Radioimmune assay of sialyltransferase and N-acetylgalactosaminyltransferase activities using specific antibodies on a 96-well filtration plate of a multiscreen assay system.

A new assay method for glycosphingolipid glycosyl-transferase activities was developed using a 96-well filtration plate of a MultiScreen assay system. An acceptor glycosphingolipid and a donor radioactive nucleotide sugar were incubated with an enzyme source in a well of the filtration plate. After incubation, both identification and quantification of the reaction product were carried out simultaneously using a specific antibody for the product which was trapped on a filtration membrane of the plate as a complex with Staphylococcus aureus protein A (IgGSorb). This assay method was used for determining the activity of cytidine 5'-monophosphate-N-acetylneuraminic acid:Lcn4Cer alpha 2----6sialyltransferase and uridine 5'-diphosphate-N-acetyl galactosamine:GM3 N-acetylgalactosaminyltransferase. In addition to the simple and rapid identification and quantification of the product, this method proved to be as reliable and sensitive as the previously published assay procedures. Furthermore, this assay method can be used with a high concentration of detergent which should not be used in the other procedures described previously using enzyme-linked immunosorbent assay methods on a 96-well multiplate even if the enzyme reaction might require a certain percentage of the detergent concentration.     

Direct assay of glycosphingolipid glycosyltransferase activities on thin-layer chromatogram

A modified method for the determination of glycosphingolipid glycosyltransferase activity using high-performance thin-layer chromatographic (HPTLC) plates has been developed. An acceptor glycosphingolipid was chromatographed on an HPTLC plate and was incubated with an enzyme mixture and an appropriate radioactive sugar nucleotide. After incubation, the plate was washed with phosphate buffer and 2% Tween 80. The radiolabeled reaction product was scrapped off the plate and the radioactivity determined using a liquid scintillation counter or, alternatively, the plate was exposed to an X-ray film to reveal the radioactive product. We have used this assay method to determine the activities of rat brain cytidine 5'-monophosphate-N-acetylneuraminic acid: LacCer-, GM3-, GM1-, or GD3-sialyltransferases. This method is sensitive, fast, and reliable and is capable of assaying simultaneously the activities of glycosyltransferases with multiple acceptor specificity. It should be useful in monitoring the enzyme activities present in various column fractions during chromatographic fractionation of glycosyltransferases with different substrate specificities.     

Isolation of two glycolipid transfer proteins from bovine brain: reactivity toward gangliosides and neutral glycosphingolipids

Two glycolipid transfer proteins that catalyze the transfer of gangliosides and neutral glycosphingolipids from phosphatidylcholine vesicles to erythrocyte ghosts have been isolated from calf brain. Purification procedures included differential centrifugation, precipitation at pH 5.1, ammonium sulfate precipitation, and gel filtration on Sephadex G-50 and G-75. The final stage employed fast protein liquid chromatography (Mono S), producing two peaks of activity. Apparent purity of the major peak (TP I) was approximately 85-90%, as judged by sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis. That of the minor fraction (TP II) was less. The major band of both fractions had a molecular mass of approximately 20,000 daltons. Both proteins catalyzed the transfer of ganglioside GM1 as well as asialo-GM1, but transfer protein I was more effective with di- and trisialogangliosides. Transfer protein II appeared to be somewhat more specific for neutral glycolipids in that GA1 was transferred more rapidly than any of the gangliosides; however, lactosylceramide transfer was relatively slow. Neither protein catalyzed transfer of phosphatidylcholine.     

Purification and properties of the polypeptide chain release factor (RF-4) from an extreme thermophile, Thermus thermophilus HB8.

The polypeptide chain release factor 1 (RF-1) has been purified from an extreme thermophile, Thermus thermophilus HB8. The purification procedure included steps of aqueous two-phase partition, ammonium sulfate fractionation, and column chromatographies on DEAE-Sephadex, Sephadex G-150, and CM-Sephadex. The preparation was more than 90% pure as judged by polyacrylamide gel electrophoresis. The specific activity was about 3.3 pmol of formyl-[3H]-methionine released in 1 min at 25 degrees C per microgram of protein under the standard assay conditions using 4 pmol of the initiation complex and 1 nmol of UpApG. The molecular weight as determined by gel filtration on Sephadex G-150 and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was 58,000 and 45,000, respectively. As expected, the factor was extremely heat-stable, 50% of its activity remaining after incubation for 5 min at 84 degrees C. Several properties of the reaction catalyzed by RF-1 are also described.     

