Decrease In Apparent Molecular Weight Research Articles

Reprocessed magnetorheological elastomers with reduced carbon footprint and their piezoresistive properties

Despite the vast amount of studies based on magnetorheological elastomers (MREs), a very limited number of investigations have been initiated on their reprocessing. This paper presents a new type of recyclable MRE which is composed of thermoplastic polyurethane (TPU) and carbonyl iron particles (CI). The chosen TPU can be processed using injection moulding (IM), followed by several reprocessing cycles while preserving its properties. Numerous types of injection moulded and reprocessed MREs have been prepared for various particle concentrations. The effect of thermo-mechanical degradation on the recycled MREs has been investigated while simulating the reprocessing procedure. An apparent decrease in molecular weight was observed for all the examined matrices during the reprocessing cycles. These changes are attributed to the intermolecular bonding between the hydroxyl groups on the surface of the CI particles and the matrix which is studied in depth. The effect of reprocessing and the presence of magnetic particles is evaluated via tensile test, magnetorheology and piezoresistivity. These characterization techniques prove that the properties of our MREs are preserved at an acceptable level despite using 100% of recyclates while in real applications only up to 30% of recycled material is generally used.

Asymmetric anionic polymerization of 2,7‐bis(4‐t‐butylphenyl)fluoren‐9‐ylmethyl methacrylate and photoinduced structural transition of the obtained polymer

2,7-Bis(4-t-butylphenyl)fluoren-9-ylmethyl methacrylate (BBPFMMA) was synthesized as a new candidate of a bulky monomer suitable for asymmetric anionic polymerization (IUPAC nomenclature: asymmetric helix-chirogenic polymerization) leading to a preferred-handed helical, optically active polymer. The new monomer was polymerized using the complex of 9-fluorenyllithium and (S)-(+)-1-(2-pyrrolidinylmethyl)pyrrolidine in toluene at -78 °C. As opposed to the original intention to obtain a polymethacrylate, the monomer seemed to undergo structural transition (decomposition) giving 2,7-bis(4-t-butylphenyl)dibenzofulvene and the obtained polymer was most probably a terpolymer of BBPFMMA, 2,7-bis(4-t-butylphenyl)dibenzofulvene, and methacrylic acid. The obtained polymer showed circular dichroism (CD) spectra, indicating that chirality had been introduced to the copolymer chain during the course of polymerization. On photo irradiation, the polymer underwent a structural transition involving a remarkable decrease in apparent molecular weight measured by size-exclusion chromatography (SEC) and changes in FT-IR spectral patterns. However, the CD spectrum of the polymer was not obviously altered by irradiation, indicating that polymer chirality is not affected by light.

Developmental expression of OAP-1/Tspan-3, a member of the tetraspanin superfamily.

OSP/claudin-11-associated protein (OAP-1/Tspan-3), originally isolated by yeast two-hybrid screening using OSP/claudin-11 (oligodendrocyte-specific protein) as bait, is a member of the tetraspanin superfamily and OAP-1/Tspan-3, OSP/claudin-11, and beta1 integrin form a protein complex that seems to be involved in oligodendrocyte proliferation and migration. This study investigated the temporal and regional expression, glycosylation status, and tissue distribution of OAP-1/Tspan-3. OAP-1/Tspan-3 mRNA was expressed as a single transcript throughout brain development, with high levels of expression in the germinal zones. OAP-1/Tspan-3 protein contains N-terminal glycosylation sites in extracellular loop 2 and deglycosylation studies indicated a decrease in apparent molecular weight of OAP-1/Tspan-3, consistent with removal of N-glycans. Similar to OSP/claudin-11, OAP-1/Tspan-3 is expressed in all stages of oligodendrocyte development and in the myelin sheath. Unlike OSP/claudin-11, however, it is expressed in all cell types tested in the central nervous system (CNS), including neurons and astrocytes. The association of OAP-1/Tspan-3 with OSP/claudin-11 and beta1 integrin, its subcellular distribution as a cell surface, membrane-spanning glycoprotein, and its widespread distribution supports its potential role in cell migration, proliferation, and interactions between cells and extracellular matrix.

