Microheterogeneity of molecular forms of arginase in mammalian tissues

Two isoforms of arginase, A1 and A2, were found in rat liver, submaxillary gland and kidney as well as beef kidney. In beef liver, however, A2 was the only detectable form. Two additional forms, A3 and A4, found only in rat kidney were probably artifactitious. A1 and A2 exhibited chromatographic and immunological microheterogeneity. While A1 in rat liver and submaxillary gland was excluded by DEAE-cellulose (pH 8.3) and retained on CM-cellulose (pH 7.5), that (A'1) in beef and rat kidneys was excluded by both ion-exchangers. A2 in all tissues was retained on DEAE-cellulose, but not on CM-cellulose. Both A1 and A2 in rat liver and beef kidney, A1 from rat submaxillary gland and A2 from beef liver were precipitated by antibodies to rat and beef liver arginases. None of the forms in rat kidney (A1, A2, A3 and A4) showed any cross-reactivity to either antibody. Rat submaxillary gland A2 was precipitated by anti-rat liver arginase, but activated by anti-beef liver arginase. While the major molecular forms were A1 in rat liver and submaxillary gland and A2 in beef liver and rat kidney, the two forms occurred in equal proportions in beef kidney. It appears that different isoforms might function as components of the urea cycle in the liver of different mammals and of the arginine catabolic pathway in different extrahepatic tissues.     

The isolation and immunological properties of two arginase forms from human erythrocytes

1. Two forms of arginase were isolated from human erythrocytes; the main form adsorbed on CM-cellulose and the second form, occurring in much smaller amount, adsorbed on DEAE-cellulose. 2. The molecular weight of either arginase was 120,000 +/- 5000. 3. The erythrocyte arginases are similar in immunological properties to arginase A4 from human kidney and A2 from human liver, respectively. 4. Despite the literature data stating that human erythrocyte arginase and human liver arginase are identical, it was found that the main forms of arginase of these tissues A4 from erythrocytes and A5 from liver differ in immunological properties.     

Five forms of arginase in human tissues

Five forms of arginase, A1, A2, A3, A4, and A5, were found to be present in human tissues. The molecular weight of all these forms is the same, 120,000, but they differ in the behavior on DEAE- and CM-cellulose, electrophoretic mobility, isoelectric point, and immunochemical properties. Forms A1 from kidney and A5 from liver show complete immunological incompatibility, whereas forms A2 from liver, A3 from salivary gland, and A4 from kidney exhibit partial incompatibility with respect to each other and to forms A1 and A5.     

Sex differences in the subunits of glutathione-S-transferase isoenzyme from rat and human kidney

Glutathione-S-transferase (GST) isoenzymes were purified from cytosolic preparations from kidneys of male and female rats and kidney cortical specimens from 2 male and 1 female human subjects. GST isoenzyme expression was analyzed by SDS-PAGE, measurement of catalytic activities with specific substrates and determination of their subunits by ELISA and Western blotting using specific antibodies. GST from female rat kidneys showed a preponderance of subunits 3 and 4; levels of these isoenzymes were 3-4 times greater in females than in males. Levels of subunits 1 and 2 were 1.5-2 times greater in the male rat kidneys. Additional minor bands at 24 and 22 kD were observed in GST preparations from both male and female rat kidneys while a band at 25.3 kD was observed only in the male rat kidney. These bands did not react with antibodies to GST 1-1, GST 2-2 or GST 3-4. Both male and female human kidney samples contained GST isoenzymes comparable to the near-neutral (25-5 kD) and basic forms (25 kD) of GSTs found in human liver. In addition a 28-kD band was present in GST preparations from both male and female human kidneys. Additional bands at 29 and 25.2 kD were present only in male human kidneys. Both the kidney cytosol and the total GSTs prepared from female rats shared 2- to 4-fold greater activity with 1,2-dichloro-4-nitrobenzene, ethacrynic acid and trans-4-phenyl-3-buten-2-one than those from males. The measurement of specific subunit amounts by ELISA were in agreement with these results.(ABSTRACT TRUNCATED AT 250 WORDS)     

The subunit structure of a major glutathione S-transferase form, MT, in rat testis. Evidence for a heterodimer consisting of subunits with different isoelectric points