An antigen common to a wide range of bacteria. I. The isolation of a 'common antigen' from Pseudomonas aeruginosa

In crude water-soluble extracts of Pseudomonas aeruginosa 64 antigens can be demonstrated by crossed immunoelectrophoresis in agarose with polyvalent Pseudomonas-immunoglobulin. One of these antigens cross-reacts with antigens prepared from bacteria of a wide range of taxonomic groups. Monospecific immunoglobulins to this antigen (Common Antigen) were produced by immunization with the appropriate immunocomplex extracted from agarose. Common Antigen was purified by the combination of two fractionation methods: Precipitation of the crude extract with 18% (w/v) sodium sulfate, followed by gel filtration of the supernatant on a Sephadex G-200 column. By this method, 35% of Common Antigen from the crude extract was recovered, more than half of the fractions electrophoretically pure. Electrophoresis of reduced Common Antigen on a dodecyl sodium sulfate polyacrylamide gel revealed two protein bands with apparent molecular weights of 59-62 000 and 62-65 000, respectively. The untreated antigen, however, passed a column of Sephadex G-200 with the void volumen, indicating a substance of high molecular weight (> 4-600 000).     

High molecular weight forms of human placental lactogen: synthesis in vitro and binding to membrane receptors for lactogenic hormones.

Translation in wheat germ extracts of poly(A)-containing RNA isolated from human term placentas resulted in the synthesis of immunoreactive forms of human placental lactogen (hPL) capable of specific binding to lactogenic receptors. The minor component coelectrophoresed on sodium dodecyl sulfate-polyacrylamide gels with authentic hPL while the major component migrated with an apparent molecular weight about 3000 larger. In addition to this precursor-like molecule, even higher molecular weight forms of hPL were observed under certain conditions: (i) when the cell-free translation products were purified by precipitation with anti-hPL serum followed by dissociation of the immunoprecipitate in guanidine hydrochloride and chromatography of the solubilized material on Sephadex G-150 in the same denaturing buffer, and (ii) when the cell-free reaction mixture was analyzed by direct chromatography on Sephadex G-150 in nondenaturing buffers. Under both sets of conditions 50–75% of the radioactivity was eluted in the column void volume, suggesting it had a molecular weight of 150,000 or more. When the high molecular weight translated product was analyzed by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the radioactive components were identical to authentic hPL and the precursorlike form, suggesting the large forms are aggregates of the smaller forms. Both the very high molecular weight forms, composed primarily of the precursor-like molecule, and the less aggregated products bound to specific lactogenic hormone receptors in rat liver membrane preparations, although the larger forms exchanged less readily with unlabeled hPL than did the monomeric form of the hormone. The aggregated, receptor-bindable cell-free translation product may be similar to high molecular weight lactogens previously described in vivo.     

Production of an extracellular alkaline metalloprotease from a newly isolated, moderately halophile, Salinivibrio sp. strain AF-2004

An extracellular protease was produced under stress conditions of high temperature and high salinity by a newly isolated moderate halophile, Salinivibrio sp. strain AF-2004 in a basal medium containing peptone, beef extract, glucose and NaCl. A modification of Kunitz method was used for protease assay. The isolate was capable of producing protease in the presence of sodium chloride, sodium sulfate, sodium nitrate, sodium nitrite, potassium chloride, sodium acetate and sodium citrate. The maximum protease was secreted in the presence of 7.5 to 10% (w/v) sodium sulfate or 3% (w/v) sodium acetate (4.6 U ml⁻¹). Various carbon sources including glucose, lactose, casein and peptone were capable of inducing enzyme production. The optimum pH, temperature and aeration for enzyme production were 9.0, 32 °C and 220 rpm, respectively. The enzyme production corresponded with growth and reached a maximum level during the mid-stationary phase. Maximum protease activity was exhibited in the medium containing 1% (w/v) NaCl at 60 °C, with 18% and 41% activity reductions at temperature 50 and 70 °C, respectively. The optimum pH for enzyme activity was 8.5, with 86% and 75% residual activities at pH 10 and 6, respectively. The activity of enzyme was inhibited by EDTA. These results suggest that the protease secreted by Salinivibrio sp. strain AF-2004 is industrially important from the perspectives of its activity at a broad pH ranges (5.0–10.0), its moderate thermoactivity in addition to its high tolerance to a wide range of salt concentration (0–10% NaCl).     

