Neutral Enzyme Research Articles

Purification and properties of neutral β- N-acetylglycosaminidase from carp blood

A neutral beta-N-acetylglucosaminidase has been purified to homogeneity from carp blood by a seven-step procedure. It was localized in the cytosol of red blood cells. The purified enzyme was specific to beta-N-acetylglucosaminide and inactive to beta-N-acetylgalactosaminide. It was competitively inhibited by free N-acetylglucosamine, but not by free N-acetylgalactosamine. The optimum pH of the enzyme was 6.5, with a stable pH range of 7.0 to 11.0. The enzyme showed greater heat-lability and anodal electrophoretic mobility than acidic beta-N-acetylglucosaminidases. The Mr value, estimated by sucrose density gradient centrifugation, was 122,000, and the enzyme dissociated into two nonidentical subunits with Mr values of 66,000 and 53,000, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With respect to the major characteristics, the neutral enzyme in carp blood was supposed to be a counterpart of hexosaminidase C in human and other mammalian tissues.

Capsaicin-induced release of tachykinins: effects of enzyme inhibitors

We studied the effects of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) inhibition on the airway responses and the recovery of endogenously released substance P- and neurokinin A-like immunoreactivities (SP-LI and NKA-LI) after tracheal injection of capsaicin in isolated guinea pig lungs superfused through the trachea. Capsaicin in doses from 10(-10) to 10(-7) mol induced a dose-dependent increase in airway opening pressure and release of SP-LI and NKA-LI. Airway opening pressure changes and the recovery of SP-LI and NKA-LI were significantly greater in lungs superfused with the NEP inhibitor SCH 32615 than in control lungs. ACE inhibition with captopril did not increase the mechanical response or the recovery of SP-LI compared with lungs not receiving captopril. In lungs from guinea pigs pretreated with high doses of capsaicin 7-10 days before study, a regimen designed to deplete endogenous tachykinins, there was a significant decrease in the content and release of NKA-LI and SP-LI. There were no detectable airway effects of acute capsaicin infusion even after doses of 10(-5) mol. Because NEP is important in modulating the airway effects of endogenously released tachykinins after tracheal infusion of capsaicin, but ACE is not, it seems likely that tracheal administration of capsaicin releases tachykinins from epithelial rather than endothelial loci.

Phosphoramidon-sensitive endothelin-converting enzyme in the cytosol of cultured bovine endothelial cells

Neutral metalloproteases with endothelin-1 (ET-1) converting activity were detected in membranous and cytosolic fractions of cultured endothelial cells (EC) from bovine carotid artery in a ratio of 5:1, respectively. The cytosolic enzyme specifically and quantitatively converts big ET-1 to ET-1 (Km = 10.7 microM), but does not convert big ET-3. Like the membranous enzyme, the cytosolic enzyme is only active at pH 6.5-7.5, and is competitively inhibited by phosphoramidon (Ki = 0.79 microM). The apparent molecular weight of the cytosolic enzyme is about 540 kD, which is 5-6 times greater than that of the membranous enzyme. These results indicate the presence of two types of phosphoramidon-sensitive neutral ET-converting enzyme in vascular EC.

Endothelin converting enzyme of bovine carotid artery smooth muscles

This is the first report clearly demonstrating the presence of endothelin (ET) converting enzyme in vascular smooth muscle. Like cultured endothelial cells, noncultured vascular smooth muscle homogenate of bovine carotid arteries converts human big ET-1 to ET-1 at pH 3.0, pH 5.0 and pH 7.0, and the apparent ratio of these three activities is about 6: 5 : 1, respectively. Peptides generated during incubation of the homogenate and big ET-1 at the three pHs were identified as ET-1 by radioimmunoassay and high performance liquid chromatography. The two acid enzymes are in the cytosol (103,000xg sup) and are inhibited by pepstatin A, while the neutral enzyme is sensitive to EDTA or phosphoramidon; 73% of the neutral enzyme activity was membrane-bound and the remainder (27%) cytosolic.

