Human leucocyte alpha-L-fucosidase.

1. Human alpha-L-fucosidase (EC 3.2.1.51) was studied from leucocytes, urine and serum. 2. The leucocyte and urine enzymes are similar in many properties (KM, pH optimum, electrophoretic pattern, heat stability). 3. The serum alpha-L-fucosidase differs from the leucocyte and 4rine enzyme wit,respect to: electrophoretic pattern, pH optimum and heat stapility. 4. The molecular weight of leucocyte alpha-L-fucosidase was determined to be 80 000 +/- 5000. 5. Cu2+, Hg2+ and PCMB are strong inhibitors of leucocyte alpha-L-fucosidase. This inhibition could be completely reversed by beta-mercaptoethanol, indicating that thiol groups are essential for catalytic activity.     

An abnormal form of purine nucleoside phosphorylase in a family with a child with severe defective T-cell-and normal B-cell immunity.

1. Purine nucleoside phosphorylase and adenosine deaminase (ADA) were studied in normal red blood cells and lymphocytes and in the cells of a family with a child with a defective T-cell-and normal B-cell immunity. 2. In the propositus no purine nucleoside phosphorylase (NP) activity could be detected in her red cells and lymphocytes, while the ADA activity was somewhat increased. The NP activities of the father, mother and brother of the propositus are in the heterozygote range. The decreased activity of NP was not only found for the substrate inosine but also when guanosine or xanthosine were used as substrate. The mode of inheritance is autosomal recessive. 3. With starch gel electrophoresis no NP activity could be detected in the patient's haemolysate. The electrophoretic patterns of NP from the father, mother and brother of the patient seem to be the same as for normal NP with six bands of NP activity. 4. The nucleoside phosphorylases of the father, mother and brother of the patient were characterized by an increased KM for the substrate inosine, normal pH optimum and a decreased heat stability.     

Isolation and Characterization of an Abnormal Human Intrinsic Factor

A patient has been described previously who presented at age 13 with vitamin B(12) (B(12)) deficiency secondary to a functionally abnormal intrinsic factor (IF). IF has now been isolated from the gastric juice of the patient, his sister, and both parents, who are first cousins, by using affinity chromatography on B(12)-Sepharose. Patient IF appeared normal in terms of (a) B(12) binding, (b) mol wt, (c) total amino acid and carbohydrate composition, and (d) immunodiffusion with rabbit anti-patient and anti-normal IF sera. After adsorption with normal IF, however, anti-patient IF serum precipitated the various IFs as follows: patient IF (> 95%); mother, father, and sister IF (50%); and normal IF (< 10%). Additional adsorption with mother, father, or sister IF completely inhibited the precipitation of patient IF. The association constant determined for patient IF-B(12) and human ileal mucosal homogenates (0.1 x 10(9) M(-1)) was 60-fold lower than that determined with normal IF-B(12) (6.0 x 10(9) M(-1)). Intermediate amounts of ileal IF-B(12) binding were observed with mother, father, and sister IF-B(12). These in vitro studies were supported by multiple Schilling tests, performed with a totally gastrectomized volunteer, that gave the following mean urinary excretions of [(57)Co]B(12): free B(12) (0.5%); + patient gastric juice (2.6%); + mother or father gastric juice (17%); and + normal gastric juice (26%). These studies demonstrate that the patient is homozygous and that the mother, father, and sister are heterozygous for a structurally abnormal IF that has a markedly decreased, but not absent, affinity for ileal IF-B(12) receptors. These studies also indicate that the B(12) and ileal binding sites are located on different portions of the IF molecule.     

Complete and partial deficiencies of complement factor D in a Dutch family.

