Introduction of the alpha subunit mutation associated with the B1 variant of Tay-Sachs disease into the beta subunit produces a beta-hexosaminidase B without catalytic activity

Lysosomal beta-hexosaminidase (beta-N-acetylhexosaminidase, EC 3.2.1.52) occurs in two major isozyme forms, hexosaminidase A (alpha beta) and hexosaminidase B (beta beta). Although dimer formation is required for enzymatic activity, both subunits contain active sites which share many common substrates. However, the alpha subunit alone confers on hexosaminidase A the specificity for negatively charged substrates, e.g. GM2 ganglioside. Recently, a point mutation, producing a single amino acid substitution in the alpha subunit (Arg178-His), has been found to be associated with the B1 variant phenotype of Tay-Sachs disease (Ohno, K., and Suzuki, K. (1988) J. Neurochem. 50, 316-318). This variant is characterized by normal levels of hexosaminidase A as measured by a common artificial substrate, but an absence of activity toward alpha subunit-specific substrates. However, because of the presence of an active beta subunit in the mutant hexosaminidase A, it has not been possible to determine whether the affected alpha subunit has undergone a change in substrate specificity or become totally inactive. In order to define the full effect of the B1 mutation we have taken advantage of the common evolutionary origin of the genes coding for the alpha and beta subunits. Since the B1 mutation occurs in a region of extended identity between the two subunits, we have duplicated the Arg178-His mutation in a cDNA coding for the human beta subunit (Arg211-His). By expression of the mutant construct in monkey COS cells we have been able to examine the effect of this mutation on beta subunits which are capable of forming stable, active homodimers, an experiment that could not readily be accomplished with heterodimeric hexosaminidase A. Our data show that beta homodimers containing the Arg211-His substitution are formed and are transported into the lysosome in a manner identical to that of normal pro-hexosaminidase B. However, the mutant homodimers are processed at a slower rate and are less stable in the lysozyme. Their most striking feature was a total lack of normal hexosaminidase B activity. We conclude that while the effect of the Arg178-His substitution is not strictly limited to the active site, the severe B1 phenotype results from a totally inactive alpha-subunit in hexosaminidase A.     

Purification and characterization of beta-N-acetylhexosaminidase I from human placenta

beta-N-Acetylhexosaminidase (hexosaminidase) I, which has an intermediate charge character between those of hexosaminidases A(alpha beta 2) and B[beta beta)2), was purified 1,500-fold from human placenta by procedures including chromatographies on concanavalin A (Con A)-Sepharose and an immunoadsorbent column. The isolated hexosaminidase I was heat-stable, and antigenically cross-reactive to anti-beta chain-IgG but not to anti-alpha chain-IgG. The results of substrate specificity experiments using 3H-labeled natural substrates indicated that the hexosaminidase I hydrolyzed Gb4Cer to Gb3Cer but not GM2 to GM3. The tryptic peptide map of the hexosaminidase I was similar to that of hexosaminidase B, though some differences were observed. The hexosaminidase I after treatment with neuraminidase or endo-beta-N-acetylglucosaminidase H was partly converted to less acidic forms. Treatment of the hexosaminidase I with acid phosphatase did not change the charge character. Therefore hexosaminidase I is an acidic variant form of hexosaminidase B, possibly resulting from sialylation and the presence of phosphodiester bonds at the carbohydrate moiety.     

Cellular localization and substrate specificity of isoelectric forms of human liver neuraminidase activity.

The four major isoelectric forms of human liver neuraminidase (with pI values between 3.4 and 4.8) have been isolated by preparative isoelectric focusing and characterized with regard to their substrate specificity using glycoprotein, glycopeptide, oligosaccharide and ganglioside natural substrates. All forms exhibited a rather broad linkage specificity and were capable of hydrolyzing sialic acid glycosidically linked alpha 2-3, alpha 2-6 and alpha 2-8, although differential rates of hydrolysis of the substrates were found for each form. The most acidic form 1 (pI 3.4) was most active on sialyl-lactose, whereas form 2 (pI 3.9) and 3 (pI 4.4) were most active on the more hydrophobic ganglioside substrates. Form 4 (pI 4.8) was most active on the low-Mr hydrophilic substrates (fetuin glycopeptide, sialyl-lactose). Each form was less active on the glycoprotein fetuin than on a glycopeptide derived from fetuin. Organelle-enriched fractions were prepared from fresh human liver tissue and neuraminidase activity on 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid was recovered in plasma membrane, microsomal, lysosomal and cytosolic preparations. Isoelectric focusing of the neuraminidase activity recovered in each of these preparations resulted in significantly different isoelectric profiles (number, relative amounts and pI values of forms) for each preparation. The differential substrate specificity of the isoelectric forms and the different isoelectric focusing profiles of neuraminidase activity recovered in subcellular-enriched fractions suggest that specific isoelectric forms with broad but defined substrate specificity are enriched at separate sites within the cell.     