Endothelin-Converting Enzyme and Its in vitro and in vivo Inhibition

We investigated endothelin (ET)-converting enzyme and its localization in the vasculature. The membrane and cytosol fractions of cultured endothelial cells of bovine carotid artery contain phosphoramidon-sensitive ET-converting enzymes, and their molecular weights are about 100 and 540 kDa, respectively. The specific conversion of big ET-1 by these enzymes proceeds at pH 7.0 +/- 0.5, and it is inhibited by EDTA, o-phenanthroline, and phosphoramidon. Big ET-3 is converted by the membrane enzyme at a rate about one-tenth that of big ET-1, but it is not converted by the cytosol enzyme. Big ET-1 (but not ET-1)-induced hypertension in rats was remarkably suppressed by pretreatment with phosphoramidon, and big ET-1 (but not ET-1)-induced contraction of isolated coronary arteries, either with or without the endothelium, was substantially suppressed by phosphoramidon. These results suggest an essential role of phosphoramidon-sensitive enzyme(s) in the vascular conversion of big ET-1, and the existence of such enzymes also in nonendothelial cells. We found three converting enzymes operating at different optimal pH values in noncultured vascular smooth muscle cells; two pepstatin-sensitive, cytosolic acid proteinases and a phosphoramidon-sensitive neutral enzyme(s) in the membrane and cytosol. All of these findings strongly suggest the importance of phosphoramidon-sensitive neutral enzymes in the vascular conversion of big ET-1.

Molecular cloning and nucleotide sequence of a gene for alkaline celluase from Bacillus sp. KSM-635

A gene for alkaline cellulase from the alkalophilic Bacillus sp. KSM-635 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. Although the recombinant plasmid contained two HindIII inserts of 2.6 kb and 4.0 kb, the inserts were found to be contiguous in the Bacillus genome by hybridization analysis. Nucleotide sequences of a 2.4 kb region which was indispensable for the production of cellulase, and the flanking, 1.1 kb region, were determined. There was an open reading frame (ORF) of 2823 bp in the 3498 bp sequence determined, which encoded 941 amino acid residues. Two putative ribosome-binding sites and a sigma 43-type, promoter-like sequence were found upstream from an initiation codon in the ORF. The deduced amino-terminal sequence resembles the signal peptide of extracellular proteins. A region of amino acids, 249 to 568, of the deduced amino acid sequence of the cellulase from this organism is homologous with those of alkaline and neutral enzymes of other micro-organisms, but nine amino acid residues were found to be conserved only in the alkaline enzymes.

Calpain and calpastatin activity in the optic pathway

The levels of the neutral proteolytic enzymes calpains and their endogenous inhibitor calpastatin were determined in the retina and in the retrobulbar optic pathway in the albino rabbit. The highest level of calpains was observed in the optic nerve with decreasing levels in the optic tract and superior colliculus. The level of calpastatin in the retina was very low compared to that in the optic nerve and tract and other parts of the nervous system.

Neutral protein-degrading enzymes in experimental fracture callus: a preliminary report.

The process of endochondral fracture healing is biochemically similar to growth plate calcification. Recent studies have identified potentially important roles for proteoglycan-degrading enzymes in the growth plate. The purpose of the study described herein was to identify, in healing fractures, neutral enzyme activities capable of degrading proteoglycans and other matrix proteins. Two sets of 60 male Sprague-Dawley rats underwent the production of closed femoral fractures. Calluses were retrieved at timed intervals, and cell and matrix vesicle fractions were prepared for electron microscopy, neutral peptidase, and alkaline phosphatase assays. In another group of 10 animals, fractions were prepared from 14-day calluses and examined for proteoglycanase activity. In the cell fractions, alkaline phosphatase, alanyl-beta-naphthylamidase, aminopeptidase, and endopeptidase activities showed somewhat parallel distributions peaking at approximately 14-17 days. In the matrix vesicle fractions, similar relative distributions were observed for alkaline phosphatase and endopeptidase. However, here the peak activities occurred up to 3 days later than they did in the cell fractions. Significant proteoglycanase activity was confirmed in both cell and matrix vesicle fractions. These findings are consistent with the hypotheses that (a) neutral peptidases, by virtue of their temporal expression in parallel with alkaline phosphatase, may be involved in preparing fracture callus matrix for calcification; and (b) matrix vesicles may convey certain of these enzymes to sites of both matrix degradation and calcification, since the same activities found in cells are found in matrix vesicles a few days later. The possibility that some of these enzymes are involved in growth factor activation remains to be investigated.

Pyrene-methyl lauryl ester, a new fluorescent substrate for lipases: use for diagnosis of acid lipase deficiency in Wolman's and cholesteryl ester storage diseases.