A young man suffering from recurrent Neisseria infections was shown to lack detectable serum complement factor D hemolytic activity. Addition to the patient's serum of purified factor D to a final concentration of 1 microgram/ml resulted in full restoration of the activity of the alternative pathway. Using an enzyme-linked immunosorbent assay, it was shown that the patient's serum did not contain measurable amounts of factor D antigen either. The sister, the father, as well as the parents of the mother had factor D levels within the normal range, and the factor D level of the mother was decreased. The capacity of the patient's serum, at concentrations up to 5%, to promote phagocytosis of Escherichia coli by normal human granulocytes was low when compared to normal serum. Substitution of the patient's serum with purified factor D resulted in a full restoration of opsonic activity. This study describes the first complete deficiency of factor D, and demonstrates its possible relation to recurrent Neisseria infections.     

Serum and tissue alpha-L-fucosidase activity in the pre-clinical and clinical stages of hepatocellular carcinoma.

1. To assess the value of serum alpha-L-fucosidase (EC 3.2.1.51) as a marker for hepatocellular carcinoma, the activity was measured in patients with hepatocellular carcinoma and in control subjects. 2. Mean serum alpha-L-fucosidase activity was significantly greater in 35 patients with hepatocellular carcinoma (225 +/- 69 nkat/l) than in 35 patients with cirrhosis and 20 normal subjects (134 +/- 30 and 93 +/- 28 nkat/l, respectively). The overlap between hepatocellular carcinoma and cirrhosis, however, was such as to severely limit any value of the enzyme as a diagnostic test. 3. In four cirrhotic patients with hepatocellular carcinoma, an increased enzyme activity was detectable in samples taken up to 66 months before the tumours were diagnosed clinically. 4. The serum activity of alpha-L-fucosidase fell to within the reference range after liver transplantation for hepatocellular carcinoma in three patients and in one of these a subsequent rise was associated with tumour recurrence which was diagnosed at 8 months after the rise in activity. Ineffective cytotoxic chemotherapy was also associated with a progressive rise in serum alpha-L-fucosidase activity. 5. alpha-L-fucosidase activity in tumour tissue was significantly lower than that seen in non-tumour tissue from cirrhotic patients. These reductions may represent increased transport from the tissue and may partly account for the increased serum activity found in some patients with hepatocellular carcinoma.     

Angiotensin I-converting enzyme in human urine.

It was demonstrated that angiotensin I-converting enzyme was excreted in human urine. The mean activity of the enzyme in normal urine was found to be 0.38 +/- 0.04 (S.E.M.) units/day (n = 18) and the enzymic activity correlated well with the concentration of the excreted sodium (r = 0.76, p less than 0.005). Urinary angiotensin I-converting enzyme was partially purified. Three different molecular weights of enzyme (greater than 400 000, 290 000 and 140 000) were demonstrated by Sephadex G-200 gel filtration. The enzymic properties of these three enzymes were identical with those of angiotensin I-converting enzyme from human lung with regard to inhibitory effects (bradykinin potentiator c and Arg-Pro-Pro), Cl- dependency, pH optimum and KM value.     

A study of lysosomal enzyme activities in serum and leukocytes in chronic hepatic disease (author's transl)

Five lysosomal enzyme activities (arylsulfatase A, alpha-D-mannosidase, alpha-L-fucosidase, hexosaminidase and beta-D-galactosidase) were determined in serum and leucocytes of 30 controls and 114 patients suffering from various liver diseases, including 30 with idiopathic hemochromatosis and 34 with alcoholic cirrhosis. The results show (1) a decrease of the serum arylsulfatase A activity in idiopathic hemochromatosis, (2) an increase of this activity in cholestatic jaundice, and (3) mainly, a sharp rise of the leucocyte lysosomal enzyme activities in the liver diseases studied (chiefly idiopathic hemochromatosis and alcoholic cirrhosis). The mechanism and the meaning of these disturbances are discussed.     

The use of tears for diagnosis of GM1 gangliosidosis.

The properties of beta-galactosidase of tears were investigated and the standard assay system was accomplished. The pH optimum was 4.2. The enzyme had a KM of 8.3 X 10(-4) M. The activity was stimulated by chloride ions and slightly stabilized by bovine serum albumin. The activities of normal individuals were 205 +/- 80 (S.D.) nmol/h/ml. The activity in the tears of the patient with GM1 gangliosidosis decreased to about 20% of normal control and this disease could be diagnosed by the assay of beta-galactosidase in tears.     

