Martin I. Horowitz

Molecular species of phosphatidylcholine from rat gastric mucosa.

Phosphatidylcholine of rat gastric mucosa were found to constitute about half of the total phospholipids. The composition of 20 molecular species accounting for approx. 90% of the total phosphatidylcholine was determined by specific enzymic hydrolyses and AgNO3 thin-layer and gas-liquid chromatography. Disaturated (dipalmitoyl) phosphatidylcholine made up about 31% of the total phosphatidylcholines. Other species which occurred in significant concentrations included 16:0/18:1, 18:0/18:1, 16:0/18:2, 18:0/18:2, 16:0/18:3, 18:0/18:3, 16:0/20:4, and 18:0/20:4. These results indicate that rat gastric mucosa is similar to lung in that both contain elevated amounts of dipalmitoyl phosphatidylcholine. Other similarities between these two tissues are discussed.

Sulfatides of hog gastric mucosa.

Abstract Mono-, di- and trihexose sulfatides have been isolated from hog gastric mucosa lipids by the procedure involving column fractionations on DEAE-Sephadex, silicic acid and thin-layer chromatography. Desulfation of mono-, di- and trihexose sulfatides with methanolic HCl resulted in the formation of galactosylceramide, lactosylceramide and galactosyllactosylceramide, respectively. By partial acid hydrolysis, periodate oxidation and methylation analysis, the structures of these sulfatides were identified as: (SO3H→ 3)-Gal-(1→ 1)-Cer,(SO3H→ 3)-Gal-(1 → 4)-Glc-(1 → 1)-Cer and (SO3H → 3)-Gal-(1 → 4)-Gal-(1 → 4)-Glc-(1 → 1)-Cer, respectively.

Lysophospholipase and transacylase activities of rat gastric mucosa.

Abstract A transacylase that converts 1-palmitoyl lysophosphatidylcholine to dipalmitoyl phosphatidylcholine was demonstrated in the rat gastric mucosa. This enzyme required neither ATP or CoA nor bile salt and detergent for its activity. The enzyme preparation also exhibited powerful lysophospholipase activity. The transacylase and lysophospholipase were both located in the cytosol fraction, and their activities remained associated at a constant ratio throughout the purification steps, including the isoelectrofocusing procedure. They responded similarly with respect to the addition of metal ions, bile salt, detergent, and heat treatment. Both enzyme activities also exhibited similar apparent K m values for lysophosphatidylcholine. These observations suggest that both the lysophospholipase and transacylase activities may reside in the same enzyme.

Blood group a active ceramide hexasaccharide lacking N-acetylglucosamine isolated from hog stomach mucosa

Abstract Blood group A active ceramide hexasaccharide devoid of N -acetylglucosamme was isolated from lipid extract of hog stomach mucosa. By partial acid hydrolysis, sequential hydrolysis with glycosidases and methylation analysis the structure of this glycolipid was identified as GalNAc−(l → 3)−Gal−(l → 3)−Gal−(l → 4)−Gal−(l → 4)−Glc⊂l → l)−Cer (2↑1)α Fuc

Separation of polar lipid by column chromatography hydroxylapatite

Abstract The separation of various lipid by column chromatography on hyddroxylapatite was investigated. Selective elution of lipid classes was affected by varying the ratio of acetone to methanol in the eluant. Separations achieved compared favorably to those obtained with silicic acid or DEAE-cellulose. Column chromatography on hydroxylapatite afforded complete recovery of polar lipids, judged by the 99–100% recovery of total phosphorus. Careful examination of eluted fractions did not indicate degradation of lipids during column development.

The 1-O-galactosylceramide fatty acid esters from hog stomach

Abstract 1. 1. Three 1- O -galactosylceramide fatty acid esters (A, B, C) in which the second fatty acid moiety is attached to the 3-, 4- and 6-position of the galactose, respectively, have been isolated from hog stomach substance powder. 2. 2. The isolated cerebroside esters upon alkaline methanolysis gave methyl esters of O - acyl - linked fatty acids and cerebroside. 3. 3. Gas-liquid chromatography of the sugar moiety of permethylated cerebroside esters (A, B, C) gave 2,4,6-, 2,3,6- and 2,3,4-trimethylgalactitol, respectively. 4. 4. Treatment of the cerebroside esters with periodate resulted in destruction of the galactose in glycolipid B and C but not in A. 5. 5. Mild acid hydrolysis of the periodate-treated and reduced cerebroside ester C gave 1-monoglyceride. Threitol has been identified from the products of periodate oxidation of glycolipid B. 6. 6. Acid methanolysis of the permethylated cerebroside esters gave 3- O - methylsphingenine as the major product. 7. 7. Negligible content of the hydroxy acids indicate that the isolated glycolipids are the fatty acid esters of kerasin.