N-Acetyl-β-d-hexosaminidase component A. Different forms in human tissues and fluids

1. Hexosaminidase A of human serum was resolved into two components, a minor form with properties identical with those of the single hexosaminidase A component of human liver, and a major form with significantly different properties. 2. The major serum hexosaminidase A form was eluted from a DEAE-cellulose column at a lower salt concentration than that required to elute the liver form. 3. A multiple-pass technique was used to elute the major serum enzyme A from a Sephadex G-150 column before that of liver enzyme A. 4. Clostridium perfringens neuraminidase converted the major component of serum hexosaminidase A into a form that was held less tightly by DEAE-cellulose, but the minor component of the A enzyme of serum, and the A enzyme of liver were not affected. 5. The hexosaminidase A from tears was similar to the A enzyme from serum, whereas those from several human tissues and from urine and lymph were similar to the liver form. 6. The A enzyme from serum may be derived from the A enzyme from liver by glycosylation before secretion.     

Immunological properties of N-acetyl-β-d-glucosaminidase of normal human liver and of GM2-gangliosidosis liver

Antisera were raised to a partially purified preparation of human liver hexosaminidase and to highly purified preparations of hexosaminidase isoenzymes A and B. All the antisera precipitated the enzyme in an enzymically active form, which could be located on immunodiffusion and immunoelectrophoretic gels by using a histochemical substrate. The antisera to the purified isoenzymes were shown to react with hexosaminidase from human liver, kidney, brain and spleen, but did not cross-react with human liver beta-glucosidase, beta-galactosidase, alpha-mannosidase, beta-xylosidase, arylsulphatase or acid phosphatase. Hexosaminidases A and B were immunologically identical. The immunological properties of the hexosaminidases from livers of patients with three types of GM(2)-gangliosidoses were closely similar. No evidence could be found for cross-reacting material in enzyme-deficient states.     

Purification and subunit structure of a high-molecular-weight phosphoprotein phosphatase (phosphatase II) from rat liver

1. Phosphatase II is a form of phosphoprotein phosphatase originally found in rat liver extract; it has a molecular weight of 160 000 by gel filtration and is highly active towards phosphorylase alpha. This phosphatase has been purified 1800-fold by using DEAE-cellulos (DE-52), aminohexyl--Sepharose-4B, protamine--Sepharose-4B and Sephadex G-200 chromatography. Throughout the purification steps, the original molecular weight and substrate specificity of phosphatase II were almost perfectly preserved. 2. The product of the final purification step migrated predominantly as a single protein band on non-denaturing gel electrophoresis. Sodium dodecyl sulfate gel electorphoresis revealed that the enzyme contains two types of subunit, alpha and beta, with molecular weights of 35 000 and 69 000, respectively. When treated with 0.2 M 2-mercaptoethanol at -20 degrees C, phosphatase II was dissociated to release the catalytically active alpha subunit. The beta subunit may be catalytically inactive but interacts with the alpha subunit so that phosphatase II becomes much less susceptible than the alpha subunit to inactivation by ATP or pyrophosphate.     

