Purification of mitochondria and secretory granules isolated from pancreatic beta cells using Percoll and Sephacryl S-1000 superfine

Mitochondria and secretory granules were isolated from beta-cell-rich pancreatic islets of ob/ob mice. Crude fractions obtained by differential centrifugation were subjected to centrifugation on isotonic Percoll. The gradient medium was removed from the resulting fractions by gel filtration on Sephacryl S-1000 Superfine. When compared to the crude fractions, the purified mitochondrial fraction exhibited a 4.5-fold increase in citrate synthase activity, a 70% reduction of lysosomal arylsulfatase, and a 40% decrease of contamination with granular insulin. In the purified secretory granule fraction, the insulin content was as high as 60% of the total protein (albumin standard) with arylsulfatase unchanged and no detectable citrate synthase activity.     

Characterization of newly synthesized proteoglycans from rabbit menisci in organ culture.

Rabbit menisci were incubated with Na2 35SO4 in short-term organ culture to label newly synthesized proteoglycans. The radioactive products present in both tissue and culture medium were characterized separately with respect to distribution after ultracentrifugation in CsCl isopycnic density gradients, hydrodynamic size, interaction with hyaluronic acid, and glycosaminoglycan composition (types, size and content). Analysis of proteoglycan size by gel-filtration chromatography of the most-dense CsCl fractions (A1) on Sephacryl S-500 (associative conditions) resolved three species. A peak with Kav. approx. 0.7 was present in each chromatogram, and constituted the principal component in tissue extracts. Two other peaks with Kav. values of approx. 0.2 and 0.45 were also found. When the A1 fraction from tissue was subjected to CsCl-density-gradient ultracentrifugation under dissociative conditions, 71% of the recovered radioactivity was present in the most dense (A1D1) fraction. Incubation with hyaluronic acid of either A1 or A1D1 fraction from associative extract did not alter the apparent size of the labelled product, indicating a lack of aggregate formation. Meniscal proteoglycans showed an unusual and marked tendency to adsorb irreversibly to agarose and agarose-containing gel-filtration-chromatography media. High-pressure liquid-chromatographic analyses indicated that the sulphated glycosaminoglycans consisted of chondroitin 6-sulphate (72%), chondroitin 4-sulphate (19%) and dermatan sulphate (5%). Endo-beta-galactosidase (keratanase) digestion of the material failed to detect the presence of keratan sulphate. Of the labelled glycosaminoglycans, 95% was eluted from Sephacryl S-400 as a single symmetrical peak with a Kav. of 0.5. The results of studies with tissue extracts and culture medium were similar.     

Molecular Weight and Subunit Composition of the Sensitive to GABA-Ergic Ligands Cl<Superscript>−</Superscript>/HCO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack>-Stimulated Mg<Superscript>2+</Superscript>-ATPase from Plasma Membrane of Rat Brain

The molecular weight and subunit composition of Cl-,HCO3(-)- and picrotoxin-stimulated Mg2+-ATPase from rat brain plasma membrane solubilized in sodium deoxycholate were studied by gel filtration chromatography. The enzyme activity eluted from a Sephacryl S-300 column in a single peak associated with a protein of molecular weight approximately 300 kD and a Stokes radius of 5.4 nm. The enzyme-enriched fraction, concentrated and denatured by SDS, migrated through a Sephacryl S-200 column as three peaks with molecular weights of approximately 57, 53, and 45 kD. SDS-PAGE also showed three major protein bands with molecular weights of about 57, 53, and 48 kD. The molecular weight and subunit composition of the Cl- and HCO3(-)-stimulated Mg2+-ATPase from neuronal membrane of rat brain are similar with the molecular properties of GABA(A)-benzodiazepine receptor complex from mammalian brain but are different from those of P-type transport ATPases.     