The Use of Cellulase in Inhibiting Biofilm Formation from Organisms Commonly Found on Medical Implants

A study was made of the use of cellulase to inhibit biofilm formation by a pathogenic bacterium commonly found in medical implants. A Pseudomonas aeruginosa biofilm was grown on glass slides in a parallel flow chamber for 4 d with glucose as the nutrient source. Biofilm development was assessed by measuring the colony forming units (CFU) and biomass areal density. Biofilm was grown at pH 5 and 7 in the presence of three different cellulase concentrations, 9.4, 37.6 and 75.2 units mlm 1. In addition, a control study using deactivated cellulase was performed. The results show that cellulase is effective in partially inhibiting biomass and CFU formation by P. aeruginosa on glass surfaces. The effect of cellulase depended on concentration and was more effective at pH 5 than pH 7. The experiment was further extended by investigating the effect of cellulase on the apparent molecular weight of purified P. aeruginosa exopolysaccharides (EPS). The observation of EPS using size exclusion chromatography showed a decrease in apparent molecular weight when incubated with enzyme. An increase in the amount of reducing sugar with time when the purified EPS were incubated with enzyme also supports the hypothesis that cellulase degrades the EPS of P. aeruginosa. While cellulase does not provide total inhibition of biofilm formation, it is possible that the enzyme could be used in combination with other treatments or in combinations with other enzymes to increase effectiveness.

Syndecan-1 is up-regulated in ras-transformed intestinal epithelial cells.

The syndecans, a family of cell-surface heparan sulphate proteoglycans, have been proposed to mediate cellular interactions with extracellular effector molecules, such as growth factors and components of the extracellular matrix, during critical phases of development. Transcripts of all four syndecans are expressed at varying levels in the developing rat intestine and in a series of immature rat intestinal epithelial cell lines. In addition, we report the novel finding that, in the intestinal epithelial cell lines, expression of syndecan-1 transcript is up-regulated by transformation with activated H-ras. This is in contrast to other cell lines in which ras transformation is associated with a decrease in syndecan-1 levels. The observed increase in the syndecan-1 occurs as a result of increased transcription and can be correlated with the degree of transformation of the IEC-18 cells. Transformation is also associated with a decrease in apparent molecular weight and increased shedding of the proteoglycan into the culture medium. Increased shedding of syndecan-1 into the culture medium after transformation with H-ras may contribute to the disruption of proteoglycan interactions with the extracellular matrix, leading to alterations in cell adhesion and organization.

Murine Granulocytic Cell Adhesion to Bone Marrow Hemonectin Is Mediated by Mannose and Galactose

Hemonectin (HN) is a bone marrow (BM) protein that promotes specific attachment of immature granulocytes and their precursors within the BM. We report that HN is a glycoprotein containing both mannose and galactose residues, and provide evidence that these carbohydrates mediate granulocytic cell adhesion to HN. Carbohydrate structure was determined by digoxigenin-conjugated lectin binding to HN and indicated the presence of mannose, galactose, sialic acid, and the absence of fucose-linked oligosaccharides. The role of carbohydrates in mediating cell adhesion was examined by chemical and enzymatic deglycosylation. Deglycosylation of HN with trifluoromethanesulfonic acid, which cleaves N- and O-linked oligosaccharides, inhibits 66% of cell attachment to HN, and results in an apparent decrease in molecular weight from 60 to 50 kD. Enzymatic deglycosylation with endo-B-N-acetylglucosaminidase H, which hydrolyzes specific N-linked mannose residues, inhibits 30% of cell adhesion to HN. Finally, the role of these specific sugars in hemonectin-mediated cell adhesion was confirmed with neoglycoprotein blocking. Preincubation of BM cells with mannosyl- and galactosyl-BSA probes produces a dose-dependent inhibition of cell attachment to HN, whereas fucosyl-BSA does not inhibit cell adhesion to HN. These results show that mannose and galactose partially mediate adhesion of BM granulocytes to HN.