A major glutathione S-transferase form (pI 5.7) in rat testis (MT) purified by S-hexyl-glutathione affinity chromatography, followed by chromatofocusing, showed two polypeptide of pI 6.7 (Yn1) and 6.0 (Yn2), having apparently the same molecular mass of 26 kDa on two-dimensional gel electrophoresis. Rechromatofocusing of the MT preparation after 4 M guanidine hydrochloride treatment revealed two additional protein peaks (pI 6.2 and 5.4). These were identified as the two homodimers consisting of the subunits of MT, Yn1Yn1 and Yn2Yn2, respectively. Furthermore, MT could be reconstituted from Yn1Yn1 and Yn2Yn2. These results indicate that MT is a heterodimer, Yn1Yn2, consisting of subunits with very similar molecular masses but different isoelectric points. The Yn1Yn1 form had glutathione S-transferase activities towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene. However, the Yn2Yn2 form had no activity towards any of the substrates examined. N-terminal amino acid sequences of subunits Yn1 and Yn2 revealed differences at two positions in the first 20 residues; the amino acid compositions of these subunits were also similar but not identical, indicating that these two subunits are different in the primary structure. Subunits Yn1 and Yn2 are immunologically related to each other and also to subunits 3 (Yb1) and 4 (Yb2) but they are not identical. These four subunits also showed a high degree of similarity in N-terminal amino acid sequences. Subunits Yn1 and Yn2 seem to belong to the rat GST 3-4 family or class mu. Subunits Yn1 and 4 can make a heterodimer, which is detectable not only in rat testis, but also in the heart, kidney and lung. The Yn1Yn1 form was not detected in the testis, but is present in rat brain [Tsuchida et al. (1987) Eur. J. Biochem. 170, 159-164]. The Yn2Yn2 form seemed to differ from GST 5-5 and may be a new form of rat glutathione S-transferase.     

臭鼩血清蛋白和六种组织乳酸脱氢酶同工酶的研究

本实验对臭鼩的血清蛋白及心肌、骨骼肌、肾脏、脾脏、肝脏,睾丸6种组织器官的乳酸脱氢酶(LDH)同工酶进行了聚丙烯酰胺凝胶盘状电泳的分析研究。臭鼩血清蛋白存在15—17条带,各组织的LDH同工酶均由5条带构成,其中心肌LDH-1、LDH-2和肾脏LDH-1各出现1条亚带。     

Multiple forms of gamma-butyrobetaine hydroxylase (EC 1.14.11.1).

gamma-Butyrobetaine hydroxylase [4-trimethylaminobutyrate, 2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating), EC 1.14.11.1] from human kidney was resolved into three forms by chromatofocusing. After further chromatography on an anion-exchanger, each form appeared as a single band on electrophoresis in polyacrylamide gel containing sodium dodecyl sulphate. The isoelectric points of isoenzymes 1, 2 and 3 were 5.6, 5.7 and 5.8 respectively, as estimated by isoelectric focusing. Their specific activities were 17-29 mu kat/g of protein. The concentrations of the three isoenzymes were about equal, possibly slightly lower for isoenzyme 1. The requirement for Fe2+ and the Km values for gamma-butyrobetaine and 2-oxoglutarate were about the same for the different enzyme forms. L- and D-Carnitine caused decarboxylation of 2-oxoglutarate to the same extent (8 and 29%) with the three forms. The enzyme forms had the same mass, 64 kDa, as determined by gel filtration in nondenaturing media. The same subunit mass, 42 kDa, was obtained for the multiple forms by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Isoenzyme 2 was resolved into two protein bands by isoelectric focusing in polyacrylamide gels containing urea. Isoenzyme 1 contained only one of these bands and isoenzyme 3 the other. The three enzyme forms of gamma-butyrobetaine hydroxylase thus appear to be dimeric combinations of two subunits differing in charge but not in size. gamma-Butyrobetaine hydroxylase from crude extracts of human, rat and calf liver was also separated into multiple forms by a chromatofocusing technique. The isoenzyme pattern was the same in human liver and kidney. The technique used to resolve the mammalian enzymes gave no evidence for the presence of multiple forms of the bacterial enzyme from Pseudomonas sp. AK 1.     

Mouse hepatic glutathione transferase isoenzymes and their differential induction by anticarcinogens. Specificities of butylated hydroxyanisole and bisethylxanthogen as inducers of glutathione transferases in male and female CD-1 mice.