The glycolipids of bovine serum

Abstract 1. 1. Fractionation of bovine serum lipids on a silicic acid column gave two glycolipid-containing fractions eluted with acetone-methanol 9:1 (v/v) and methanol. 2. 2. The fraction eluted with acetone-methanol contained mono-, di-, tri-, and tetrahexoside ceramides. 3. 3. Long-chain base analyses of glycosyl ceramides revealed the presence of a number of different bases, both saturated and unsaturated. The principal bases found in order of abundance were sphingenine, heptadecasphinganine, sphinganine, eicosaphingenine and hexadecasphinganine. Sphingenine as the fraction of total base increased with the size of the hexoside part of glycolipid. 4. 4. Palmitic, isostearic and stearic acids were the major fatty acid components of the bovine serum glycolipids. 5. 5. The fraction eluted with methanoi contained complex glycolipids including the J hapten. The alkaline lability and the chromatographic behavior of the J hapten glycolipid are in keeping with our earlier findings that the glycerol and phosphorus are the integral parts of the J hapten structure.

Nonenzymatic modifications of amino sugars in vitro

Browning reactions of amino sugars were observed in a variety of sterile pH buffers at 25-37 degrees C. These reactions were signaled by an increase in absorbance at 273 nm, followed by an increase in absorbance at 320-360 nm. The reactions were maximal at pH 7.0 in phosphate buffer. Acidic solutions (pH less than 2.2) of 50 mM D-glucosamine hydrochloride gave only a negligible reaction and 2-acetamido-2-deoxy-D-glucose was unreactive. Half of the D-glucosamine in a 100 mM solution in sterile 0.2 M sodium phosphate buffer, pH 7.4, at 37 degrees C decomposed or was transformed in 27 h. A comparison of reactivity in generating A273 and A340 chromophores showed D-mannosamine greater than D-galactosamine greater than D-glucosamine. Permanganate oxidation of incubated glucosamine solutions afforded a compound which chromatographed like 2,5-pyrazinedicarboxylic acid and gave the same ultraviolet absorption spectrum. This, together with fractionating and thin-layer chromatography of the products of glucosamine incubation, suggests that 2,5-bis(tetrahydroxybutyl)pyrazine is formed as one of the products of autocondensation of D-glucosamine in accord with the report of Candiano et al. (1988, Carbohydr. Res. 184, 67-75) on products formed in glucosamine-lysine incubation mixtures. Formation of products absorbing at 325-360 nm was inhibited by the chelator diethylene-triaminepentaacetic acid. This suggests that the later reactions may be mediated by a metal-stimulated free radical mechanism. After 4 days incubation high molecular weight products with absorbance maxima at 273 nm and 325-360 nm were detected. Some of these were retained by dialysis membranes of molecular weight cut-off greater than 3500 and greater than 12,000.

Chondroitin sulfate-degrading enzymes in human polymorphonuclear leukocytes: Characteristics and evidence for concerted mechanism

Abstract Human polymorphonuclear leukocyte-derived enzymes, β-glucuronidase, β- N -acetylgalactosaminidase, and aryl sulfatase were studied for their ability to degrade chondroitin sulfate. Evidence is presented which implies a concerted, synergistic mechanism of action for these enzymes in glycosaminoglycan degradation excluding the possibility of endoglycosidase activity. Conclusions are based upon results derived from pH optimum, inhibitor studies, kinetics, and partial purification of these enzymes. The data presented here demonstrate that the polymorphonuclear leukocytes contain all of the enzymes necessary for the solubilization and complete degradation of the chondroitin-4-sulfate of cartilage matrix.

Solubilization and chemical and immunochemical characterization of sparingly soluble canine gastric mucin

Abstract 1. 1. Sparingly soluble mucin obtained from anacid secretion of canine Heidenhain pouch was solubilized by the use of Na 2 SO 3 . The solubilized mucin was then fractionated by Sephadex G-200 chromatography which yielded high molecular weight glycoproteins in the excluded and serum proteins in the included fractions. Immunoelectrophoresis of the glycoprotein fraction gave a single arc of immunoprecipitate. 2. 2. The isolated glycoprotein fraction was fractionated further into several individual glycoproteins and a protein on a DEAE-cellulose column by stepwise elution with graded concentrations of NaCl. Analysis by immunodiffusion indicated that the two principal glycoproteins which were eluted at 0.2 and 0.3 M NaCl contained antigenic factors in common. Spur formation at the intersections of the arcs of immunoprecipitate of the minor glycoproteins and the principal glycoproteins suggest that the minor glycoproteins contain determinants which are serologically related though not identical to those of the major glycoproteins of the sparingly soluble mucin. The protein isolated from the sparingly soluble mucin failed to give immunoprecipitate when reacted with antiserum to the sparingly soluble mucin. 3. 3. The carbohydrates present in the isolated glycoproteins were N -acetyl glucosamine, N -acetyl galactosamine, galactose, fucose and N -acetyl neuraminic acid. Serine, threonine and proline constituted more than half the amino acid residues of the glycoproteins and alkaline borohydride cleavage study indicated that O -glycosidic linkage exists between N -acetyl hexosamine and serine and threonine of the peptide core of glycoprotein. 4. 4. When the sparingly soluble mucin was alkylated first with N -ethylmaleinide and the solubilized with Na 2 35 SO 3 , the 35 S radioactivity was associated with the isolated glycoproteins suggesting that the insolubility of the sparingly soluble mucin may be due at least in part, to the intermolecular disulfide bonds. From the amount of 35 SO 3 2− taken up by the alkylated glycoproteins, we estimate that a minimum of r μmole of cystine may have been cleaved per 57 mg of protein in the glycoprotein.