Biosynthesis and characterization of lipid-linked sugars and glycoproteins in aorta.

A particulate enzyme from bovine aorta catalyzes the incorporation of mannose from GDP-D-[14C]mannose into three products as follows: 1. Most of the radioactivity which is incorporated in short term incubations is into a product that is soluble in CHCl3/CH3OH (2/1, v/v). This product was purified by chromatography on DEAE-cellulose and Sephadex LH-20. The purified glycolipid was stable to alkaline saponification but released [14C]mannose when subjected to mild acid hydrolysis (1/2 = 7 min at 100 degrees in 0.01 N HCl). The purified glycolipid had the same mobility on silica gel plates in an acidic, basic, or neutral solvent system as did glycolipid had the same mobility on silica gel plates in an acidic, basic, or neutral solvent system as did authentic dolichyl mannopyranosyl phosphate. The synthesis of the 14C-mannolipid was reversed by the addition of GDP and Mg2+. 2. [14C]mannose is also incorporated, although at a slower rate into products which are soluble in CHCl3/CH3OH/H2O (1/1/0.3, v/v). When the 1/10.3 soluble material was chromatographed on Avicel plates, it gave rise to three distinct radioactive bands which appear to be lipid-linked oligosaccharides. Mild acid hydrolysis of the 1/10.3 soluble material released water-soluble, neutral 14C-oligosaccharides which eluted from Sephadex G-50 in two or three peaks between the standards cytochrome c and GDP-mannose...     

Preparation from keratin sulfate of substrates for the measurement of 2-acetamido-2-deoxy-D-glucose 6-sulfate sulfatase and (1 goes to 3)-N-acetyl-beta-D-glucosaminidase

An extract of bacterial cells Pseudomonas sp. IFO-13309 grown on medium containing 0.1% bovine cornea keratan sulfate of low sulfate content degraded exhaustively bovine cornea keratan sulfate to give 2-acetamido-2-deoxy-beta-D-gluco-pyranosyl 6-sulfate-(1 goes to 3)-D-galactose, isolated by gel filtration on Sephadex G-25 and purified by preparative paper chromatography. This was reduced with sodium borotritide to give 2-acetamido-2-deoxy-beta-D-glucopyranosyl 6-sulfate-(1 goes to 3)-D-[1-3H]galactitol, purified by gel filtration on Sephadex G-15, which was an excellent substrate for the measurement of 2-acetamido-2-deoxy-D-glucose 6-sulfate sulfatase. The reduced, radioactive monosulfated disaccharide was desulfated with methanolic 70mM hydrogen chloride and purified by gel filtration on Sephadex G-15 to give O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1 goes to 3)-D-[1-3H]galactitol, which allowed the measurement of (1 goes to 3)-N-acetyl-beta-D-glucosaminidase. This enzyme may participate in the normal degradation of keratan sulfate.     

Heterogeneity of big-big hPRL in hyperprolactinemia

Sera from a patient with macroprolactinoma (case 1) and from a hyperprolactinemic woman with regular menstruation (case 2) were analyzed for prolactin activity by gel filtration using Sephadex G-100, Sephadex G-200 and TSK G3000SW columns. The chromatographic profile by Sephadex G-100 showed that the percentage of immunoreactive big-big hPRL was 10.7% in case 1 and 64.1% in case 2. On Sephadex G-200 and TSK G3000SW columns, the molecular weight of big-big hPRL was estimated to be more than 500,000 daltons (big-big1 hPRL) in case 1 and approximately 250,000-300,000 daltons (big-big2 hPRL) in case 2. Big-big1 hPRL in case 1 was converted to big and little hPRLs when the serum was treated with 2-mercaptoethanol (2-ME), but part of the big-big2 hPRL in case 2 was converted to a larger molecule. Radioactive big-big hPRL generated by mixing labeled hPRL with the serum from case 1 was eluted with the void volume on Sephadex G-100 column and was not converted to the other molecular forms after 2-ME treatment. There were two radioactive big-big hPRL on TSK G3000SW column and these estimated molecular weights were more than 300,000 daltons. The data demonstrated the existence of at least two forms of big-big hPRL in the serum and indicated that radioactive big-big hPRL may be different from these hPRLs in the serum.     