Human immunodeficiency virus type 1 envelope glycoprotein is modified by O-linked oligosaccharides

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein has been shown to be extensively modified by N-linked glycosylation; however, the presence of O-linked carbohydrates on the glycoprotein has not been firmly established. We have found that enzymatic deglycosylation of the HIV-1 envelope glycoprotein with neuraminidase and O-glycosidase results in a decrease in the apparent molecular weight of the envelope glycoprotein. This result was observed in both vaccinia virus recombinant-derived envelope glycoproteins and glycoproteins derived from the IIIB, SG3, and HXB2, strains of HIV-1. The decrease in molecular weight was also observed when the envelope glycoprotein had been deglycosylated with N-glycanase F after treatment with neuraminidase and O-glycosidase, indicating that the decrease in apparent molecular weight was not attributable to the removal of N-linked carbohydrate. Treatment with neuraminidase, O-glycosidase, and N-glycanase F was found to be necessary to remove all radiolabel from [3H]glucosamine-labelled envelope glycoprotein, a result seen for both recombinant and HIV-1-derived envelope glycoprotein. [3H]glucosamine-labelled carbohydrates liberated by O-glycosidase treatment were separated by paper chromatography and were found to be of a size consistent with O-linked oligosaccharides. We, therefore, conclude that the HIV-1 envelope glycoprotein is modified by the addition of O-linked carbohydrates.

Cell Wall Dissolution in Ripening Kiwifruit (Actinidia deliciosa)

Pectic polysaccharides solubilized in vivo during ripening, were isolated using phenol, acetic acid, and water (PAW) from the outer pericarp of kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang and A.R. Ferguson var deliciosa ;Hayward') before and after postharvest ethylene treatment. Insoluble polysaccharides of the cell wall materials (CWMs) were solubilized in vitro by chemical extraction with 0.05 molar cyclohexane-trans-1,2-diamine tetraacetate (CDTA), 0.05 molar Na(2)CO(3), 6 molar guanidinium thiocyanate, and 4 molar KOH. The Na(2)CO(3)-soluble fraction decreased by 26%, and the CDTA-soluble fraction increased by 54% 1 day after ethylene treatment. Concomitantly, an increase in the pectic polymer content of the PAW-soluble fraction occurred without loss of galactose from the cell wall. The molecular weight of the PAW-soluble pectic fraction 1 day after ethylene treatment was similar to that of the Na(2)CO(3)-soluble fraction before ethylene treatment. Four days after ethylene treatment, 60% of cell wall polyuronide was solubilized, and 50% of the galactose was lost from the CWM, but the degree of galactosylation and molecular weight of pectic polymers remaining in the CWMs did not decrease. The exception was the CDTA-soluble fraction which showed an apparent decrease in molecular weight during ripening. Concurrently, the PAW-soluble pectic fraction showed a 20-fold reduction in molecular weight. The results suggest that considerable solubilization of the pectic polymers occurred during ripening without changes to their primary structure or degree of polymerization. Following solubilization, the polymers then became susceptible to depolymerization and degalactosidation. Pectolytic enzymes such as endopolygalacturonase and beta-galactosidase were therefore implicated in the degradation of solubilized cell wall pectic polymers but not the initial solubilization of the bulk of the pectic polymers in vivo.

Glycosaminoglycan conformations and changes on periodate oxidation

The progressive periodate oxidation of glycosaminoglycans (GAG), including hyaluronate (HA), chondroitins (CH) (chondroitin, chondroitin 4- and 6-sulfate), dermatan sulfate (DS), and keratan sulfate (KS), were monitored by CD and high performance liquid chromatography (HPLC) using a size-exclusion column. The rate of oxidation also was measured and calculated using first- and second-order kinetics, and the data appear to fit better with first-order kinetics. In both HA and CH, the n - pi amide band at 208 nm decreases in intensity upon oxidation, but in HA it becomes positive after 16 h of periodate treatment. In CH, the band disappears, and the pi - pi amide band below 200 nm becomes optically active. Concomitantly, a second negative band near 290 nm appears for these two oxidized GAG. Oxidation causes a slight change in the CD of DS. It ordinarily displays a very weak n - pi band at 210 nm, but instead shows an intense pi - pi amide band near 190 nm. CD of KS remains unaffected by periodate. Kinetic studies, however, show a higher oxidation rate for DS than HA and CH. With the exception of KS, all other oxidized polymers shown an apparent decrease in molecular weight (higher peak retention time) in HPLC analysis. Both CD and HPLC results have been attributed to a major conformational change of HA and CH, and a minor one for DS. The ease and extent of periodate oxidation as well as the changes in molecular properties following periodate treatment are critically dependent on the configuration of the individual GAG rather than the oxidation rate. There is a distinct difference in the conformational change between HA and CH, as manifested by their dichroic behavior, that was attributed to the equatorial disposition of C-4 hydroxyl group in HA and axial disposition CH.