Oligosaccharide structure and amino acid sequence of the major glycopeptides of mature human beta-hexosaminidase

Human beta-hexosaminidase (EC 3.2.1.52) is a lysosomal enzyme that hydrolyzes terminal N-acetylhexosamines from GM2 ganglioside, oligosaccharides, and other carbohydrate-containing macromolecules. There are two major forms of hexosaminidase: hexosaminidase A, with the structure alpha(beta a beta b), and hexosaminidase B, 2(beta a beta b). Like other lysosomal proteins, hexosaminidase is targeted to its destination via glycosylation and processing in the rough endoplasmic reticulum and Golgi apparatus. Phosphorylation of specific mannose residues allows binding of the protein to the phosphomannosyl receptor and transfer to the lysosome. In order to define the structure and placement of the oligosaccharides in mature hexosaminidase and thus identify candidate mannose 6-phosphate recipient sites, the major tryptic/chymotryptic glycopeptides from each isozyme were purified by reverse-phase high-performance liquid chromatography. Two major concanavalin A binding glycopeptides, localized to the beta b chain, and one non concanavalin A binding glycopeptide, localized to the beta a chain, were found associated with the beta-subunit in both hexosaminidase A and hexosaminidase B. A single major concanavalin A binding glycopeptide was found to be associated with the alpha subunit of hexosaminidase A. The oligosaccharide structures were determined by nuclear magnetic resonance spectrometry. Two of them, the alpha and one of the beta b glycans, contained a Man3-GlcNAc2 structure, while the remaining one on the beta b chain was composed of a mixture of Man5-7-GlcNAc2 glycans. The unique glycopeptide associated with the beta a chain contained a single GlcNAc residue. Thus, all three mature polypeptides comprising the alpha and beta subunits of hexosaminidase contain carbohydrate, the structures of which have the appearance of being partially degraded in the lysosome. In the alpha chain we found only one possible site for in vivo phosphorylation. In the beta it is unclear if only one or all three of the sites could have contained phosphate. However, mature placental hexosaminidase A and B can be rephosphorylated in vitro. This requires the presence of an oligosaccharide containing an alpha 1,2-linked mannose residue. Only the single Man6-7 (of the Man5-7-GlcNAc2 glycans) containing site on the beta b chain retains this type of residue. Therefore, this site may act as the sole in vitro substrate in both of the mature isozymes for the phosphotransferase.     

A low-molecular-weight protein cross-reacting with human liver N-acetyl-β-d-glucosaminidase

Antisera were raised to preparations of hexosaminidase isoenzymes A and B purified from human liver. Protein that cross-reacted with the liver hexosaminidase was detected by an antibody-consumption method. A cross-reacting protein with a low molecular weight (20000) was partially characterized and purified from control human liver. This protein is also present in the liver of patients with Tay-Sachs disease or with Sandhoff's disease. Hexosaminidases A and B gave an immunological reaction of partial identity with the low-molecular-weight protein. The possible identity of the low-molecular-weight cross-reacting protein as a subunit of hexosaminidase is discussed.     

Structure and evolution of artiodactyla haptoglobins.

1. Artiodactyla haptoglobins (Hps), goat, sheep and cattle (family Bovidae), and pig (family Suidae) were structurally characterized. 2. The polymeric Hp systems of goat, sheep and cattle were similar to the polymeric human Hp system, while the monomeric system of pig was more comparable to the monomeric human form. 3. All members of the Artiodactyla (family Bovidae) examined exhibited a large polypeptide subunit, comparable to that of the beta subunit of human Hp. 4. In addition, a small subunit, similar in molecular weight to the human alpha 2 subunit, was demonstrated. Pig Hp was shown to have two subunits, one slightly larger than the human beta subunit and the other intermediate in size to the human alpha 1 and alpha 2 subunits. 5. Immunoelectrophoretic and immunodiffusion studies indicated complete cross reactivity among the polymeric Artiodactyla Hps. 6. The polymeric Hps do not, however, cross react with the monomeric pig Hp.     

Evidence for two different active sites on human beta-hexosaminidase A. Interaction of GM2 activator protein with beta-hexosaminidase A

Competition experiments were carried out on the hydrolysis of different substrates by beta-hexosaminidase A isolated from human liver. The results show that ganglioside GM2 in the presence of the GM2 activator protein and a new synthetic substrate, 4-methylumbelliferyl-beta-N-acetylglucosaminide 6-sulfate, are hydrolyzed at the same active site on the alpha subunit of beta-hexosaminidase A, whereas 4-methylumbelliferyl-beta-N-acetylglucosaminide is degraded predominantly by a different active site on the beta-subunit. This finding provides for the first time a possible molecular basis for the observation that, in variant B1 of the GM2 gangliosidoses, beta-hexosaminidase A has lost its activity toward GM2 ganglioside and the sulfated artificial substrate while being still able to hydrolyze the unsulfated artificial substrate at a normal rate. Furthermore, the finding that the GM2 activator protein inhibits the degradation of the sulfated substrate by beta-hexosaminidases A and S indicates that the alpha subunit common to both isoenzymes might provide a binding site for the activator protein.     