Solubilization of peroxidase from porcine thyroids and characterization by photoaffinity labelling

Peroxidase was solubilized without proteolysis from porcine thyroid particulate fraction with the nonionic detergent, 1-O-n-octyl-beta-D-glucopyranoside. The enzyme was able to catalyze the oxidation of guaiacol and the iodination of bovine serum albumin (33 atoms of iodine per molecule protein). Binding studies performed with the partially purified enzyme indicated that the substrates thyroxine (T4) and tyrosine compete for the same binding site on the enzyme. Dissociation constants of 0.9 nM and 0.5 nM were found for T4 and tyrosine, respectively. After photoaffinity labelling with underivatized 125I-labelled T4, gel chromatography on Sephacryl S-1000 revealed a relative molecular weight of about 100 000 for the solubilized enzyme. The peroxidase activity and haem-absorbance peak coeluted from the Sephacryl S-1000 column. SDS-polyacrylamide gel electrophoresis under reducing conditions indicated two major radiolabelled polypeptides, Mr 83 000 and Mr 42 600, as well as a smaller peak at Mr 15 400. The 15 400 molecular weight species is probably not part of the peroxidase complex, since it could partially be removed by Sephadex G-25 prechromatography . Further analyses confirmed that the partially purified enzyme is a haemoprotein absorbing maximally at 412 nm. The Soret band is shifted to 423 nm by reducing agents and the haem-cyanide complex has a maximum absorbance at 416 nm.     

Immobilization of phospholipid vesicles and protein-lipid vesicles containing red cell membrane proteins on octyl derivatives of large-pore gels

For improved immobilization of phospholipid vesicles and protein-lipid vesicles (cf. Sandberg, M., Lundahl, P., Greijer, E. and Belew, M. (1987) Biochim. Biophys. Acta 924, 185-192) and for chromatographic experiments with vesicles containing membrane protein, we have prepared octyl sulfide derivatives of the large-pore gels Sephacryl S-1000 and Sepharose 2B with ligand concentrations up to 14 and 5 mumol/ml gel, respectively. The Sephacryl derivatives allowed higher flow rates, gave higher rates of adsorption and showed equally high or higher capacities than the Sepharose adsorbents. 'Small', 'medium' and 'large' vesicles of radii approx. 20, 50 and 100 nm showed distribution coefficients on Sephacryl S-1000 of 0.7, 0.5 and 0.05, respectively and could be immobilized on octyl sulfide-Sephacryl S-1000 in amounts corresponding to 110, 40 and 20 mumol of phospholipids per ml gel, respectively. 'Small' vesicles became absorbed onto this gel at a rate of 1.5 mumol of phospholipids per min per ml gel until 60 mumol of phospholipids had become immobilized, whereas the initial adsorption rate was about 0.4 mumol.min-1.ml-1 on octyl sulfide-Sepharose 4B (see reference above) and on octyl sulfide-Sepharose 2B. Lower ligand concentrations gave lower capacities for 'small' vesicles. When vesicles entrapping calcein were immobilized on octyl sulfide-Sephacryl S-1000 some calcein was released during the adsorption process. For 'small' and 'medium' vesicles, respectively, the leakage was 75 and 25% at a ligand concentration of 14 mumol/ml but only 3 and 2% at 5 mumol/ml. The internal volumes of immobilized 'small' and 'medium' vesicles were estimated at 0.97 and 2.9 microliters per mumol of phospholipid by determination of entrapped calcein, which could indicate vesicle radii 20 and 50 nm, respectively. The total volumes of immobilized 'medium' lipid vesicles and 'medium' protein-lipid vesicles containing integral membrane proteins from human red cells, were estimated at 2.9 and 2.0 microliters/mumol, respectively, by chromatography of D- and L-[14C]glucose and calcein on the octyl sulfide-Sephacryl S-1000 column before and after immobilization. These volumes are roughly consistent with the internal volume of the vesicles. A zone of D-glucose eluted 90 microliters later than a zone of L-glucose on a 4- or 5-ml column of octyl sulfide-Sephacryl S-1000 with immobilized 'medium' protein-lipid vesicles containing the glucose transporter from human red cells, probably since part of the internal vesicle volume was accessible to the D-glucose but not to the L-glucose. This indicates that the glucose transporter was active in the immobilized vesicles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solubilization, purification, and characterization of a truncated form of rat hepatic squalene synthetase.