Purification and characterization of human factor II

Human plasma Factor II has been purified approximately 800-fold by a combination of barium citrate adsorption, ion-exchange chromatography and preparative polyacrylamide gel electrophoresis. The procedure is relatively simple and results in excellent yields of purified Factor II essentially free of Factor X activity. The purified factor behaved as a single component by analytical polyacrylamide gel disc electrophoresis at pH 8.9. No Factor V, VII or IX activity was detected in the purified Factor II. Its molecular weight was 7200±3000 as determined by analytical ultracentrifugation, electrophoresis in the presence of sodium dodecyl sulfate and gel filtration on Bio-Gel P-200. An apparent molecular weight of 90 000–100 000 was observed on calibrated columns of Sephadex G-100, G-150, and G-200. The specific activity of human factor II was approximately 1300 N.I.H. units/mg as determined by the two-stage assay and 7 Ortho units/mg by the one stage assay. The purified protein contained by weight 2.8% neutral hexose, 2.3% sialic acids and 3.1% hexosamines.     

Isolation and characterization of calcitonin from pericardium and esophagus of eel.

Calcitonin was extracted from the pericardium and esophagus of eel in quantities sufficient to permit purification and chemical characterization. Homogeneous calcitonin could be isolated by a six-step fractionation starting from acetone powder of the organs. The fractionation procedure consisted of acid extraction, gel filtration on Sephadex G-75, chromatography on SP-Sephadex C-25, gel filtration on the Sephadex G-50, chromatography on carboxymethylcellulose, and gel filtration on Sephadex G-50. Fractionation of the hormone was monitored by assay of its biological activity and from its behaviour on thin layer chromatography and polyacrylamide gel disc electrophoresis. The hormone contained 32 amino acid residues, like calcitonins from other species of animals, but its amino acid composition was different from those of previously characterized hormones. Eel calcitonin possessed almost the same, or higher, biological activity as the salmon or chicken hormone, which show the highest specific activity among calcitonins so far isolated.     

Purification and characterization of an extracellular polygalacturonase from Lactobacillus plantarum strain BA 11

Lactobacillus plantarum produced extracellular polygalacturonase in a medium containing 1.5% low methyl-pectin (w/v) and 0.5% glucose (w/v) as inducers. The enzyme was purified (approximately 70-fold) by ammonium sulphate fractionation, Sephadex G-100 gel filtration and DEAE-cellulose ion exchange chromatography. Two peaks (PG I and PG II) of enzymic activity were obtained from the DEAE-cellulose column. The molecular mass of PG I was similar to that of PG II (32 000 Da). The K _m values of PG I and PG II for sodium polypectate were calculated to be 1.63 mg/ml and 1.78 mg/ml respectively. Their isoelectric points were about pH 5.5. The pH optimum was 4.5, while the optimum temperature was 35°C for both PG I and PG II. The two purified enzymes had similar endo modes of action on polygalacturonic acid, as determined by comparison of viscosity reduction and reducing group release.     

Regulatory Factors of Lymphocyte-Lymphocyte Interaction: II. Characterization of Human Mitogenic Factor Derived from the Culture Supernatant of a Mouse-Human Hybridoma

Cell hybridization of phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes (PBL) with murine lymphoma (EL-4) provided three hybridomas (MHH-16, MHH-20, and MHH-22) which spontaneously produced human mitogenic factor (MF). MHH-16 was serially subcloned by limiting dilution procedures, which resulted in maintaining two subclones producing human MF spontaneously for more than one year (PQL-3 and PQL-5 subcloned lines). Human MF (MHH-MF) derived from supernatants of PQL-5 line cultures had a molecular weight (m.w.) of about 26,000–30,000 daltons (the major peak) with a minor peak with an m.w. of 15,000 daltons on Sephadex G-100 chromatography, and at a high concentration of NaCl (1 m), the activity of the 26,000–30,000-m.w. fraction became weak and that of the 15,000-m.w. fraction became predominant. MHH-MF had an isoelectric point of pH 5.0–6.5. On DEAE-cellulose chromatography, MHH-MF was eluted at a fairly low salt concentration (sodium phosphate buffer 0.02 M, pH 8.0, NaCl 10 mm). After periodate treatment of this MHH-MF, the mitogenic activity almost disappeared. MHH-MF was relatively unstable to heating at 56 C for 20 min. In the presence of tunicamycin (0.3μg/ml), an inhibitor of N-linked glycosylation, the synthesized MHH-MF showed a decrease in m.w. as follows: the major peak shifted from 26,000–30,000 to 23,000 daltons and the minor peak from 15,000 to 10,000 daltons on Sephadex G-100 chromatography. In internal labeling experiments with [³H]leucine, the ³H-labeled MF was partially purified, with mitogenic activity as a guide. This ³H-labeled MHH-MF fraction could be absorbed by PHA blasts but not by normal PBL. On SDS-PAGE under reducing conditions, only the radioactive peak of the 15,000-dalton fraction was recovered. MHH-MF obtained from the hybridoma culture supernatants may be a dimer of the 15,000-dalton fraction and a glycoprotein.     