Role of beta Arg211 in the active site of human beta-hexosaminidase B

Tay-Sachs or Sandhoff disease results from a deficiency of either the alpha- or the beta-subunits of beta-hexosaminidase A, respectively. These evolutionarily related subunits have been grouped with the "Family 20" glycosidases. Molecular modeling of human hexosaminidase has been carried out on the basis of the three-dimensional structure of a bacterial member of Family 20, Serratia marcescens chitobiase. The primary sequence identity between the two enzymes is only 26% and restricted to their active site regions; therefore, the validity of this model must be determined experimentally. Because human hexosaminidase cannot be functionally expressed in bacteria, characterization of mutagenized hexosaminidase must be carried out using eukaryotic cell expression systems that all produce endogenous hexosaminidase activity. Even small amounts of endogenous enzyme can interfere with accurate K(m) or V(max) determinations. We report the expression, purification, and characterization of a C-terminal His(6)-tag precursor form of hexosaminidase B that is 99.99% free of endogenous enzyme from the host cells. Control experiments are reported confirming that the kinetic parameters of the His(6)-tag precursor are the same as the untagged precursor, which in turn are identical to the mature isoenzyme. Using highly purified wild-type and Arg(211)Lys-substituted hexosaminidase B, we reexamine the role of Arg(211) in the active site. As we previously reported, this very conservative substitution nevertheless reduces k(cat) by 500-fold. However, the removal of all endogenous activity has now allowed us to detect a 10-fold increase in K(m) that was not apparent in our previous study. That this increase in K(m) reflects a decrease in the strength of substrate binding was confirmed by the inability of the mutant isozyme to efficiently bind an immobilized substrate analogue, i.e., a hexosaminidase affinity column. Thus, Arg(211) is involved in substrate binding, as predicted by the chitobiase model, as well as catalysis.     

Identification of the 6-sulfate binding site unique to alpha-subunit-containing isozymes of human beta-hexosaminidase

In humans, beta-hexosaminidase A (alphabeta) is required to hydrolyze GM2 ganglioside. A deficiency of either the alpha- or beta-subunit leads to a severe neurological disease, Tay-Sachs or Sandhoff disease, respectively. In mammals beta-hexosaminidase B (betabeta) and S (alphaalpha) are other major and minor isozymes. The primary structures of the alpha- and beta-subunits are 60% identical, but only the alpha-containing isozymes can efficiently hydrolyze beta-linked GlcNAc-6-SO(4) from natural or artificial substrates. Hexosaminidase has been grouped with glycosidases in family 20. A molecular model of the active site of the human hexosaminidase has been generated from the crystal structure of a family 20 bacterial chitobiase. We now use the chitobiase structure to identify residues close to the carbon-6 oxygen of NAG-A, the nonreducing beta-GlcNAc residue of its bound substrate. The chitobiase side chains in the best interactive positions align with alpha-Asn(423)Arg(424) and beta-Asp(453)Leu(454). The change in charge from positive in alpha to negative in beta is consistent with the lower K(m) of hexosaminidase S, and the much higher K(m) and lower pH optimum of hexosaminidase B, toward sulfated versus unsulfated substrates. In vitro mutagenesis, CHO cell expression, and kinetic analyses of an alphaArg(424)Lys hexosaminidase S detected little change in V(max) but a 2-fold increase in K(m) for the sulfated substrate. Its K(m) for the nonsulfated substrate was unaffected. When alphaAsn(423) was converted to Asp, again only the K(m) for the sulfated substrate was changed, increasing by 6-fold. Neutralization of the charge on alphaArg(424) by substituting Gln produced a hexosaminidase S with a K(m) decrease of 3-fold and a V(max) increased by 6-fold for the unsulfated substrate, parameters nearly identical to those of hexosaminidase B at pH 4.2. As well, for the sulfated substrate at pH 4.2 its K(m) was increased 9-fold and its V(max) decreased 1.5-fold, values very similar to those of hexosaminidase B obtained at pH 3.0, where its betaAsp(453) becomes protonated.     