Primary amniotic fluid cell, skin fibroblast and liver alpha-L-fucosidase and its relation to cystic fibrosis.

Cultured skin fibroblast and primary amniotic fluid cell alpha-L-fucosidase had a double optimum of pH 5.0 and 6.0. Alpha-L-fucosidase was largely bound as a single peak to DEAE-cellulose at pH 6.6. Sucrose density isoelectric focusing revealed up to seven components with pI values of 4.9, 5.2, 5.4, 5.8, 6.1, 6.5 and 7.1 with their apparent KM values (77--500 mumol/l) being higher than that (57 mumol/l) of the unfocused enzyme. Liver, skin fibroblast and amniotic fluid cell alpha-L-fucosidase was separated into two peaks by gel filtration. Peak one was more active and stable at low pH and more thermostable at 50 degrees C than peak two, while both peaks had an apparent KM of 52 mumol/l. Apart from the different proportions of the peaks separated by gel filtration, the results for the three tissues were similar. The properties of alpha-L-fucosidase studied were similar for control and cystic fibrosis liver or skin fibroblasts.     

A fluorimetric assay for angiotensin-I converting enzyme in human serum.

A fluorimetric method is described for a simple, sensitive and reproducible assay for angiotensin-I converting enzyme in human and guinea pig sera. The very weak fluorescent substrate o-aminobenzoylglycyl-p-nitro-L-phenylalanyl-L-proline is enzymatically hydrolyzed, producing the highly fluorescent o-aminobenzoylglycine that is quantitatively determined by spectrofluorimetry. Dependence of activity on substrate concentration, amount of serum, time of incubation and pH were investigated. The KM value for the substrate is 0.1 and 0.032 mM for the human and guinea pig serum enzyme, respectively. The mean value of serum angiotensin-I converting enzyme for 16 normal adult persons was 2.56 +/- 0.10 (S.E.) with a standard deviation of 0.81 nmol/min/ml serum.     

Evidence for both a regulatory mutation and a structural mutation in a family with maple syrup urine disease

Maple syrup urine disease (MSUD) results from a deficiency of branched chain alpha-ketoacid dehydrogenase (BCKDH). We have studied the etiology of MSUD by determining the enzyme activity, protein, and mRNA levels of BCKDH in fibroblasts from a classic MSUD patient and his parents. By enzymatic amplification of the patient's mRNA followed by cloning and DNA sequencing, we have identified a T to A transversion that alters a tyrosine to an asparagine at residue 394 of the E1 alpha subunit. Amplification of both mRNA and genomic DNA, in combination with allele-specific oligonucleotide hybridization, demonstrated that the father was heterozygous for this mutant allele. The mother was homozygous for the allele encoding the normal Tyr394, but expressed only about half of the normal level of mRNA and protein. The patient was genetically heterozygous for this altered allele, although only the abnormal allele was expressed as mRNA. We conclude that the patient was a compound heterozygote, inheriting an allele encoding an abnormal E1 alpha from the father, and an allele from the mother containing a cis-acting defect in regulation which abolished the expression of one of the E1 alpha alleles. Our results revealed for the first time that a case of MSUD was caused by structural and regulatory mutations involving the E1 alpha subunit.     

A case of GM1-gangliosidosis type I: glycosphingolipid profiles of urine and transformed lymphocytes and beta-D-galactosidase activities in peripheral lymphocytes, cultured skin fibroblasts and transformed lymphocytes

A female infant with early-onset GM1-gangliosidosis type I was investigated. The lymphocytes, transformed lymphocytes and cultured skin fibroblasts of the patient were demonstrated to have severe beta-D-galactosidase deficiency. The beta-D-galactosidase activities of these cells from the patient's father and mother were at the lower limit of the normal range. The oligosaccharide accumulation in urine of the patient showed the typical type I GM1-gangliosidosis pattern, but no GM1 ganglioside was detected in the patient's urine or transformed lymphocytes. The clinical features were compatible with infantile GM1-gangliosidosis. The mixture of homogenates from the cultured fibroblasts or transformed lymphocytes of the patient and controls showed no complementation of beta-D-galactosidase activity against the controls.     