Fluorescent pyrene-methyl lauryl ester (PMLes) was synthesized and used for the determination of cellular lipase activities in lymphoblasts and fibroblasts from normal subjects and from patients affected with Wolman's or cholesteryl ester storage diseases (both exhibiting a deficiency of the lysosomal acid lipase). The hydrolysis of PMLes by acid lipase could be followed directly in a spectrofluorometer; this was possible because of the very high fluorescence emission of pyrene-methanol at 378 nm (monomeric form) in aqueous medium, whereas the substrate has practically no monomeric emission at 378 nm but emits only at 475 nm (excimeric form) in the experimental conditions used: this property permitted us to use PMLes as a fluorogenic substrate. In an alternative procedure, the enzymatic reaction could be determined after partition of the reaction mixture in a biphasic system of heptane and aqueous ethanol; the residual undegraded substrate partitioned into the upper heptane phase and the fluorescence of the product (i.e. pyrene-methanol) was read in the lower aqueous-ethanolic phase, at 378 nm. PMLes was hydrolyzed in extracts of normal lymphoblasts and fibroblasts by at least two lipases, one acidic lipase (pH 4.0) and a second more neutral enzyme (pH 6.5). The acidic lipase activity was practically absent in lymphoblasts and fibroblasts from Wolman's or cholesteryl ester storage diseases. This demonstrates that the fluorescent PMLes is hydrolyzed by the lysosomal acid lipase and can be used as a very sensitive fluorogenic substrate which permits direct recording of product formation and is suitable for the enzymatic diagnosis of either of these diseases.

Glutathione transferase from bovine placenta. Preparation, biochemical characterization, crystallization, and preliminary crystallographic analysis of a neutral class PI enzyme.

A method was developed to purify glutathione transferase from bovine placenta by affinity chromatography and fast protein liquid chromatography. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate gel electrophoresis and isoelectric focusing. The dimeric enzyme is composed of identical subunits with a molecular weight of 23,000; its isoelectric point is 6.9. In contrast to previously described isoenzymes of glutathione transferase, the protein we have purified exists in two forms, an active reduced form and a less active oxidized form. These can be reversibly transformed into each other but behave differently in sedimentation analysis, gel chromatography, and gel electrophoresis. These differences may reflect a change in the molecular shape of glutathione transferase. Chemical modification with iodoacetate, iodoacetamide (presumably of thiol groups), phenylglyoxal, and butadione (presumably of arginyl groups), and their inhibitory effects on the activity were investigated. From substrate specificity studies and N-terminal sequence analysis it is obvious that this glutathione transferase must belong to the isoenzyme class pi. The purified enzyme could be crystallized from 1.4 M ammonium sulfate solution, pH 8.0, in the presence of S-hexyl-glutathione. The crystals are tetragonal, with space group P4(1)2(1)2 or P4(3)2(1)2. The cell constants are a = b = 6.1 nm, c = 23.7 nm, alpha = beta = gamma = 90 degrees. The crystals diffract to 0.26-nm resolution and are suitable for x-ray structure analysis.

Hydrolysis of fluorescent pyrenetriacylglycerols by lipases from human stomach and gastric juice

Fluorescent triacylglycerols containing pyrenedecanoic (P 10) and pyrenebutanoic (P 4) acids were synthesized and their hydrolysis by lipases from human gastric juice and stomach homogenate was investigated. The existence in stomach homogenate of four different lipolytic enzymes hydrolyzing fluorescent triacylglycerols is suggested by the comparison of various enzymatic properties: acyl chain length specificity, heat inactivation and effect of detergents (Triton X-100 and taurocholate), serum albumin, diethyl- para-nitrophenyl phosphate (E 600) and other inhibitors. (1) The acid pH4-lipase hydrolyzes P 10-triacylglycerols but not P 4-triacylglycerol and exhibited the characteristic properties of the lysosomal lipase: the maximal activating effect of detergents occurs at relatively high concentrations (the substrate /detergent optimal molar ratios were 1:5 and 1:25 for triacylglycerols / taurocholate and triacylglycerols / Triton X-100, respectively); its activity was strongly inhibited by para-chloromercuribenzoate (2.5 mmol/l), but was not significantly affected by serum albumin and E 600 (10 −2 mmol/l). (2) The neutral pH7-lipase hydrolyzes P 10-triacylglycerols but not P 4-triacylglycerol. It is resistant to E 600 and heat-stable, similarly to the acid pH4-lipase, but it is well discriminated from the acid enzyme by its substrate / detergent optimal molar ratios (1:2 and 1:3 for triacylglycerols / taurocholate and triacylglycerols / Triton X-100, respectively), whereas higher detergent concentrations, optimal for the acid lipase, are strongly inhibitory for the neutral enzyme. (3) The pH5-lipase present in gastric juice as well as in stomach homogenate exhibited properties obviously discriminating it from the other lipolytic enzymes from stomach homogenate: broad substrate specificity for P 10- as well as P 4-triacylglycerols, activation by low concentrations of amphiphiles (with optimal ratios triacylglycerols / taurocholate, triacylglycerols / taurocholate and triacylglycerols / phosphatidylcholine around 1:1, 1:3 and 1:0.1, respectively), heat-lability, strong activation by serum albumin and inhibition by E 600 (10 −2 mmol/l). This pH5-lipase is the sole lipolytic enzyme present in gastric juice and is probably identical with the well-known ‘gastric’ lipase. (4) A pH7.5-enzyme is characterized by its specificity for P 4-triacylglycerols, its heat-lability at 50°C and its strong inhibition by E 600 (10 −2 mmol/l).