Characterization of Leishmania donovani acid phosphatases

A crude membrane fraction from promastigotes of Leishmania donovani grown in a liquid culture medium containing 20% fetal calf serum was prepared by freeze-thawing, centrifugation (200,000 X g, 30 min), and extraction with 2% (w/v) sodium cholate. After removal of the bile salt by chromatography on a Sephadex G-75 column, the solubilized membrane protein fraction, rich in acid phosphatase activity, was chromatographed on columns containing concanavalin A-Sepharose, QAE-Sephadex, and Sephadex G-150 and G-100. Three distinct acid phosphatases were resolved: the major phosphatase activity (70% of the total) was L-(+)-tartrate-resistant (designated ACP-P1) and corresponds to the acid phosphatase localized to the outer surface of the parasite's plasma membrane; the other two phosphatases (ACP-P2 and ACP-P3) account for the remaining 30% of the particulate acid phosphatase activity, and both of these enzymes are L-(+)-tartrate-sensitive. Using a combination of sucrose density gradient centrifugation, gel filtration chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was determined that ACP-P1 is a 128,000-dalton protein composed of two subunits of 65,000-68,000 daltons. ACP-P1 has an isoelectric point of 4.1, a pH optimum of 5.5, hydrolyzes fructose 1,6-diphosphate, but no other sugar phosphates and dephosphorylates phosphotyrosine, yeast mannan, and the phosphorylated form of rat liver pyruvate kinase. ACP-P2 (pI, 5.4) and ACP-P3 (pI, 7.1) with molecular masses of 132,000 and 108,000 daltons, respectively, are both tartrate-sensitive and are distinguished from each other on the basis of their sensitivity to inhibition by polyanionic molybdenum complexes. These two phosphatases also have their pH optima in the pH 5.0-6.0 range, but have a considerably broader substrate specificity than ACP-P1.     

Clostripain-catalyzed re-formation of a peptide bond in a cytochrome C fragment complex

Enzymatically-catalyzed condensation of cytochrome c fragments, ferrous heme fragment (1-38) and apofragment (39-104), has allowed the back-conversion of cytochrome c complex to native cytochrome c. The conversion was accomplished in 90% (v/v) glycerol, a solvent which has been shown to decrease the ionization of the terminal alpha-carboxyl group liberated during hydrolysis of a peptide bond. The effect on the pK is probably the main reason the thermodynamic obstacle to re-synthesis is minimized. A 30% conversion to cytochrome c was obtained. The cytochrome c product was distinguished from the non-covalent complex and separated fragments by molecular weight analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis, by elution from Sephadex G-50 and sulfopropyl-Sephadex in the presence of denaturant, by amino acid analysis of the product purified under complex-dissociation conditions, and by spectral analysis of the absorption bands of the heme. This method provides an opportunity to study the covalent rather than the complex form of cytochrome c analogs.     

Detection of enteroviruses in groundwater with the polymerase chain reaction.

