acid 2-sulfate residues to a 2,5-anhydrouronic acid. This product is an artifact of the reaction conditions, and its formation represents one of several pathways followed in the acid-catalyzed cleavage of the glycosidic bond of the sulfated L-idosyluronic acid residues.

Animals↗
PubMed1976

Nearest neighbor analysis of heparin: identification and quantitation of the products formed by selective depolymerization procedures.

Heparin was carboxyl-reduced with sodium boro[3H]hydride and converted to a mixture of oligosaccharides by treatment with nitrous acid at pH 2. The oligosaccharide mixture was aldehyde-reduced with sodium boro[3H]hydride and the mixture of products, labeled both in the hexoses formed in the carboxyl-reduction step and in the reducing sugars formed in the nitrous acid reaction, was separated and analyzed. The major product, L-idosyl 2-sulfate leads to anhydro-D-mannitol 6-sulfate (I), contained 60% of hexoses derived from the hexuronic acid residues in the original heparin. A second product, which contained 15% of the hexoses derived from the hexuronic acid residues in the original heparin, was identified as a tetrasaccharide composed of two L-idosyl 2-sulfate residues, one anhydro-D-mannitol 6-sulfate residue (the reducing end),and a hydroxymethylpentose sulfate residue formed by deamination of a disulfated D-glucosamine residue without bond cleavage. Several additional disaccharides derived from the regions of the polymer which contained D-glucuronic acid residues and lower degrees of O-sulfation were also identified among the deamination products. The oligosaccharides that were obtained accounted for 100% of the original carboxyl-reduced heparin, and paper chromatographic profiles of the oligosaccharide separations can be used as a fingerprint of the heparin preparation. The properties of I were examined in greater detail. The glycosidic bond of the L-idosyl 2-sulfate residue was found to be extremely labile to 0.1 N HCl at 100 degrees C, hydrolyzing with a t 1/2 of 18 min to give high yields of L-idose 2-sulfate and anhydro-D-mannitol 6-sulfate. L-Idofuranose was also identified as an intermediate in the conversion of L-idose 2-sulfate to L-idosan. The acid lability of the L-idosyl 2-sulfate bond in I offers a new route for the selective cleavage of carboxyl-reduced heparin.

Animals↗