Rat hepatic microsomal squalene synthetase (EC 2.5.1.21) was induced 25-fold by feeding rats with diet containing the hydroxymethylglutaryl-coenzyme A reductase inhibitor, fluvastatin, and cholestyramine, a bile acid sequestrant. A soluble squalene synthetase protein with an estimated mass of 32-35 kDa, as determined by gel filtration chromatography on Sephacryl S-200 column, was solubilized out of the microsomes by controlled proteolysis with trypsin. Approximately 25% of the activity was recovered in a soluble form. The enzyme was purified to homogeneity utilizing a series of column chromatography purification steps on DEAE-cellulose, hydroxylapatite, and phenyl-Sepharose sequentially. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Initial kinetic analysis indicated an S0.5 values for trans-farnesyl diphosphate of 1.0 microM and for NADPH of 40 microM. The Vmax with respect to trans-farnesyl diphosphate was calculated at 1.2 mumol/min/mg. NADH also serves as substrate for the reaction with S0.5 value of 800 microM. Western blot analysis utilizing rabbit antisera raised against the purified, trypsin-truncated enzyme showed a single band for the isolated solubilized enzyme at 32-33 kDa and a band for the intact microsomal enzyme at about 45-47 kDa.     

Purification and properties of glucoside 3-dehydrogenase from Flavobacterium saccharophilum

A membrane-bound glucoside 3-dehydrogenase [EC 1.1.99.13], which oxidizes validoxylamine A to the 3-keto derivative, was solubilized from the membrane fraction of Flavobacterium saccharophilum by Triton X-100 and purified about 280-fold with an overall yield of 30% from the membrane fraction by column chromatography on DEAE- and CM-Sepharose CL-6B and gel filtration on Sephacryl S-300. The purified enzyme exhibited a single protein band on disc gel electrophoresis, and FAD was shown to be the prosthetic group. The enzyme had a molecular weight of 270,000 as determined by gel filtration on Sephacryl S-300 and consisted of 4 identical subunits each with a molecular weight of 66,000. The enzyme reacted with various artificial electron acceptors such as 2,6-dichlorophenolindophenol (DCIP), phenazine methosulfate, and ferricyanide. The optimum pH for DCIP reductase activity was 6.0. The enzyme was inhibited by Hg2+ and p-chloromercuribenzoate. D-Glucose and methyl-alpha- and beta-D-glucoside showed the highest susceptibility to the enzyme, and were converted to the corresponding 3-keto sugars.     

Resolution and purification of taurine- and GABA-synthesizing decarboxylases from calf brain

The present work describes a procedure for the co-purification of cysteine sulfinate decarboxylase (CSAD) and glutamate decarboxylase (GAD) from calf brain. A crude enzyme preparation was first made from brain homogenate by acid precipitation and ammonium sulphate fractionation. Subsequent fractionation of the decarboxylase preparation by cation exchange chromatography on CM-Sepharose CL-6B revealed the existence of a specific CSAD enzyme, which has no GAD activity. The GAD activity peak was found to possess CSAD activity. Further fractionation by gel filtration on Sephacryl S-200 separated the specific CSAD activity into two enzyme forms, one of them having a molecular weight of 150,000 and the other of 71,000. GAD activity was eluted from the gel filtration column in a single peak (mol wt 330,000) and showed CSAD activity. The purification of the specific CSAD enzyme was 920-fold and that of GAD activity 850-fold as compared with the starting material, whole calf brain. SDS gel electrophoresis indicated that the purified CSAD and GAD enzymes consisted of two or more subunits. The crude decarboxylase preparation was analysed by isoelectric focusing in ultra-thin polyacrylamide gel in the pH range 3.5-10.0. The most active fraction of CSAD indicated an isoelectric point of 6.5 and that of GAD 6.8. The pH optimum for CSAD activity in the crude preparation was 7.2 and that for GAD activity 7.9.     

Separation of Escherichia coli penicillin-binding proteins into different membrane vesicles by agarose electrophoresis and sizing chromatography.

Membrane vesicles from the envelope of Escherichia coli were separated by electrophoresis through dilute agarose and by sizing chromatography through Sephacryl S-1000. These techniques revealed that proteins were associated with different subsets of vesicles. In particular, dilute agarose electrophoresis clearly separated the inner membrane penicillin-binding proteins (PBPs) into different vesicle groups. Vesicles containing PBPs 4, 6, 7, and 8 migrated rapidly through agarose; vesicles with PBPs 1a, 1b, 2, 3, and 5 eluted later. With the exception of PBP 4, which migrated with PBPs 1 through 5, chromatography through Sephacryl S-1000 was able to distinguish the same two vesicle sets, though the extent of separation was poorer than with agarose. The existence of these associations among vesicles and proteins suggests that there is an organization to the inner membrane of E. coli which is not observed when membrane vesicles are separated solely on the basis of density in sucrose gradients.     