Variant of GM2-gangliosidosis with hexosaminidase A having a severely changed substrate specificity

The levels of hexosaminidase A activity in cultivated fibroblasts of two patients with GM2-gangliosidosis were close to the normal range with 4-methylumbelliferyl-beta-D-2-acetamido-2-deoxyglucopyranoside and 4-methylumbelliferyl-beta-D-2-acetamido-2-deoxygalactopyranoside as substrates, and the enzymes were normal in most parameters analyzed. However, the enzymes of both patients were almost completely inactive against two specific substrates for hexosaminidase A, rho-nitrophenyl-6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside, and ganglioside GM2 in the presence of GM2-activator. Fibroblast extracts of both patients showed normal hexosaminidase B and GM2-activator activity, the latter was strongly decreased in two cases with variant AB. It is suggested that human hexosaminidase A may contain two different active sites which might be inactivated separately by different mutations.     

The alpha4 regulatory subunit exerts opposing allosteric effects on protein phosphatases PP6 and PP2A

The protein Ser/Thr phosphatase family contains three enzymes called PP2A, PP4, and PP6 with separate biological functions inferred from genetics of the yeast homologues Pph21/22, Pph3, and Sit4. These catalytic subunits associate with a common subunit called alpha4 (related to yeast Tap42). Here, we characterized recombinant PP6 and PP2A catalytic monomers and alpha4.phosphatase heterodimers. Monomeric PP6 and PP2A showed identical kinetics using either p-nitrophenyl phosphate (pNPP) or 32P-myelin basic protein (MBP) as substrates, with matching Km and Vmax values. Using pNPP as substrate, PP6 and PP2A gave the same IC50 with active site inhibitors okadaic acid, microcystin-LR, calyculin A, and cantharidin. However, with MBP as substrate, PP6 was inhibited at 5-fold lower concentrations of toxins relative to PP2A, suggesting PP6 might be a preferred in vivo target of toxins. Heterodimeric alpha4.PP6 and alpha4.PP2A were starkly different. With MBP as substrate the alpha4.PP2A heterodimer had a 100-fold higher Vmax than alpha4.PP6, and neither heterodimer was active with pNPP. Thus, these phosphatases are distinguished by their different responses to allosteric binding of the common regulatory subunit alpha4. Transient expression of alpha4 differentially increased or decreased phosphorylation of endogenous phosphoproteins, consistent with opposing effects on PP2A and PP6.     

Proteolytic processing of pro-alpha and pro-beta precursors from human beta-hexosaminidase. Generation of the mature alpha and beta a beta b subunits

There are two major isozymes of human lysosomal beta-hexosaminidase (beta-N-acetylhexosaminidase, EC 3.2.1.52), hexosaminidase A, alpha(beta a beta b), and hexosaminidase B, 2(beta a beta b). The alpha subunit contains a single polypeptide chain, while the beta subunit is composed of two nonidentical chains (beta a and beta b) derived from a common pro-beta precursor. The mature subunits, like those of most lysosomal enzymes, are produced through the proteolytic processing of propolypeptides once they enter the lysosome. In order to define the structure of the alpha and beta subunits generated in the lysosome, the alpha, beta a, and beta b polypeptides of hexosaminidase A and B were separated by a combination of molecular sieve and ion exchange high performance liquid chromatography, and amino-terminal sequences were determined. These were localized to the deduced amino acid sequences of previously isolated cDNAs coding for the prepro-alpha and beta polypeptides. From this analysis, the sites of hydrolysis generating the mature alpha, beta a, and beta b chains from hexosaminidase A and B could be determined. First, the signal peptide, required for processing of the pre-propolypeptides through the rough endoplasmic reticulum was predicted from the first in-frame Met residue on the cDNA. Second, amino acid sequencing defined the amino termini of the mature polypeptide chains and identified the pro-sequences removed from both the pro-alpha and pro-beta polypeptides. Third, an internal cleavage resulted in the removal of a tetrapeptide, Arg-Gln-Asn-Lys, and tripeptide, Arg-Gln-Asn, from the pro-beta chain of hexosaminidase A and B, respectively , to generate the beta b and beta a chains. This result localized the beta b and beta a chains to the amino-terminal and carboxyl-terminal halves of the pro-beta sequence, respectively. Finally, we previously reported minimal or no carboxyl-terminal processing of the pro-beta chain in the lysosome. On the other hand, we suggest that there is trimming at the carboxyl terminus of the pro-alpha chain based on comparison of molecular weights of deglycosylated alpha with the isolated beta b and beta a chains comprising the mature beta subunit with those predicted from the cDNA. Thus, in the lysosome the pro forms of hexosaminidase A and B undergo extensive proteolytic processing which, while specific in nature, has the appearance of removing easily accessible, nonessential domains, rather than contributing to biosynthetic maturation of function.     