Proteolytic fragmentation and peptide mapping of human carboxyamidomethylated tracheobronchial mucin

Human tracheobronchial mucin was isolated from lung mucosal gel by chromatography on Sepharose 4B in the presence of dissociating and reducing agents, and its thiol residues were carboxyamidomethylated with iodo[1(-14)C]acetamide. The 14C-carboxyamido-methylated mucin was purified by chromatography on Sepharose 2B. No low molecular weight components were detected by molecular sieve chromatography or polyacrylamide gel electrophoresis in the presence of dissociating and reducing agents or by analytical density centrifugation in CsCl/guanidinium chloride. After digestion of the purified 14C-mucin with trypsin-L-1-tosylamido-2-phenylethyl chloromethyl ketone, three fractions (TR-1, TR-2, and TR-3) were observed by chromatography on Sepharose 4B. TR-1, a 260-kDa mucin glycopeptide fragment, contained all of the neutral hexose and blood group activity and 20% of the radioactivity in the undigested mucin. TR-1 was refractory to a second incubation with trypsin but could be digested by papain or Pronase to a smaller mucin glycopeptide fraction, as judged by the slight decrease in apparent molecular weight on Sepharose CL-4B. These mucin glycopeptides contained approximately 50% of the radioactivity in the TR-1 fraction, indicating that the glycosylated domains of carboxyamidomethylated tracheobronchial mucin contained thiol residues. The remainder of the radioactivity from papain or Pronase digests of TR-1 eluted, like the TR-3 fractions, in the salt fraction on Sepharose CL-4B. Peptide mapping of the nonglycosylated TR-3 fraction by TLC and high voltage electrophoresis yielded six principal and several less intensely stained ninhydrin reactive components, with the radiolabel concentrated in one of the latter peptides. Peptide purification of the TR-3 fraction by high pressure liquid chromatography on a C18 reverse phase column demonstrated the presence of four major peptides, with TR-3A being the dominant component. The TR-3D peptide contained S-carboxy-aminomethylcysteine and had 69% sequence similarity to the sgs-7 salivary glue protein of Drosophila.

Mutations affecting assembly and stability of tubulin: evidence for a nonessential beta-tubulin in CHO cells.

Eight strains of Chinese hamster ovary (CHO) cells having an assembly-defective beta-tubulin were found among revertants of strain Cmd 4, a mutant with a conditional lethal mutation in a beta-tubulin gene (F. Cabral, M. E. Sobel, and M. M. Gottesman, Cell 20:29-36, 1980). The altered beta-tubulins in these strains have electrophoretically silent alterations or, in some cases, an increase or a decrease in apparent molecular weight based on their migration in two-dimensional gels. The identity of these variant proteins as beta-tubulin was confirmed by peptide mapping, which also revealed the loss of distinct methionine-containing peptides in the assembly-defective beta-tubulins of lower apparent molecular weight. The altered mobility of these beta-tubulin polypeptides was not the result of a posttranslational modification, since the altered species could be labeled in very short incubations with [35S]methionine and were found among in vitro-translated polypeptides by using purified mRNA. In at least one strain, an altered DNA restriction fragment could be demonstrated, suggesting that an alteration occurred in one of the structural genes for beta-tubulin. Assembly-defective beta-tubulin was unstable and turned over with a half-life of only 1 to 2 h in exponentially growing cells. This rapid degradation of a tubulin gene product resulted in approximately 30% lower steady-state levels of both alpha- and beta-tubulin yet did not affect the growth rate of the cells or the distribution of the microtubules as judged by immunofluorescence microscopy. These results argue that CHO cells possess a beta-tubulin gene product that is not essential for survival.

Mutations affecting assembly and stability of tubulin: evidence for a nonessential beta-tubulin in CHO cells.