Immunochemical specificity of placental NADPH cytochrome c (P-450) reductase in neoplastic and non-neoplastic human tissue.

NADPH cytochrome c (P-450) reductase was purified from human placental microsomes using a combination of affinity and gel filtration chromatography. Affinity chromatography using agarose-hexane-adenosine 2'5 diphosphate resulted in two protein bands being detected by SDS-PAGE of approximate MwS 68 and 75 kDa. Fractions containing the two proteins were pooled, and then resolved using Sephacryl S-200. Both of the purified proteins displayed enzyme activity, measured by their ability to reduce cytochrome c. The 75 kDa protein obtained was used to immunize three female New Zealand white rabbits. The IgG fraction was partly purified from rabbit sera which suppressed placental microsomal NADPH cytochrome c reductase activity by > 80% using 33% ammonium sulphate. The procured antibody suppressed androstenedione aromatase activity in microsomal preparations of human placental and breast adipose tissue, and NADPH cytochrome c reductase activity in prostate (benign and malignant), MDA-MB-231 breast cancer cells, breast adipose, Hep G2 hepatoma cells and placental microsomal preparations. The extent of NADPH cytochrome c reductase inhibition varied in the order of malignant prostate < benign prostate < MDA < breast adipose < Hep G2 < placenta. The results suggest that human placental NADPH cytochrome c (P-450) reductase shares common antigenic epitopes pertinent to its capability of reducing cytochrome c in all of the above-mentioned tissues. In attempting to associate possible changes in NADPH cytochrome c reductase activity imposed by neoplasia to the obtained immunochemical cross reactivity and enzyme activity results, it was noted that microsomes obtained from MDA cells exhibited enzyme activity significantly less than that of breast adipose microsomes (1.6 and 8.1 nmol/min/mg protein, respectively) and by comparison showed 6% less homology towards the placental antibody. The results obtained for benign and malignant prostate showed no significant difference between the neoplastic states as adjudged by enzyme activity and immunochemical assays.     

Human placental steryl-sulfatase. Enzyme purification, production of antisera, and immunoblotting reactions with normal and sulfatase-deficient placentas

The steryl-sulfatase of normal human placental microsomes was solubilized and enriched about 350-fold. Chromatography on Sepharose 6B of the purified enzyme preparation revealed a single protein peak which eluted according to an apparent molecular mass of 270 +/- 30 kDa; when electrophorized on sodium dodecyl sulfate polyacrylamide gel the sulfatase migrated according to a molecular mass of 64 +/- 4 kDa. Estrogensulfatase activity was co-purified with the steryl-sulfatase activity; obviously, both activities belong to the same enzyme species. The purified sulfatase was injected into three rabbits. Antisera produced by the rabbits yielded a single sharp immunoprecipitation line in Ouchterlony double diffusion experiments when tested with the isolated sulfatase or with a solubilized microsomal fraction of normal placentas. The activity of sulfatase preparations incubated with antiserum was precipitated by addition of polyethylene glycol followed by centrifugation; none of the antibodies reacting with the sulfatase therefore appeared to interfere with its enzymatic activity. Using these antisera, steryl-sulfatase protein could be detected by immunoblotting analysis in solubilized microsomal fractions of normal placentas but not in solubilized microsomal fractions of three steryl-sulfatase activity-deficient placentas. This finding argues in favour of human placental steryl-sulfatase deficiency being due to extremely diminished or absent enzyme protein in the placenta.     