GSH transferase isoenzymes of class Mu (two forms), class Pi (one form) and class Alpha (two forms) were purified from liver cytosols of female CD-1 mice pretreated with an anticarcinogenic inducer, 2(3)-t-butyl-4-hydroxyanisole. GSH transferases GT-8.7, GT-8.8a and GT-8.8b, GT-9.0, GT-9.3, GT-10.3 and GT-10.6 contained a minimum of six types of subunits distinguishable by structural, catalytic and immunological characteristics. H.p.l.c. analysis of the subunit compositions of affinity-purified GSH transferases from liver cytosols of induced and non-induced male and female CD-1 mice showed that two anticarcinogenic compounds, 2(3)-t-butyl-4-hydroxyanisole and bisethylxanthogen, differed markedly in their specificities as inducers of GSH transferase.     

A study of the heterogenous structure of guinea pig lysosomal beta-mannosidase using a polyclonal antibody

Lysosomal beta-mannosidase (EC 3.2.11.25) has a functional size of 120-150 kDa, but the enzyme purified from guinea pig liver (GPL) reportedly gave a single band corresponding to a molecular mass of 110 kDa. In order to investigate the subunit structure and tissue-specific expression of beta-mannosidase, we prepared a polyclonal antibody against GPL beta-mannosidase in rabbits which immunoprecipitated beta-mannosidase activity, free from other lysosomal hydrolase activity. Following storage at -20 degrees C and SDS polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol, a sample of purified GPL beta-mannosidase gave a major Coomassie blue staining band at 97 kDa. This was confirmed by Western blot analysis, which also revealed a faster moving 37 kDa protein. In contrast, Western blot analysis of fresh GPL homogenate prepared in the presence of proteinase inhibitors showed a major band at 150 kDa. Upon freezing and thawing, we observed immunoreactive bands at 120 and 20 kDa and finally, immunoreactive bands at 97, 37 and 20 kDa. The formation of the 97, 37 and 20 kDa forms from the 150 kDa species was accelerated by an n-butanol/ether extraction of the associated lipids, suggesting some tight hydrophobic association of these subunits. In contrast to liver, both fresh and freeze-thawed preparations of guinea pig kidney (GPK) yielded only the 97, 37 and 20 kDa subunit forms confirming that these are the major beta-mannosidase subunits. Endo-F treatment converted both the liver and kidney 97 kDa into a 91 kDa form and the 37 kDa form into a 35 kDa form, whereas the 20 kDa form was unaffected. Total beta-mannosidase activity, as measured with the synthetic substrate 4MU-beta-mannoside was unaffected by dissociation of the 150 form into the 97, 37 and 20 kDa subunits, suggesting that these are the functional forms of the enzyme rather than proteolytic degradation products.     

Purification and characterization of glutathione transferases with an activity toward nitroglycerin from human aorta and heart. Multiplicity of the human class Mu forms

Although recent studies suggest involvement of glutathione transferase (GST) of blood vessels in vasodilation by nitroglycerin, GST forms in blood vessels remain to be studied. In this study, three GST forms (pI values 8.3, 6.6, and 4.8) were purified from human aorta and four (pI values 6.0, 5.6, 5.3, and 4.6) from the heart by affinity chromatography followed by chromatofocusing. The major form of both aorta (pI 4.8) and heart (pI 4.6) was identified as GST-pi, and the other five forms were immunologically related to GST-mu, suggesting that the five belong to the Mu class. Among nine human GST forms, including three in the Alpha class purified from the liver, GST-mu, aorta pI 8.3 form, and GST-I (a form of the Alpha class, corresponding to GST-epsilon (B1B1)) showed high activities toward nitroglycerin, 1.08, 0.85, and 0.78 units/mg protein, respectively. GST-pi did not exhibit the activity. The Km values of the aorta form (pI 8.3) for glutathione (GSH) and nitroglycerin were calculated as 0.12 and 1.1 mM, respectively. The Km values of GST-mu and GST-I for GSH were 0.29 and 0.09 mM, and those for nitroglycerin were 2.5 and 0.3 mM, respectively. The activity of the pI 8.3 form as well as GST-mu toward nitroglycerin was inhibited by bromosulfophthalein, which is known to inhibit the relaxation of rabbit aorta induced by nitroglycerin, at the lower concentration (IC50, 2 microM) than was GST-I (IC50, 32 microM). Two-dimensional gel electrophoresis and N-terminal amino acid sequence analysis revealed that five forms in the Mu class are homo- or heterodimers of five different subunits named M1 (pI 7.0/Mr 27,000), M2 (6.6/27,000), M3 (6.0/27,000), N1 (6.5/26,500), and N2 (5.9/26,500). The subunit structures of the five forms are as follows: pI 8.3 form, M1M2; 6.6 form, M2N1; 6.0 form, M3M3; 5.6 form, M3N2; and 5.3 form, N2N2. M3 and N2 seem to correspond to the subunits of GST-mu, and -4 (Board, P. G., Suzuki, T., and Shaw, D. C. (1988) Biochim. Biophys. Acta 953, 214-217), respectively. These subunits except N1 are different from each other at two or three positions in the first 20 residues of N-terminal amino acid sequence. These results indicate the presence of five different subunits in the human Mu class and also suggest that GST-M1M2 and -M2N1 found in the aorta are involved in the expression of the pharmacologic effect of nitroglycerin.     