A case of hereditary angioneurotic edema provoked by functional insufficiency of C1-inactivator

The hemolytic activity of complement, the concentration of C1-inactivator, of C1q and C4 components of complement as well as the activity of contact-activated kallikrein and the functional activity of C1-inactivator were studied in the plasma of a female patient with hereditary angioneurotic edema (HAE), of her mother, sister and normal donors. The hemolytic activity of complement, the concentration of C1-inactivator and C1q in the blood plasma of the persons examined turned out unchanged as compared with the same parameters in donors. Meanwhile in the patient with HAE and her mother with symptoms similar to HAE, the concentration of C4 appeared lower than that in the patient's sister not suffering from the disease and in donors. The activity of contact-activated kallikrein in the plasma of the patient's sister and mother exceeded the activity of the enzyme in donors. The least activity of plasma kallikrein was recorded in the proband. The functional activity of C1-inactivator in the proband constituted 50-60% of the activity of that parameter in her sister and donors. The magnitude of the functional activity of C1-inactivator in the patient's mother approximated its magnitude in the proband. Therefore, the development of HAE in the given case was mainly provoked by functional insufficiency of C1-inactivator. The magnitude of the spontaneous TAME-esterase kallikrein-like activity in the blood plasma of the patient with HAE remained within normal.     

Purification and serological studies of human alpha-L-fucosidase in the normal and fucosidosis states.

An antiserum has been raised to purified alpha-L-fucosidase. Levels of cross-reaction with serum of two unrelated fucosidosis patients and normal individuals with low activity are consistent with the presence of very low amounts of normal enzyme. Similarly no cross-reacting material could be found in cultured fibroblasts from fucosidosis patients. It is deduced that in these cases there is no production of mutant enzyme in quantities comparable to normal levels. Some observations on the interrelations of fucosidases I and II are reported.     

An unusual hemoglobin anomaly and its relation to alpha-thalassemia and hemoglobin-H disease

A Chinese family with hemoglobin H in the propositus has been reinvestigated. Although the original propositus is now deceased, a sister has the same hematological manifestations. Her hemoglobin, like that of the deceased sister, contains hemoglobins A, H, and Bart's. In addition, however, two minor components have been detected. These minor components appear to have abnormal alpha-chains and are also present in the maternal grandmother, the mother, a maternal aunt, and three other siblings but only in about one-tenth the amount. One of the minor components may be the same as Hb-Thai (25). The father has the characteristics of classical alpha-thalassemia. These results are discussed in relation to current concepts of alpha-thalassemia as they relate to "silent" and "classical" alpha-thalassemia and to possible multiple alpha-chain loci.     

Human serum alpha-L-fucosidase

Human serum alpha-L-fucosidase has been purified 241 200-fold with 35% yield by an affinity chromatographic procedure utilizing agarose-epsilon-aminocaproyl-fucosamine. Isoelectric focusing of the purified enzyme indicated the presence of several forms, with the form at pI 5.0 comprising the majority of the activity. Assay of the purified alpha-L-fucosidase showed only trace amounts of contaminating glycosidases present, with beta-galactosidase being the largest contamnant (0.5% by activity). Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated the presence of two subunits with very similar molecular weights (56 500 and 54 000). Using the p-nitrophenyl substrate, the purified serum alpha-L-fucosidase has an apparent Michaelis constant of 0.52 mM and a broad pH optimum centered around pH 4.8 with a second, minor optimum at pH 6.1. Gel filtration on Sepharose 6-B indicated an apparent molecular weight of 296 000 +/- 30 000. Preincubation with antibodies made previously against purified liver alpha-L-fucosidase led to quantitative immunoprecipitation of the purified serum alpha-L-fucosidase. Assay of the purified serum alpha-L-fucosidase for sialic acid indicated the presence of 1.7 microgram sialic acid per 100 microgram enzyme, about twice that previously found for the purified liver enzyme.     