Enzymatic Lactose Hydrolysis for Prevention of Lactose Crystallization in a Whey Spread

ABSTRACTThe enzymatic lactose hydrolysis for elimination of sandiness was studied in a whey‐buttermilk spread of previously optimized composition (18% fat, 12% protein, 17% lactose). Soluble enzyme preparations of either the acid (Aspergillus) or the neutral (Kluyveromyces) type were suitable for the minimum 30% hydrolysis required to prevent the lactose crystallization. Two types of acid enzymes used at either 1 mg or 2 mg per g hydrolyzed mixture produced the desired effect after 2 hr of hydrolysis at 30°C. Similar results were obtained with two types of neutral enzymes at 2 mg per g hydrolyzed mixture after either 2 or 4hr of hydrolysis at 30°C.

Electrophoretic study of enzymes from cereal aphid populations. V. Spatial and temporal genetic similarity of holocyclic populations of the bird-cherry oat aphid, Rhopalosiphum padi (L.) (Hemiptera: Aphididae), in Britain

AbstractLarge colonies of holocyclic Rhopalosiphum padi (L.) were sampled in May-June in 1985 and 1986 from the principal host, Prunus padus, in various parts of Britain, mainly in the south-east. They were tested electrophoretically using nine enzymes representing 14 putative genetic loci. Only two loci (14%) were polymorphic (GOT, glutamate-oxaloacetate transaminase, and SORDH, sorbitol dehydrogenase), the remainder being monomorphic. Allele and genotype frequencies at each locus were generally similar both spatially and temporally, and genotype frequencies were close to Hardy-Weinberg expectations. The low level of genetic variability may reflect population bottlenecks which have purged most of the variation and which have been imposed by the distribution of P. padus, especially in the south of Britain where the tree is generally scarce. In addition, it is probable that adverse climatic conditions directly affect winter survival of anholocyclic populations of the aphid and thus also reduce variability. In the long term, such bottlenecks would tend to preclude the maintenance of effectively neutral enzyme variants that arise within populations. The similarity of allele and genotype frequencies may reflect the highly migratory behaviour of R. padi. Thus, in southern Britain, where P. padus is generally much rarer in comparison with the north of the country, R. padi inter-population gene flow is still sufficient to alleviate the influences of genetic drift and possible directional selection on the allelic variation that remains.

Different susceptibilities of platelet phospholipids to various phospholipases and modifications induced by thrombin. Possible evidence of rearrangement of lipid domains

On the membrane surface of the human platelet, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were hydrolyzed to different extents by the snake venom phospholipases A 2 of varying p I values. The susceptibility of platelet phospholipids to basic phospholipase A 2 of Naja nigricollis (p I 10.6) has been reported (Wang et al. (1986) Biochim. Biophys. Acta 856, 244–258). The susceptibilities of platelet phospholipids to acidic phospholipase A 2 of Naja naja atra (p I 5.2) and to neutral phospholipase A 2 of Hemachatus haemachatus (p I 7.3) were investigated in this study. In gel-filtered platelets, acidic phospholipase A 2 hydrolyzed 35% PC and 10% PE, while neutral phospholipase A 2 hydrolyzed 18% PC and 3% PE. In thrombin-induced shape-changed platelets, acidic phospholipase A 2 hydrolyzed 20% PC and 10% PE, while neutral phospholipase A 2 hydrolyzed 15% PC and 6% PE. In thrombin-activated platelets, acidic phospholipase A 2 hydrolyzed 25% PC and 7% PE, while neutral phospholipase A 2 hydrolyzed 25% PC and 10% PE. Sequential lipid hydrolysis experiments showed that basic phospholipase A 2 of Naja nigricollis could hydrolyze the remaining PC and PE in the membrane previously treated with the neutral enzyme. The results may mean that: (1) the PC and the PE domains exist on the platelet membrane surface; and (2) the lipid domains on the membrane surface of resting platelets are rearranged by thrombin.