Characterization of multiple forms of carbonyl reductase from chicken liver.

Three enzyme forms (CR1, CR2 and CR3) of carbonyl reductase were purified from chicken liver with using 4-benzoylpyridine as a substrate. CR1 was a dimeric enzyme composed of two identical 25-kD subunits. CR2 and CR3 were monomeric enzymes whose molecular weights were both 32 kD. CR1 exhibited 17 beta-hydroxysteroid dehydrogenase activity as well as carbonyl reductase activity in the presence of both NADP(H) and NAD(H). CR2 and CR3 had similar properties with regard to substrate specificity and inhibitor sensitivity. They could exhibit the activity only with NADPH and had no hydroxysteroid dehydrogenase activity. CR2 and CR3 cross-reacted with anti-chicken kidney carbonyl reductase antibody, though CR1 did not. The results suggest that CR1 is a hydroxysteroid dehydrogenase, and CR2 and CR3 are similar to each other and to the kidney enzymes.     

Purification and characterization of glutathione S-transferases of human kidney.

Several forms of glutathione S-transferase (GST) are present in human kidney, and the overall isoenzyme pattern of kidney differs significantly from those of other human tissues. All the three major classes of GST isoenzymes (alpha, mu and pi) are present in significant amounts in kidney, indicating that GST1, GST2 and GST3 gene loci are expressed in this tissue. More than one form of GST is present in each of these classes of enzymes, and individual variations are observed for these classes. The structural, immunological and functional properties of GST isoenzymes of three classes differ significantly from each other, whereas the isoenzymes belonging to the same class have similar properties. All the cationic GST isoenzymes of human kidney except for GST 9.1 are heterodimers of 26,500-Mr and 24,500-Mr subunits. GST 9.1 is a dimer of 24,500-Mr subunits. All the cationic isoenzymes of kidney GST cross-react with antibodies raised against a mixture of GST alpha, beta, gamma, delta and epsilon isoenzymes of liver. GST 6.6 and GST 5.5 of kidney are dimers of 26,500-Mr subunits and are immunologically similar to GST psi of liver. Unlike other human tissues, kidney has at least two isoenzymes (pI 4.7 and 4.9) associated with the GST3 locus. Both these isoenzymes are dimers of 22,500-Mr subunits and are immunologically similar to GST pi of placenta. Some of the isoenzymes of kidney do not correspond to known GST isoenzymes from other human tissues and may be specific to this tissue.     

Expression of an enzymatically active Yb3 glutathione S-transferase in Escherichia coli and identification of its natural form in rat brain

Glutathione S-transferases containing Yb3 subunits are relatively uncommon forms that are expressed in a tissue-specific manner and have not been identified unequivocally or characterized. A cDNA clone containing the entire coding sequence of Yb3 glutathione S-transferase mRNA was incorporated into a pIN-III expression vector used to transform Escherichia coli. A fusion Yb3-protein containing 14 additional amino acid residues at its N terminus was purified to homogeneity. Recombinant Yb3 was enzymatically active with both 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates but lacked glutathione peroxidase activity. Substrate specificity patterns of recombinant Yb3 were more limited than those of glutathione S-transferase isoenzymes containing Yb1- or Yb2-type subunits. Peptides corresponding to unique amino acid sequences of Yb3 as well as a peptide from a region of homology with Yb1 and Yb2 subunits were synthesized. These synthetic peptides were used to raise antibodies specific to Yb3 and others that cross-reacted with all Yb forms. Immunoblotting was utilized to identify the natural counterpart of recombinant Yb3 among rat glutathione transferases. Brain and testis glutathione S-transferases were rich in Yb3 subunits, but very little was found in liver or kidney. Physical properties, substrate specificities, and binding patterns of the recombinant protein paralleled properties of the natural isoenzyme isolated from brain.     