Stereospecific oxidation of secondary alcohols by human alcohol dehydrogenases

The human liver alpha alpha alcohol dehydrogenase exhibits a different substrate specificity and stereospecificity for secondary alcohols than the human beta 1 beta 1, and gamma 1 gamma 1 or horse liver alcohol dehydrogenases. All of the enzymes efficiently oxidize primary alcohols, but alpha alpha oxidizes secondary alcohols far more efficiently than human beta 1 beta 1 and gamma 1 gamma 1 or horse liver alcohol dehydrogenase. Specifically, alpha alpha oxidizes four- and five-carbon secondary alcohols with efficiencies 0.06-2.2 times that of primary homologs and oxidizes these secondary alcohols with efficiencies up to 3 orders of magnitude greater than those of the three other isoenzymes. Whereas the human beta 1 beta 1, gamma 1 gamma 1 and horse isoenzymes show a distinct preference toward (S)-(+)-3-methyl-2-butanol, the alpha alpha isoenzyme prefers (R)-(-)-3-methyl-2-butanol. Computer-simulated graphics demonstrate that the horse subunit accommodates (S)-(+)-3-methyl-2-butanol within the active site much better than the opposite stereoisomer, primarily due to steric hindrance caused by Phe-93. Human alpha may accommodate (R)-(-)-3-methyl-2-butanol better than (S)-(+)-3-methyl-2-butanol because of close contacts between the latter and Thr-48. These observations suggest that substitutions at positions 93 and 48 in the active site of human liver alcohol dehydrogenase isoenzymes may determine their substrate specificity for secondary alcohols.     

Photoinactivation and photoaffinity labeling of tryptophan synthase alpha 2 beta 2 complex by the product analogue 6-azido-L-tryptophan

The photoaffinity reagent 6-azido-L-tryptophan was synthesized by chemical methods. It binds reversibly in the dark to the alpha 2 beta 2 complex of tryptophan synthase of Escherichia coli and forms a quinonoid intermediate with enzyme-bound pyridoxal phosphate (lambda max = 476 nm). The absorbance of this chromophore has been used for spectrophotometric titrations to determine the binding of 6-azido-L-tryptophan (the half-saturation value [S]0.5 = 6.3 microM). Photolysis of the quinonoid form of the alpha 2 beta 2 complex results in time-dependent inactivation of the beta 2 subunit but not of the alpha subunit. The extent of photoinactivation is directly proportional to the absorbance at 476 nm of the quinonoid intermediate prior to photolysis. The substrate L-serine is a competitive inhibitor of 6-azido-L-tryptophan binding and photoinactivation. The competitive inhibitors L-tryptophan, D-tryptophan, and oxindolyl-L-alanine also protect against photoinactivation. The results demonstrate that 6-azido-L-tryptophan is a quasi-substrate for the alpha 2 beta 2 complex of tryptophan synthase and that photolysis of the enzyme-quasi-substrate quinonoid intermediate results in photoinactivation. The modified alpha 2 beta 2 complex retains its ability to bind pyridoxal phosphate and to cleave indole-3-glycerol phosphate, a reaction catalyzed by the alpha subunit. 6-Azido-L-tryptophan (side-chain 1,2,3-14C3 labeled) was synthesized enzymatically from 6-azidoindole and uniformly labeled L-[14C]serine by the alpha 2 beta 2 complex of tryptophan synthase on a preparative scale and has been isolated. Incorporation of 14C label from 6-azido-L-[14C]tryptophan is stoichiometric with inactivation. Our finding that most of the incorporated 14C label is bound in an unstable linkage suggests that an active site carboxyl residue is the major site of photoaffinity labeling by 6-azido-L-tryptophan.     