Large disulfide-stabilized proteoglycan complexes are synthesized by the epidermis of axolotl embryos

Proteoglycans (PGs) synthesized by the epidermis during stages crucial to the subepidermal migration of neural crest cells in the trunk of the axolotl (Ambystoma mexicanum, Urodela, Amphibia) embryo were studied. The glycosaminoglycan chains were biosynthetically labeled with [35S]sulfate in vitro during a period corresponding to the onset of migration. After extraction with guanidine HCl, the radiolabeled PGs were separated according to size by molecular-sieve chromatography on Sepharose CL-2B under dissociative conditions. This resulted in the separation of high-molecular-weight PGs, which eluted in the void volume, and low-molecular-weight PGs, eluting in a broad peak with a mean Kav of 0.7. The large PGs were also found to elute in the void volume when chromatographed on a Sephacryl S-1000 column. The low-molecular-weight PGs contained heparan sulfate and chondroitin sulfate (CS) and were not further characterized. The glycosaminoglycan component of the high-molecular-weight PG was completely degraded by chondroitinase ABC, while a large portion was resistant to chondroitinase AC, indicating the presence of dermatan sulfate (DS). These CS/DS chains were of unusually large size (Mr approximately 150,000) as estimated by chromatography on Sepharose CL-4B, relating the elution position to hyaluronan standards. Moreover, the chains were found to have a lower surface charge density than standard CS, and may therefore be undersulfated. After reduction and alkylation the high-molecular-weight PGs were included on both Sepharose CL-2B and Sephacryl S-1000 columns, eluting at Kav 0.2 and 0.4, respectively. Hence, the high-molecular-weight material appears to consist of large PG complexes, stabilized by intermolecular disulfide bonds. A CS/DSPG of similar size as the reduced monomeric form of the high-molecular-weight PG was found in small amounts in the total extract of 35S-labeled material.     

Purification and characterization of spermidine N1-acetyltransferase from chick duodenum

We have reported that spermidine N1-acetyltransferase has a larger role than ornithine decarboxylase in putrescine synthesis in chick duodenum induced by 1 alpha,25-dihydroxycholecalciferol (calcitriol) [Shinki, T., Kadofuku, T., Sato, T. and Suda, T. (1986) J. Biol. Chem. 261, 11712-11716]. In the present study, spermidine N1-acetyltransferase was purified from the duodenal cytosol of calcitriol-treated chicks to homogeneity judged by SDS/polyacrylamide gel electrophoresis. The purified enzyme converted spermidine only to N1-acetyl-spermidine. The apparent molecular mass of the purified spermidine N1-acetyltransferase was found to be 36 kDa by gel filtration on Sephacryl S-200 and 18 kDa by SDS/polyacrylamide gel electrophoresis. When duodenal crude 105,000 x g extracts were directly applied to a Sephacryl S-200 column without prior purification, three peaks with spermidine N1-acetyltransferase activity appeared. The first peak was in the void volume, the second peak was in the fraction corresponding to an apparent molecular mass of 70 kDa, and the third peak was in the fraction corresponding to 36 kDa. These results suggest that spermidine N1-acetyltransferase exists as a dimer of the 18 kDa subunits and is stabilized in (a) form(s) bound to other components or proteins in intact cells.     

SIMULTANEOUS ISOLATION OF PURIFIED MICROSOMAL and MYELIN FRACTIONS FROM RAT SPINAL CORD

Abstract— The fraction that sediments between 2 × 10⁵ g -min and 6 × 10⁶ g -min from dilute dispersions of rat brain in 0.32 m -sucrose is a microsomal fraction with very little contamination by myelin. A crude microsomal fraction prepared in the same way from rat spinal cord contains more myelin than microsomes. Centrifugation of the crude microsomal fraction in 0.85 m -sucrose gave a floating fraction, an infranatant fraction (purified microsomes) and a small pellet. The purified microsomes contained very little myelin as judged by electron microscopy and polyacrylamide gel electrophoresis. The lipid composition resembled that of spinal cord myelin except that the purified microsomes contained relatively less cholesterol and ethanolamine plasmalogens. The content of galactolipids was much greater in spinal cord microsomes than in brain microsomes. The spinal cord CDP-ethanol-amine:diglyceride ethanolaminephosphotransferase activity (EC 2.7.8.1) was concentrated in the purified microsomes.
A spinal cord myelin fraction isolated from the 2 × 10⁵ g -min pellet was quite pure as judged by electron microscopy, enzyme activities and polyacrylamide gel electrophoresis. No NADPH-cyto-chrome c reductase activity (EC 1.6.2.3) could be detected in the purified myelin. The ethanolaminephosphotransferase specific activity was about 5% of that found in the purified microsomal fraction. The protein content was 25% by weight for spinal cord myelin and 31% for brain myelin. Of the total spinal cord 2',3'-cyclic nucleotide-3'-phosphohydrolase activity, 16% was lost from the crude myelin during purification, 21% was recovered in the purified myelin, and 11% was found in the floating fraction from the crude microsomes. The purified myelin and microsomal fractions from spinal cord were relatively pure. Additional myelin was recovered in the floating fraction from the crude microsomes.     