On the control of arginine metabolism in chicken kidney and liver.

Arginases have been found to be located on the external side of the inner mitochondrial membrane of chicken kidney and liver. Transamidinase has been detected within the liver mitochondrial matrix space. Arginases and transamidinase act upon two different intracellular arginine pools. Penetration of arginine into the matrix space occurs only in respring mitochondria and in the presence of anions such as acetate and phosphate; D-arginine, L-ornithine, D-'ornithine and L-lysine penetrate with the same modalities. L-Histidine penetrates only kidney mitochondria. Because of transamidinase compartmentation, the rate of creatine synthesis is influenced by the rate of penetration of arginine into the mitochondria.     

Comparison of human alkaline phosphatase isoenzymes. Structural evidence for three protein classes.

The structural relationships among human alkaline phosphatase isoenzymes from placenta, bone, kidney, liver and intestine were investigated by using three criteria. 1. Immunochemical characterization by using monospecific antisera prepared against either the placental isoenzyme or the liver isoenzyme distinguishes two antigenic groups: bone, kidney and liver isoenzymes cross-react with anti-(liver isoenzyme) serum, and the intestinal and placental isoenzymes cross-react with the anti-(placental isoenzyme) antiserum. 2. High-resolution two-dimensional electrophoresis of the 32P-labelled denatured subunits of each enzyme distinguishes three groups of alkaline phosphatase: (a) the liver, bone and kidney isoenzymes, each with a unique isoelectric point in the native form, can be converted into a single form by treatment with neuraminidase; (b) the placental isoenzyme, whose position also shifts after removal of sialic acid; and (c) the intestinal isoenzyme, which is distinct from all other phosphatases and is unaffected by neuraminidase digestion. 3. Finally, we compare the primary structure of each enzyme by partial proteolytic-peptide 'mapping' in dodecyl sulphate/polyacrylamide gels. These results confirm the primary structural identity of liver and kidney isoenzymes and the non-identity of the placental and intestinal forms. These data provide direct experimental support for the existence of at least three alkaline phosphatase genes.     

Arginase isoforms in frog and lizard tissues

Isoforms of arginase in the liver and kidney tissues of the ureotelic frog (Rana tigerina) and uricotelic lizard (Calotes versicolor) were fractionated by DEAE-cellulose chromatography (pH 8.3). Four molecular forms, designated as A'1, A2, A3 and A4 based on the KCl concentration required for their elution from the ion-exchange column, were detected in lizard liver, while only two forms were found in lizard kidney (A3 and A4) and frog liver and kidney (A2 and A3). No major differences were found in the pH optimum, substrate affinity and molecular weight of the isoenzymes. The isoforms in lizard tissues were either totally unaffected or only partially immunoprecipitated by antibodies raised against rat liver and beef liver arginases, but those in frog tissues were significantly activated by the two antibodies. While the physiological importance of the presence of four isoforms in lizard liver remains enigmatic, different sets of isoenzymes were present in the liver of the two ureotelic vertebrates, rat and frog. Hence, it appeared that a given mode of nitrotelism was not associated with a specific set of isoenzymes. Also, the data were not consistent with the generally held view that a basic isoform of arginase served as a component of the urea cycle in liver and a neutral/slightly acidic form functions in the synthesis of proline, glutamate and polyamines in extra-hepatic tissues. The isoforms appeared to show considerable functional overlap.     