Characterization of acid and neutral α-mannosidases in bull semen and reproductive organs

1. 1. Acid and neutral α-mannosidase activities were studied in the bull reproductive tissues, isolated spermatozoa, epididymal and seminal vesicle secretion and seminal plasma. 2. 2. The acid enzyme in the seminal plasma mainly derived from the epididymal secretion, while the neutral one was enriched in the sperm cells. The latter activity in the seminal plasma appears to be due to an enzyme released from the cytoplasmic droplets in the epididymis. 3. 3. The acid enzyme had a molecular weight of 220,000–320,000, pI 7.3–6.0 and an optimum at pH 4.0. It was sensitive to swainsonine but was stimulated by Zn 2+. 4. 4. The neutral enzyme had a molecular weight of 360,000–460,000, pI 5.4-4.7 and showed double optima at pH 5.5 and 6.0–7.0. It was resistant to swainsonine but was markedly activated by Co 2+ or Fe 2+. The neutral enzyme was also more sensitive to thermal inactivation than the acid one.

First trimester diagnosis of Pompe's disease (glycogenosis type II) with normal outcome: assay of acid alpha-glucosidase in chorionic villous biopsy using antibodies.

Prenatal diagnosis of glycogenosis type II was performed by direct assay of acid alpha-glucosidase (EC 3.2.1.20) in chorionic villous biopsy obtained by transcervical cannula aspiration from a pregnancy at risk in the 10th week of gestation. The exact value of the enzyme activity estimated by the use of antibody preparations for purified human liver acid alpha-glucosidase was in the heterozygous range, and so the homozygous enzyme deficiency could be excluded. The subsequent analysis of cells cultured from amniocentesis sampling in the 18th week of gestation resulted in a similar outcome. The study with antibodies showed that in 23 control chorionic villi obtained during gestational ages between 7-13 weeks, 1-15% of the total alpha-glucosidase activity at pH 4.0 were due to renal or neutral enzyme. This indicates that it may be important to employ antibodies for prenatal diagnosis using chorionic villous sampling. A healthy and unaffected boy was born. The biochemical values obtained from an umbilical blood specimen were in accordance with the results of the prenatal diagnosis.

Neutral proteases in human osteoarthritic synovium.

The activities of neutral collagenolytic enzymes (CE) and neutral proteoglycan-degrading enzymes (PE) in the synovial membranes of osteoarthritis (OA) patients were determined. The total neutral metallo-CE activity showed a significantly higher level of activity when the membranes of OA patients were compared with those of controls. The severely and moderately inflamed synovia had significantly more enzyme activity than did either mildly inflamed or control synovia. Steroids reduced the total metallo-CE activity. In specimens with severe inflammation, the active form of the neutral metallo-CE was significantly elevated over that found in controls. The serine-CE activity was also significantly elevated in OA synovia with severe inflammation and synovial hypertrophy. The total and active neutral metallo-PE was significantly elevated in synovial membranes of OA patients with severe inflammation. Moreover, the serine-PE showed much more activity in OA patients than in controls. The enzyme activity remained at a significantly high level in the OA synovium, regardless of the presence or absence of macroscopic synovial hypertrophy or the histologic grading of the synovium (mild, moderate, severe). Our data indicate that, in OA, an increased level of neutral proteases in the synovia could be involved in the local tissue destruction of the periarticular structures. Because of the very high level of serine proteases, their diffusion may render plausible a degradative action on the cartilage surface.

Different forms of human liver glutathione S-transferases arise from dimeric combinations of at least four immunologically and functionally distinct subunits.