Eight strains of Chinese hamster ovary (CHO) cells having an assembly-defective beta-tubulin were found among revertants of strain Cmd 4, a mutant with a conditional lethal mutation in a beta-tubulin gene (F. Cabral, M. E. Sobel, and M. M. Gottesman, Cell 20:29-36, 1980). The altered beta-tubulins in these strains have electrophoretically silent alterations or, in some cases, an increase or a decrease in apparent molecular weight based on their migration in two-dimensional gels. The identity of these variant proteins as beta-tubulin was confirmed by peptide mapping, which also revealed the loss of distinct methionine-containing peptides in the assembly-defective beta-tubulins of lower apparent molecular weight. The altered mobility of these beta-tubulin polypeptides was not the result of a posttranslational modification, since the altered species could be labeled in very short incubations with [35S]methionine and were found among in vitro-translated polypeptides by using purified mRNA. In at least one strain, an altered DNA restriction fragment could be demonstrated, suggesting that an alteration occurred in one of the structural genes for beta-tubulin. Assembly-defective beta-tubulin was unstable and turned over with a half-life of only 1 to 2 h in exponentially growing cells. This rapid degradation of a tubulin gene product resulted in approximately 30% lower steady-state levels of both alpha- and beta-tubulin yet did not affect the growth rate of the cells or the distribution of the microtubules as judged by immunofluorescence microscopy. These results argue that CHO cells possess a beta-tubulin gene product that is not essential for survival.

Transepithelial transport of epidermal growth factor by absorptive cells of suckling rat ileum.

Epidermal growth factor (EGF), an acid-stable peptide present in rodent and human milk, is absorbed and promotes intestinal growth when fed to suckling rats. To determine whether absorptive cells of suckling rat ileum conduct selective transepithelial transport of EGF, we followed uptake of 125I-EGF from ileal loops by autoradiography and biochemical methods. Specific binding sites for 125I-EGF were localized by electron microscope autoradiography on apical membranes of ileal epithelial sheets in vitro. During uptake in vivo, radiolabeled molecules were concentrated in apical endosomal compartments and were also associated with lysosomal vacuoles, basolateral cell surfaces, and lamina propria. Excess cold EGF reduced basolateral label by 44% and TCA precipitable serum label by 38%. After 30 and 60 min of continuous uptake, radiolabeled molecules in epithelium, denuded mucosa, blood, and liver were analyzed under reducing conditions by reversed-phase high-pressure liquid chromatography (HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although considerable degradation of 125I-EGF occurred after uptake from the lumen, a portion of radiolabel in epithelium and mucosa represented 125I-EGF which eluted somewhat more rapidly from C18 HPLC columns and showed a slight decrease in apparent molecular weight by SDS-PAGE. All radiolabel in blood and liver represented breakdown products. Thus, EGF is selectively transported across the ileal epithelium in suckling rats but is modified during transport. Milk EGF may accumulate in the lamina propria where it could influence growth and maturation of the suckling intestine.

Regulation of low density lipoprotein receptor synthesis in cultured luteinized human granulosa cells by human chorionic gonadotropin and 8-bromo-cyclic AMP.

We investigated the regulation of synthesis of low density lipoprotein (LDL) receptor in cultured luteinized human granulosa cells using a monoclonal antibody recognizing the human LDL receptor (IgG-C7). Cells cultured under serum-free conditions were treated with human chorionic gonadotropin (hCG) or 8-bromo-cAMP alone or in combination with aminoglutethimide (to block conversion of cholesterol to steroid hormones) and 5-cholesten-3 beta, 25-diol (25-hydroxycholesterol, a potent suppressor of LDL receptor expression in human fibroblasts) and pulse-labeled with [35S]methionine. A labeled protein immunoisolated with IgG-C7 was identified as the mature LDL receptor in 7.5% sodium dodecyl sulfate-polyacrylamide gels on the basis of an apparent molecular mass of 160 kDa, absence of the protein from immunoisolates prepared with a monoclonal antibody against an irrelevant antigen, and an apparent decrease in molecular weight of the mature receptor upon treatment with neuraminidase or electrophoresis under nonreducing conditions. hCG and 8-bromo-cAMP consistently increased the incorporation of radioactivity into the mature LDL receptor by 2-6-fold. The effect of hCG on LDL receptor synthesis was observed with as little as 10 mIU of hCG/ml and was apparent within 2 h of addition of the hormone. A combination of 25-hydroxycholesterol and aminoglutethimide resulted in a 60% suppression of label incorporation into mature LDL receptor compared to untreated cells. This would suggest some regulation of LDL receptor synthesis by negative feedback of sterol. However, both hCG and 8-bromo-cAMP increased label incorporation into the LDL receptor in the face of these agents. We conclude that in human granulosa cells, hCG, through the intermediacy of cAMP, rapidly increases LDL receptor synthesis by a mechanism which is, at least in part, independent of alterations in cellular cholesterol balance.