The genetic system of the L-type pyruvate kinase forms in man. Subunit structure, interrelation and kinetic characteristics of the pyruvate kinase enzymes from erythrocytes and liver

Pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from human liver and red cells has been purified to homogeneity; its subunit structure and some of its kinetic characteristics have been studied. The influence of a partial proteolysis by trypsin on the subunit structure, the isozymic pattern and the kinetic characteristics of red cell and liver enzyme have been investigated. From the results of this study we may conclude that: 1. Liver (L-type) pyruvate kinase is composed of 4 identical L subunits while the major form of erythrocyte enzyme (PK-R2) is a heterotetramer designated as L2L2', the molecular weight of L' being slightly higher than that of L subunits (63 000 and 58 000 respectively). Pyruvate kinase PK-R1, predominant in the erythroblasts and the young red cells, is composed of four identical L' subunits. 2. A mild tryptic attack is able to transform PK-R1 into PK-R2, then PK-R2 into pyruvate kinase L (PK-L). The same proteolytic treatment transforms the L' subunits into L ones. 3. Consequently L-type pyruvate kinase seems to be initially synthesized in the erythroid precursors as an L4' enzyme secondarily partially proteolysed into L2L2'. In liver a very active proteolytic system would be responsible for the total transformation into L4 pyruvate kinase. 4. L4' enzyme exhibits Michaelis-Menten kinetic behaviour with an apparent Michaelis constant of 3.8 mM whereas L4 enzyme shows both positive and negative homotropic interactions towards phosphoenolpyruvate and has [S] 0.5 of 1.2 mM. The characteristics of L2L2' are roughly intermediate between those of L4' and of L4. Fructose 1,6-biphosphate decreases [S]0.5 for these three pyruvate kinase forms without suppressing the differences in the apparent affinity for phosphoenolpyruvate of these enzymes. 5. L4 pyruvate kinase is more inhibited by Mg-ATP than L4', with L2L2' in the intermediate range. 6. Tryptic treatment of each enzyme form studied transforms its kinetic behaviour into that observed for L4.     

Biosynthesis, import and processing of precursor polypeptides of mammalian mitochondrial pyruvate dehydrogenase complex.

An immunological analysis has been conducted of early events in the biosynthesis, import and assembly of the mammalian pyruvate dehydrogenase complex (PDC). For this purpose, monospecific polyclonal antisera were produced against the intact assembly from ox heart, Mr 8.5 x 10(6), and each of its component polypeptides, E1 alpha, E1 beta, E2, E3 and protein X. Optimal detergent-based incubation mixtures were developed for obtaining clean immunoprecipitation of PDC polypeptides and their precursors from [35S]methionine-labelled extracts of PK-15 (pig kidney), NBL-1 (bovine kidney) and BRL (Buffalo Rat liver) cells. In PK-15 cells, independent higher Mr species, corresponding to precursors of the E2, E1 alpha and E1 beta subunits of PDC, could be detected by immune precipitation and fluorography after incubation of intact cells for 4 h with [35S]methionine and 1-2 mM-2,4-dinitrophenol or 10-15 microM-carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Similar precursor states could be observed in uncoupler-treated BRL or NBL-1 cells. Pre-E1 alpha, pre-E1 beta and also pre-E3, have signal sequences in the Mr range 1500-3000 while pre-E2 contains a long additional segment of Mr 7000-9000. All of these forms exhibit similar kinetics of processing to the mature subunits with a transit time of 10-12 min. In NBL-1 cells, E3 is present in the immune complexes formed with anti-PDC serum whereas this is not the case in PK-15 cells. Thus, there are significant variations in the affinity of lipoamide dehydrogenase (E3) for the E2 core structure in different species. Pre-E1 alpha accumulates only poorly in PK-15 cells and is aberrantly processed on removal of uncoupler. This precursor is markedly more stable in NBL-1 and BRL cells. The lack of detection of a precursor form of component X is also discussed.     

Heterogeneity of acid beta-galactosidase from rabbit kidney

1. Rabbit kidney acid beta-galactosidase can be resolved into three peaks (named A3, A2 and A1) by gel-filtration chromatography. Their estimated molecular weights were: more than 250,000, 150,000 and 17,000 respectively. 2. The purified acid form appeared as a single band of protein (Mr = 28,000) on electrophoresis in the presence of sodium dodecyl sulphate, suggesting that forms A3 and A2 are multimeric forms of beta-galactosidase A1. 3. Treatment with neuraminidase from Clostridium perfringens converts form A3 into a more basic form. This phenomenon occurs also when this form is stored for a week at 4 degrees C and parallels its disaggregation. 4. The data suggest that the sialic acids present in the multimeric forms are involved in the aggregation of the acidic form of beta-galactosidase.