Standard methods for the detection of enteroviruses in environmental samples involve the use of cell culture, which is expensive and time-consuming. The polymerase chain reaction (PCR) is an attractive method for the detection of enteroviruses in water because primary cell culture is not needed and the increased sensitivity of PCR allows detection of the low numbers of target DNAs and RNAs usually found in environmental samples. However, environmental samples often contain substances that inhibit PCR amplification of target DNA and RNA. Procedures that remove substances that interfere with the amplification process need to be developed if PCR is to be successfully applied to environmental samples. An RNA-PCR assay for the detection of enteroviruses in water was developed and used to test a variety of groundwater concentrates and humic acid solutions seeded with poliovirus type 1. The groundwater samples and humic acid solutions were treated with Sephadex G-50, Sephadex G-100, Sephadex G-200, Chelex-100 resin, and a mixed bed resin to remove PCR-inhibitory material from the samples. Sephadex G-100 in combination with Chelex-100 was found to be very effective in removing inhibitory factors for the detection of enteroviruses in groundwater concentrates by PCR. Viruses were detected in two of the groundwater concentrates by the RNA-PCR assay after treatment with Sephadex G-100 plus Chelex-100. This was confirmed by tissue culture, suggesting that the treatment protocol and, subsequently, the RNA-PCR assay are applicable for the detection of enteroviruses in environmental samples.     

Purification and Characterization of Extracellular Amylolytic Enzymes from the Yeast Filobasidium capsuligenum

The extracellular amylolytic system of Filobasidium capsuligenum consisted of an alpha-amylase (1,4-alpha-d-glucan glucanhydrolase, EC 3.2.1.1) and two forms of glucoamylase (1,4-alpha-d-glucan glucohydrolase, EC 3.2.1.3). The enzymes were purified by ammonium sulfate fractionation, repeated ion-exchange chromatography (DEAE-Sephadex A-50), and gel filtration (Sephadex G-25, Sephadex G-100 sf). alpha-Amylase had an optimum pH of 5.6 and an optimum temperature of 50 degrees C but was rapidly inactivated at higher temperature. The molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 64,000. An acarbose concentration of 20 mug/ml was required for 50% inhibition of the alpha-amylase. Both glucoamylases are glycoproteins of identical molecular weight (60,000) and produce only glucose by exohydrolysis. The debranching activity of the glucoamylases was evidenced with substrates containing alpha-1,6 linkages. The pH optima were 5.0 to 5.6 for glucoamylase I and 4.8 to 5.3 for glucoamylase II. Glucoamylase I had a higher optimum temperature (55 degrees C) than glucoamylase II (50 degrees C) and was also more resistant to thermal inactivation. Only low acarbose concentrations (<0.1 mug/ml) were required to reduce the activity of the glucoamylases by 50%.     

Further studies on bovine serum amylase. 3. Affinity chromatography

The major bovine serum isoamylases controlled by the AmI locus have been examined by gel filtration. On Sephadex G-200 the isoamylases can be resolved into two classes. The AmI A and AmI B have apparent molecular weights of 307,000 daltons whilst the AmI C isozyme has an apparent molecular weight of 44,400 daltons. The separation of the isozymes into two classes according to their elution behaviour on Sephadex G-200 has been shown to be an affinity separation. All three AmI isozymes are eluted from a non-dextran media (BioGel A1.5m) with apparent molecular weights of 417,000 daltons. The affinity separation on Sephadex G-200 has been shown to be inhibited by the addition of 1% (w/v) maltose to the elution buffer. In the presence of 1% (w/v) maltose all three AmI isozymes are coeluted from Sephadex G-200 with apparent molecular weights of 321,000 daltons. The maltase and amylase activities of the AmI isozymes were eluted coincidentally under all the conditions studied.     

Further studies on bovine serum amylase. 3. Affinity chromatography

The major bovine serum isoamylases controlled by the AmI locus have been examined by gel filtration. On Sephadex G-200 the isoamylases can be resolved into two classes. The AmI A and AmI B have apparent molecular weights of 307 000 daltons whilst the AmI C isozyme has an apparent molecular weight of 44 400 daltons. The separation of the isozymes into two classes according to their elution behaviour on Sephadex G-200 has been shown to be an affinity separation. All three AmI isozymes are eluted from a non-dextran media (BioGel A1.5m) with apparent molecular weights of 417 000 daltons. The affinity separation on Sephadex G-200 has been shown to be inhibited by the addition of 1% (w/v) maltose to the elution buffer. In the presence of 1 % (w/v) maltose all three AmI isozymes are coeluted from Sephadex G-200 with apparent molecular weights of 321000 daltons. The maltase and amylase activities of the AmI isozymes were eluted coincidentally under all the conditions studied.