Structural characterization and the determination of negative cooperativity in the tight binding of 2-carboxyarabinitol bisphosphate to higher plant ribulose bisphosphate carboxylase.

When CO2/Mg2+-activated spinach leaf ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) is incubated with the transition-state analog 2-carboxyarabinitol 1,5-bisphosphate, an essentially irreversible complex is formed. The extreme stability of this quaternary complex has allowed the use of native analytical isoelectric focusing, anion-exchange chromatography, and nondenaturing polyacrylamide gel electrophoresis to probe the mechanism of the binding process and the effects of ligand tight-binding on the structure of the protein molecule. Changes in the chromatographic and electrophoretic properties of the enzyme upon tight binding of the inhibitor reveal that the ligand induces a conformational reorganization which extends to the surface of the protein molecule and, at saturation, results in a 16% decrease in apparent molecular weight. Analysis of ligand binding by isoelectric focusing shows that (i) incubating the protein with a stoichiometric molar concentration of ligand (site basis) results in an apparently charge homogeneous enzyme population with an isoelectric point of 4.9, and (ii) substoichiometric levels of ligand produce differential effects on each of the charge microheterogeneous native enzyme forms. These stoichiometry-dependent changes in electrofocusing band patterns were employed as a probe of cooperativity in the ligand tight-binding process. The tight-binding reaction was shown to be negatively cooperative.

Role of singlet oxygen in the degradation of hyaluronic acid

To investigate the effect of singlet oxygen on the molecular properties of hyaluronic acid, the polymer was irradiated in the presence of a dye sensitizer for singlet oxygen. Viscosity and circular dichroism techniques were used to monitor these changes. The relative viscosity of the polymer solution decreased steadily with increasing duration of irradiation, indicating an apparent decrease in molecular weight of hyaluronic acid. Circular dichroism measurements of the irradiated sample, however, did not show any appreciable change in the secondary structure, but do suggest that the generated singlet oxygen changes the tertiary structure and that this change is followed by a minor depolymerization.

Effect of carboxypeptidase digestion of the human choriogonadotropin molecule on its thyrotropic activity.

Digestion of hCG by a mixture of carboxypeptidases B and Y results in an enzyme dose- and incubation time-dependent increase in its ability to stimulate the adenylate cyclase (AC) of human thyroid membranes. Treated under the same conditions [3 h at 37 C; enzyme to hCG ratio, 0.04 (wt/wt)], partially and highly purified hCG preparations display an increase of about 300% in thyroid AC-stimulating activity, while TSH displays a 30% decrease. In contrast, carboxypeptidase digestion of hCG under these conditions has no significant effect on its activity in the rat testis AC assay. The carboxypeptidase digestion results in cleavage of carboxyl-terminal amino acid residues 142--145 from the hCG beta-subunit; digestion of the hCG alpha-subunit is much less effective, as the carboxy-terminal amino acid residue 92 is removed from only about 13% of the hCG molecules. In accord with the results of amino acid analysis, a slight, if any, decrease in apparent molecular weight is found by gel filtration and polyacrylamide gel electrophoresis. In addition, carboxypeptidase digestion results in antigenic alterations of the molecule, as shown by a flatter slope of the dose-response curve in a hCG RIA and a 70% decrease in potency in a RIA that uses an antiserum to the hCG beta carboxy-terminal peptide. These data demonstrate that partial digestion of the hCG molecule with carboxypeptidase results in an increase in human thyroid AC-stimulating activity, with retention of the rat testis AC-stimulating activity.

