W. Marx

Preparation of 3'-phosphoadenylyl sulfate in substrate quantities using mastocytoma enzymes.

Abstract A method was developed for the enzymic preparation and purification of 3′-phosphoadenylyl sulfate labeled with radioactive sulfur. The proposed procedure includes incubation of mouse mast cell tumor (Furth) high-speed supernatant as a source of sulfate-activating enzymes with ATP and sulfate-35S in the presence of an ATP-generating system and other cofactors. Experimental conditions were established for maximal production of the active sulfate. The radioactive 3′-phosphoadenylyl sulfate synthesized was isolated by ion-exchange column chromatography on ECTEOLA formate. The ammonium bicarbonate used as eluent was removed by lyophilization.

Paper chromatography of heparin and related sulfated mucopolysaccharides.

A paper chromatographic system was developed for the resolution of mixtures of sulfated mucopolysaccharides. The effect of varying the ammonium formate buffer/isopropanol ratio in the developing solution on the RF values of heparin and chondroitin sulfate is presented. A complete separation of heparin from chondroitin sulfate was obtained with three solvent mixtures containing different proportions of buffer and isopropanol. One of these solvent mixtures, ammonium formate buffer-isopropanol (65:35) v/v, resolved commercial beef heparin into two principal components, on “fresh” paper. This system was used to compare beef heparin with mouse mast cell tumor heparin, rat heparin, sheep lung heparin, chondroitin sulfate, and β-heparin. A common metachromatic component with an RF of approximately 0.5 was0found in all of the heparin preparations chromatographed with this solvent mixture. “Aging” of the filter paper resulted in the loss of the ability of the buffer-isopropanol mixture (65:35) v/v to resolve the beef heparin. Increase of the buffer-isopropanol ratio to (70:30) v/v restored the resolution of the beef heparin on “aged” paper. Heparitin sulfate and α-heparin monosulfate were compared to beef heparin using the latter system.

Sulfation of N-desulfoheparin and heparan sulfate by a purified enzyme from mastocytoma

Abstract 3′-Phosphoadenylylsulfate:N-desulfoheparin sulfotransferase from a postmicrosomal particulate fraction of Furth mouse mast cell tumor was solubilized by snake venom phospholipase and purified further by DEAE-cellulose chromatography. The purified enzyme was free of endogenous sulfate acceptor and 3′-phosphoadenylylsulfate sulfohydrolase activity. The sulfotransferase catalyzed sulfate transfer from 3′-phosphoadenylylsulfate-35S not only to N-desulfoheparin, but also to heparan sulfate, dermatan sulfate, and a glycosaminoglycan prepared from mast cell tumor. Heparin, chondroitin-4-sulfate, and mixed isomers of chondroitin sulfate from cartilage were practically inactive. The reaction products of N-desulfoheparin and heparan sulfate were found to be primarily N-sulfated. Sulfation of heparan sulfate was more rapid and more extensive than that of N-desulfoheparin, although the former contained fewer free amino groups. Synthetic N-acetylheparin and N-succinylheparin exhibited only very low sulfate-accepting ability. Apparently free amino groups are required for the enzymatic N-sulfation of glycosaminoglycans and some special structural features facilitate sulfate transfer to the amino groups of heparan sulfate.

Purification and properties of ATP-sulfurylase from Furth mouse mastocytoma.

Abstract 1. 1.|The enzyme, ATP-sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) which catalyzes the reaction, ATP + inorganic sulfate ⇌ adenylyl sulfate + inorganic pyrophosphate, was purified from a high-speed supernatant of mouse mastocytoma (Furth) by (NH4)2SO4 fractionation, hydroxylapatite column chromatography, and Geon resin electrophoresis. The purification resulted in a 545-fold increase in specific activity. 2. 2.|The purified enzyme exhibited a pH optimum of 8.5, and it was free of inorganic pyrophosphatase (EC 3.6.1.1), ATP phosphohydrolase (EC 3.6.1.3), adenylylsulfate kinase (EC 2.7.1.25) and adenylylsulfate sulfohydrolase. 3. 3.|The enzyme did not show an absolute requirement for metal ions, but its activity was increased by Mn2+, Mg2+, and Zn2+. EDTA was a powerful inhibitor of the ATP-sulfurylase; this inhibition was reversed by Mn2+, Mg2+, Cu2+, Co2+ and Zn2+. 2 mM solution of Ni2+, Ba2+ and Ca2+ inhibited the enzyme. 4. 4.|The enzyme reacted only with adenylylsulfate or deoxyadenylylsulfate and inorganic pyrophosphate as substrates (ATP sulfurylase reaction in reverse). Nucleotidylsulfates other than adenylylsulfate inhibited the reaction, when added to the latter as substrate. 5. 5.|The reaction catalyzed by the mastocytoma ATP-sulfurylase exhibited saturation phenomena, but it did not strictly follow Michaelis-Menten kinetics.