cAMP-dependent protein kinase substrates in platelets. Evidence that thrombolamban, a 22,000 dalton substrate, and the Ca++-ATPase are not associated proteins

Inhibition of platelet function by cAMP is due at least in part to a reduction in the agonist stimulated increase in cytoplasmic calcium during cell activation. This inhibition is also associated with cAMP-dependent phosphorylation of thrombolamban, a 22 kDa phosphoprotein which is present in the same membrane fraction as the calcium-dependent ATPase. Phosphorylation of this protein has been correlated with increased uptake of calcium by microsomal membranes. The present study was undertaken to examine the interaction of thrombolamban with the Ca++-ATPase in order to assess the possibility that the increased calcium uptake was by a direct effect of thrombolamban on Ca++-ATPase activity or that thrombolamban was a component of the Ca++-ATPase. Several approaches were utilized to assess the interaction of thrombolamban with the microsomal Ca++-ATPase. Gel filtration of labeled microsomes solubilized under non-denaturing conditions showed a major peak of radioactivity (Kav 0.64) corresponding to thrombolamban which was well separated from the Ca++-ATPase activity (Kav 0.09). Chemical cross-linking studies using partially purified thrombolamban and intact microsomes showed incorporation of the phosphoprotein into a 147,000 dalton complex. Indirect immunostaining with an anti-Ca++-ATPase antibody failed to demonstrate the Ca++-ATPase in the 147,000 dalton complex. Recombination of the phosphorylated thrombolamban with the Ca++-ATPase had no effect on Ca++-ATPase activity. These results indicate that, under the conditions used in these experiments, there was no apparent interaction between thrombolamban and the microsomal Ca++-ATPase. We conclude that thrombolamban is covalently bound to the Ca++-ATPase.     

Entrapment of lipid vesicles and membrane protein-lipid vesicles in gel bead pores

Phospholipid vesicles were entrapped in gel beads of Sepharose 6B and Sephacryl S-1000 during vesicle preparation by dialysis. Egg-yolk phospholipids solubilized with cholate or octyl glucoside were dialysed together with gel beads for 2.5 days in a flat dialysis bag. Some vesicles were formed in gel bead pores and vesicles of sufficient size became trapped. Red cell membrane protein-phospholipid vesicles could be immobilized in the same way. Non-trapped vesicles were carefully removed by chromatographic procedures and by centrifugation. The amount of entrapped vesicles increased with the initial lipid concentration and was dependent on the relative sizes of vesicles and gel pores. The largest amount of trapped vesicles, corresponding to 9.5 mumol of phospholipids per ml gel, was achieved when Sepharose 6B gel beads were dialysed with cholate-solubilized lipids at a concentration of 50 mM. In this case the vesicles had an average diameter of 60 nm and an internal volume of 15 microliters/ml gel. The amount of vesicles trapped in Sephacryl S-1000 gel beads upon dialysis under the same conditions was smaller: 2.2 mumol of phospholipids per ml gel. Probably most of the gel pores were too large to trap such vesicles. Larger vesicles, with an average diameter of 230 nm, were entrapped in the Sephacryl S-1000 matrix in an amount corresponding to 3.0 mumol phospholipids per ml gel upon dialysis of the gel beads and octyl glucoside-solubilized lipids at a concentration of 20 mM. The internal volume of these vesicles was 22 microliters/ml gel. The yield of immobilized phospholipids was up to 19%. The entrapped vesicles were somewhat unstable: 9% of the phospholipids were released during 9 days of storage at 4 degrees C. By the dialysis entrapment method vesicles can be immobilized in the gel beads without using hydrophobic ligands or covalent coupling.     