Four immunologically distinct subunits were characterized in glutathione (GSH) S-transferases of human liver. Five cationic enzymes (pI 8.9, 8.5, 8.3, 8.2 and 8.0) have an apparently similar subunit composition, and are dimers of 26 500-Mr (A) and 24 500-Mr (B) subunits. A neutral enzyme, pI 6.8, is a dimer of B-type subunits. One of the anionic enzymes, pI 5.5, is also a dimer of 26 500-Mr subunits. However, the 26 500-Mr subunits of this anionic enzyme form are immunologically distinct from the A subunits of the cationic enzymes, and have been designated as A'. Immunoabsorption studies with the neutral enzyme, BB, and the antibodies raised against the cationic enzymes (AB) indicate that A and B subunits are immunologically distinct. Hybridization in vitro of the A and B subunits of the cationic enzymes (AB) results in the expected binary combinations of AA, AB and BB. Studies with the hybridized enzyme forms indicate that only the A subunits express GSH peroxidase activity. A' subunits have maximum affinity for p-nitrobenzyl chloride and p-nitrophenyl acetate, and the B subunits have highest activity towards 1-chloro-2,4-dinitrobenzene. The other anionic form, pI 4.5, present in liver is a heterodimer of 22 500-Mr (C) and B subunits. The C subunits of this enzyme are probably the same as the 22 500-Mr subunits present in human lung and placental GSH transferases. The distinct immunological nature of B and C subunits was also demonstrated by immunoaffinity and subunit-hybridization studies. The results of two-dimensional polyacrylamide-gel-electrophoretic analyses indicate that in human liver GSH transferases, three charge isomers of Mr 26 500 (A type), two charge isomers of Mr 24 500 (B type) and two charge isomers of Mr 22 500 (C type) subunits are present.

Effects of 1-hydroxyethylidene-1,1 bisphosphonate and (chloro-4 phenyl) thiomethylene bisphosphonic acid (SR 41319) on the mononuclear cell factor-mediated release of neutral proteinases by articular chondrocytes and synovial cells

Articular chondrocytes and synovial cells were stimulated to produce collagenase, neutral casein and proteoglycan-degrading proteinases by conditioned medium from human peripheral blood mononuclear cells. Collagenase, neutral casein and proteoglycan-degrading proteinase secretion was inhibited by SR 41319, a new bisphosphonate, in a concentration-dependent manner. Complete inhibition was achieved at about 0.3 mM. EHDP exhibited the same general profile but was about 10-fold less active and never completely inhibited the enzyme secretion. When added before MCF, SR 41319 had a protective effect against subsequent activation of the cells by MCF. SR 41319 also inhibited the increase of enzyme secretion by cells previously stimulated with MCF. The results suggest that the ability of SR 41319 to inhibit the MCF-mediated secretion of neutral enzymes involved in cartilage destruction could be valuable in the management of connective tissue damage in rheumatoid arthritis.

Acid and neutral alpha-glucosidase in the reproductive organs and seminal plasma of the bull.

A synthetic substrate (p-nitrophenyl-alpha-D-glucopyranoside) was used to measure the acid and neutral alpha-glucosidase activity in bull seminal plasma, spermatozoa and in homogenates of bull reproductive organs. Marked differences were observed in the activities of these enzymes in the various tissues studied. Epididymis and particularly its caput region contained the highest specific activity of acid alpha-glucosidase. The activity of neutral alpha-glucosidase was highest in testis and in different parts of the epididymis. Seminal plasma, spermatozoa and seminal vesicle secretion contained only the acid enzyme activity. After fractionation with anion exchange chromatography in HPLC (Mono Q) and chromatofocussing, acid alpha-glucosidase activity of seminal plasma was recovered in two fractions with different pI values. The corresponding activities were found in the secretion of seminal vesicles, which thus form the major secretory source of seminal plasma acid alpha-glucosidase. In the fractionation with gel filtration on Sepharose 6B, the acid alpha-glucosidase had a smaller molecular weight than did the neutral enzyme. In anion exchange chromatography and chromatofocussing the testicular and epididymal homogenates each contained two acid and two neutral isoenzymes. In both fractionations the elution pattern of acid alpha-glucosidase was clearly different from that of the enzymes in seminal plasma. The pH optimum of acid alpha-glucosidase ranged from 3.75 to 4.5 and that of the neutral enzyme from 6.5 to 7.0. The neutral activity was more sensitive to many divalent metal ions and differences were also observed in the response of the enzymes to different concentrations of turanose and KCl.