Biosynthesis of heparin. Separation of subcellular mastocytoma fractions involved in the incorporation of sulfate-35S into the heparin fraction.

Abstract The abilities of various subcellular fractions of the Furth mouse mastocytoma to activate sulfate and to incorporate sulfate into the heparin fraction were studied. The enzymes catalyzing the formation of 3′-phosphoadenylylsulfate, located in the soluble portion, were separated from the system responsible for sulfate transfer to the heparin fraction which was found to be associated with a “postmicrosomal” particulate fraction. The pH optima for these two processes were found at 7.7 and 6.4, respectively. Incubation of the “postmicrosomal” particles with either purified or crude radioactive 3′-phosphoadenylylsulfate, (the latter formed by incubation of the high speed supernatant with sulfate-35S, ATP, and Mg++) resulted in a substantial incorporation of radioactivity into the heparin fraction. Some of the tumor fractions were found to contain 3′-phosphoadenylylsulfate-hydrolase activity.

3'-Phosphoadenylylsulfate:N-desulfoheparin sulfotransferase associated with a postmicrosomal particulate mastocytoma fraction

Abstract An enzyme catalyzing the transfer of sulfate from 3′-phosphoadenylylsulfate to N -desulfoheparin was purified about 27-fold from Furth mouse mast cell tumor. The enzyme activity was associated with a “postmicrosomal” particulate fraction. A significant transfer of sulfate was observed primarily to the amino groups of N -desulfoheparin. The rate of the reaction was highest between pH 6.7 and 7.2, and it was strongly influenced by the ionic strength and nature of the buffer medium. The sulfate transfer was accelerated by Mg ++ but inhibited by p -chloromercuribenzoate, phenylmercuric acetate, and Cu ++ and Zn ++ . The enzyme catalyzed also sulfation of heparan sulfate, and, to a much more limited extent, of chondroitin 4-sulfate and dermatansulfate; but heparin and p -nitrophenol were practically inactive as sulfate acceptors. The sulfotransferase preparation contained some 3′-phosphoadenylylsulfate sulfohydrolase, but it was essentially free of sulfate activating enzymes.

Enzyme-substrate complexes of ATP-sulfurylase from mouse mastocytoma

Abstract 1. 1.|A highly purified mouse mastocytoma ATP-sulfurylase (ATP: sulfate adenylyltransferase, EC 2.7.7.4) preparation was observed to bind ATP, when incubated with the triphosphate. Upon addition of inorganic sulfate to the enzyme-ATP complex, the enzyme-bound ATP was converted to enzyme-bound adenylylsulfate. 2. 2.|When the enzyme-adenylylsulfate complex was incubated in the presence of adenylylsulfate kinase and ATP, a partial conversion of the bound adenylylsulfate to free 3′-phosphoadenylylsulfate was noted. 3. 3.|On the basis of these results a reaction sequence was suggested for the mechanism of sulfate activation, with ATP-sulfurylase-ATP and ATP-sulfurylaseadenylylsulfate complexes as intermediates.

Two forms of ATP sulfurylase in Furth mouse mastocytoma

Abstract ATP sulfurylase of Furth mouse mastocytoma, partially purified by ammonium sulfate fractionation and gel filtration on Sepharose-4B is resolved into two distinct peaks upon chromatography on DEAE-cellulose column. The heat inactivation profiles of the two enzyme forms are different, but other properties so far studied are similar such as K m values, pH optima, and activities toward deoxy APS. Both forms are influenced in similar manner by ionic strength, metal ions and EDTA.

Sulfation of cholesterol in rat kidney and liver

Abstract Supernatant fractions obtained by high speed centrifugation of rat kidney and liver homogenates incorporated sulfate- 35 S into an endogenous lipid. The radioactive product was identified as cholesterol sulfate- 35 S by thin-layer chromatography and by co-crystallization with authentic cholesterol sulfate. More sulfate- 35 S was incorporated into the lipid fraction when non-radioactive cholesterol was added to the incubation mixture. When tritium labeled cholesterol was added to the supernatant fraction from kidney, tritium labeled cholesterol sulfate was formed. Following parenteral administration of sulfate- 35 S, cholesterol sulfate- 35 S was identified as the predominant radioactive lipid in the kidney and liver. These studies demonstrate the presence of cholesterol sulfotransferase activity in rat kidney and liver.

Biosynthesis of heparin evidence for the transfer of radioactive sulfate to small-molecular-weight acceptors

The incorporation of [35S]sulfate into the heparin fraction was studied in a high-speed supernatant from the Furth mass-cell tumor. The rate of incorporation was found to be linear over a 45-min period. Preincubation prior to the addition of carrier-free [35S]-sulfate caused a marked decrease in labeling of the heparin fraction. These results were interpreted to indicate that a dialyzable sulfate acceptor system was depleted during the preincubation period. The data support the hypothesis that sulfate is incorporated primarily into small-molecular-weight heparin precursors which are then polymerized to form heparin.