Further characterization of two forms of N-acetyl-α-galactosaminidase from human liver

1. 1. 2 form of N-acetyl-α-galactosaminidase (EC 3.2.1.22) can be isolated from human liver using Sepharose 4B-concanavalin A chromatography, followed by CM-cellulose chromatography. Both forms hydrolyse not only p-nitrophenyl- N-acetyl-α-galactosaminide but also p-nitrophenyl-α-galactoside. These two forms were formerly known as α-galactosidase B and A-like α-galactosidase (Schram, A.W., Hamers, M.N., Brouwer-Kelder, B., Donker-Koopman, W.E. and Tager, J.M. (1977) Biochim. Biophys. Acta 482, 125–137; Schram, A.W., Hamers, M.N. and Tager, J.M. (1977) Biochim. Biophys. Acta 482, 138–144). 2. 2. In fresh normal liver, most of the N-acetyl-α-galactosaminidase activity is found in the fractions containing α-galactosidase B and a minor portion in the fractions containing α-galactosidase A. However, the minor peak of N-acetyl-α-galactosaminidase activity does not coincide with the peak of α-galactosidase A activity. Both peaks of N-acetyl-α-galactosaminidase activity can be precipitated by antiserum raised against α-galactosidase B but not by antiserum raised against α-galactosidase A. 3. 3. In fresh Fabry liver (which contains no α-galactosidase A) more than 98% of the N-acetyl-α-galactosaminidase activity was recovered in the fractions containing α-galactosidase B. Less than 2% was found in the fractions containing A-like α-galactosidase. Upon aging of the liver, the yield of the latter form increased (to almost 25% after 17 months storage at −20°C). 4. 4. The conversion of N-acetyl-α-galactosaminidase to the second form upon aging was accompanied by a decrease in apparent molecular weight from 110 000 ± 5000 to 99 000 ± 3000, as measured by Sephacryl S-200 chromatography. 5. 5. The kinetic properties of the enzyme change slightly upon aging. In contrast, there is no effect of aging on the pH optimum of the enzyme. 6. 6. This is concluded that only one lysosomal N-acetyl-α-galactosaminidase is present in human liver and that one of the two forms of N-acetyl-α-galactosaminidase observed in extracts of liver is formed by chemical modification of the enzyme during storage of the liver or during storage of the purified preparation.

Association of polyacrylonitrile prepared by low‐temperature, solution polymerization with an organometallic catalyst

AbstractThe weight‐average molecular weights of polymers of acrylonitrile prepared by a free‐radical initiator and an organometallic catalyst have been determined by lightscattering measurements in N,N‐dimethylformamide, dimethyl sulfoxide, and dimethylacetamide at 25°C. and in dimethyl sulfoxide at 140°C. The apparent molecular weights of the polymers prepared with the NaAlEt3S(i‐Pr) catalyst in DMF at −78°C. (referred to as high‐melting polymers) changed from 54,800, 82,700, and 480,000 when measured in DMF at 25°C. to 36,000, 41,600, and 225,000 when measured in DMSO at 140°C., whereas the molecular weights of the free‐radical polymers remained unchanged. Furthermore, from results obtained in DMSO at 140°C., The intrinsic viscosity–molecular‐weight relationships were found to be identical for the high‐melting and the free‐radical polymer and in substantial agreement with an equation reported by Cleland and Stockmayer. The apparent decrease in molecular weight of the high‐melting polymer from 25 to 140°C. indicates rather clearly that the high‐melting polymers are associated in DMF at 25°C. The “aggregates,” even though present only at low concentrations, raised the weight‐average molecular weight markedly but affected the number‐average molecular weight only slightly, thus giving a high M̄w/M̄n ratio. It appears likely that when temperature and solvent are such that association does not occur, linear PAN's will have approximately the same intrinsic viscosity–molecular weight relationship (subject of course to slight change by polydispersity). The often reported abnormal molecular weight of samples prepared by solution polymerization especially at low temperatures, may be attributed to branching, or to an association, as reported here. The nature of association of PAN in dilute solution is also discussed.