Diagnosis and carrier detection of Tay-Sachs disease: direct determination of hexosaminidase A using 4-methylumbelliferyl derivatives of beta-N-acetylglucosamine-6-sulfate and beta-N-acetylgalactosamine-6-sulfate.

4-Methylumbelliferyl-6-sulfo-2-acetamido-2-deoxy derivatives of beta-D glucopyranoside and beta-D-galactopyranoside were prepared by direct sulfation of the commonly used unsulfated derivatives. Both sulfated substrates were highly specific for hexosaminidase A, and in fractionated serum, cells, and tissue preparations, less than 2.5% of these activities were associated with hexosaminidase B and the intermediate isozyme fractions. Serum and leukocytes from patients with infantile Tay-Sachs disease, including a patient with thermolabile hexosaminidase B, had less than 2% of noncarrier activities. Carrier values were clearly separated from those of noncarriers, and no problems were encountered in utilizing sera from pregnant women. The % hexosaminidase A values as derived from the ratio between the activities toward the sulfated and unsulfated substrates in the same specimen were comparable to those obtained by the heat-inactivation method (except for subjects with thermolabile hexosaminidase B) and may be helpful in genotype determination in borderline cases.     

The subunits of human hexosaminidase A.

Previous studies of the subunit structure of hexosaminidase gave ambiguous results, but suggested that the enzyme was composed of six equally sized subunits. Dissociation of hexosaminidase A with p-chloromercuribenzoate produces an alkylated fragment with mol.wt. approx. 50000, which is converted into hexosaminidase S by treatment with dithiothreitol. Treatment of native hexosaminidase A with sodium dodecylsulphate results in the formation of a large and a small fragment. However, although the native enzyme has a sedimentation coefficient of 5.8S, dissociation by S-carboxymethylation and maleic anhydride treatment results in subunits exhibiting a single schlieren boundary on analytical ultracentrifugation with a sedimentation coefficient of 2.18S. These results indicate that the enzyme is composed of four subunits, each with molwt. approx. 25000-27000. The mol.wt. of the native enzymes is calculated to be approx. 110000. Our data are consistent with the subunit structures of hexosaminidases A, B and S as being alpha2beta2, beta4 and alpha4 respectively.     

Substrate selectivity and sensitivity to inhibition by FK506 and cyclosporin A of calcineurin heterodimers composed of the alpha or beta catalytic subunit.

The calcineurin (CaN) alpha and beta catalytic subunit isoforms are coexpressed within almost all cell types. The enzymatic properties of CaN heterodimers comprised of the regulatory B subunit (CnB) with either the alpha or beta catalytic subunit were compared using in vitro phosphatase assays. CaN containing the alpha isoform (CnA alpha) has lower K(m) and higher V(max) values than CaN containing the beta isoform (CnA beta) toward the PO4-RII, PO4-DARPP-32(20-38) peptides, and p-nitrophenylphosphate (pNPP). CaN heterodimers containing the alpha or beta catalytic subunit isoform displayed identical calmodulin dissociation rates. Similar inhibition curves for each CaN heterodimer were obtained with the CaN autoinhibitory peptide (CaP) and cyclophilin A/cyclosporin A (CyPA/CsA) using each peptide substrate at K(m) concentrations, except for a five- to ninefold higher IC50 value measured for CaN containing the beta isoform with p-nitrophenylphosphate as substrate. No difference in stimulation of phosphatase activity toward p-nitrophenylphosphate by FKBP12/FK506 was observed. At low concentrations of FKBP12/FK506, CaN containing the alpha isoform is more sensitive to inhibition than CaN containing the beta isoform using the phosphopeptide substrates. Higher concentrations of FKBP12/FK506 are required for maximal inhibition of beta CaN using PO4-DARPP-32(20-38) as substrate. The functional differences conferred upon CaN by the alpha or beta catalytic subunit isoforms suggest that the alpha:beta and CaN:substrate ratios may determine the levels of CaN phosphatase activity toward specific substrates within tissues and specific cell types. These findings also indicate that the alpha and beta catalytic subunit isoforms give rise to substrate-dependent differences in sensitivity toward FKBP12/FK506.