Sulfated proteoglycans and sulfated proteins in guinea pig megakaryocytes and platelets in vivo. Relevance to megakaryocyte maturation and platelet activation

This study has examined changes in proteoglycan synthesis during megakaryocyte maturation in vivo. Guinea pigs were injected with Na235SO4, and megakaryocytes and platelets were isolated from 3 h to 5 days later. The proteoglycans and other sulfated molecules in both cells were characterized at each time point by gel filtration, ion-exchange chromatography, gel electrophoresis, and chemical and enzymatic digestions. Two populations of chondroitin 6-sulfate proteoglycans were found by DEAE-Sephacel chromatography. The major fraction was eluted with 4 M guanidine hydrochloride and the minor fraction with 4 M guanidine HCl, 2% Triton X-100. The Kav of the major proteoglycan peak in the platelets at 1 day after injection was 0.18-0.20 on Sepharose CL-6B and decreased gradually to 0.12 by 3 days, when proteoglycan radioactivity per cell was maximal. The peak for megakaryocyte proteoglycans at 3 h was broad, with Kav = 0.1-0.2. The appearance of different portions of the proteoglycan peak in platelets coincided with their disappearance from megakaryocytes. Proteoglycan size was a function of glycosaminoglycan chain length. The proteoglycans eluted with Triton X-100 from DEAE-Sephacel (Kav = 0.04-0.07 on Sepharose CL-6B) were not labeled in platelets until 2 days after injection. Our data suggest that megakaryocytes synthesize different-sized chondroitin sulfate proteoglycans at different stages of development. The proteoglycans of the major fraction were released from platelets in response to thrombin, and a small amount was released by ADP. The proteoglycans of the Triton X-100 eluate were not released by thrombin or ADP. About 20% of the sulfate radioactivity was incorporated into molecules that appear to be sulfated proteins and were not released by thrombin or ADP.     

Purification and properties of 3-ketovalidoxylamine A C-N lyase from Flavobacterium saccharophilum

3-Ketovalidoxylamine A C-N lyase was purified about 900-fold from the cell-free extract of Flavobacterium saccharophilum by ammonium sulfate fractionation, column chromatography on CM cellulose and gel filtration on Sephacryl S-200. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 36,000 by gel filtration on Sephacryl S-200 and by SDS polyacrylamide gel electrophoresis, indicating that the enzyme is a monomer. The optimum pH was found at 9.0. The enzyme activity was inhibited by EDTA or ethyleneglycol bis(beta-aminoethylether)-N,N'-tetraacetic acid and the inhibition was reversed by Ca2+ ion. The enzyme was able to eliminate p-nitroaniline or p-nitrophenol from p-nitrophenyl-3-ketovalidamine (IV) or p-nitrophenyl-alpha-D-3-ketoglucoside (VI), but not from p-nitrophenyl-1-epi-3-ketovalidamine or p-nitrophenyl-beta-D-3-ketoglucoside. Apparent Km values for IV and VI were 0.24 mM and 0.5 mM, respectively.     

Chromatographic properties of Sephacryl S-300 Superfine.

Sephacryl S-300 Superfine is a new gel filtration medium that consists of allyldextran crosslinked with N,N'-methylenebisacrylamide. It was shown to have a separation range for proteins that extends to 1--2 x 10(6) dalton. The gel filtration properties were characterized by fractionation of human serum and of bovine colostrum. Thyroglobulin from pig could be isolated in one step in a very pure form. The flow properties were characterized by the relationship between flow rate and pressure drop. It was shown that Sephacryl has very good flow properties in comparison with Sephadex G-200 and that it can be used in large-scale columns.     

Comparative studies of rodent anionic arylsulfatases

Approximately 25 and 40%, respectively, of murine (Mus musculus) and rat (Rattus norvegicus) hepatic arylsulfatase (EC 3.1.6.1) activity eluted from DEAE-ion exchange resins under high salt conditions. This high salt fraction contained arylsulfatase A and an enzyme which was immunologically similar to arylsulfatase B. The latter enzyme was thermostable, resistant to inhibition by silver, completely inhibited by phosphate, displayed linear kinetics, and had a higher pH optimum than arylsulfatase A. Anionic arylsulfatase B also hydrolyzed chondroitin-4-SO4 heptasaccharide. Sephacryl S-300 gel filtration resolved anionic arylsulfatase B into 55 and 115 kd fractions. Rodent arylsulfatase A activity was grossly underestimated when 4-methyl-umbelliferyl sulfate was employed as substrate.