Detection of heterozygotes for fructose 1,6-diphosphatase deficiency by measuring fructose 1,6-diphosphatase activity in their cultured peripheral lymphocytes

The fructose 1,6-diphosphatase activities in peripheral lymphocytes from the parents of a patient with fructose 1,6-diphosphatase deficiency were lower than the mean value of normal controls, but the value of the mother overlapped lower values for normal controls. The fructose 1,6-diphosphatase activities in lymphocytes of normal adults and the parents increased progressively during in vitro culture, but no enzyme activity could be detected in the lymphocytes of the patient even after culture. None of the values for the parents overlapped those of normal controls on either day 5 or 10 of culture. Thus, it seems probable that heterozygotes for fructose 1,6-diphosphatase deficiency can be distinguished from normal individuals by measuring the fructose 1,6-diphosphatase activity in their cultured lymphocytes.     

A splice junction mutation causes deletion of a 72-base exon from the mRNA for lysosomal acid lipase in a patient with cholesteryl ester storage disease.

The genetic defect leading to cholesteryl ester storage disease (CESD) has been determined in a 12-yr-old patient. Lysosomal acid lipase (LAL) activity in cultured skin fibroblasts was reduced to approximately 9% of control fibroblasts. Plasma cholesterol (255 mg/dl) and LDL-cholesterol (215 mg/dl) were elevated whereas HDL-cholesterol was reduced (19 mg/dl). Triglycerides were moderately elevated (141 mg/dl). There were no clinical abnormalities with the exception of hepatosplenomegaly. Both parents have reduced LAL activity in white blood cells. PCR analysis of the LAL mRNA from the propositus revealed a single slightly smaller mRNA species in skin fibroblasts as well as in leukocytes. The mother of the patient and his older brother had two mRNA species: one of normal size and one of the same size as the propositus. The father has a LAL mRNA of normal size only. Sequence analysis of a PCR-amplified cDNA fragment showed a 72-bp in-frame deletion resulting in the loss of the codons for amino acids 254-277. Analysis of genomic DNA revealed that the 72 bp represent an exon, indicating that the deletion in the mRNA is caused by defective splicing. Sequence analysis of the patient's genomic DNA revealed a G-->A substitution in the last nucleotide of the 72-bp exon in one of his alleles. The mutant allele was shown to cosegregate with the truncated mRNA in the pedigree, providing further evidence that the G-->A substitution causes aberrant splicing and exon skipping. No normal-sized mRNA is detectable in the propositus even though he is not homozygous for the splice site mutation. This can be only accounted for by assuming that he is a compound heterozygote with a null allele inherited from his father. In summary, the data presented provide evidence that deletion of the codons for amino acids 254-277 in the LAL mRNA in combination with a null allele cause the clinical expression of CESD in our patient.     

Leucocyte alpha-1,4- and alpha-1,6-glucosidase activities towards oligosaccharides in late onset glycogenosis type II

We describe the partial characterization and some properties of leucocyte alpha-glucosidase towards disaccharides with the alpha-1,4 (maltose) and alpha-1,6-glucosidic linkage (isomaltose) and tetrasaccharides with the alpha-1,4 (maltotetraose) and alpha-1,6-glucosidic linkage (tetrasaccharide, Glc alpha 1----6Glc alpha 1----4Glc alpha 1----4Glc, which was isolated from the urine of a patient with glycogenosis type II). Leucocyte alpha-glucosidase showed optimal activity towards all four oligosaccharides under two conditions, acidic (pH 4.0-4.5) and neutral (pH 6.0-6.5) regions. Our comparative studies on enzyme kinetics showed that leucocyte alpha-glucosidase was able to hydrolyze both the 1----4 isomers and the 1---6 isomers at acidic and neutral pH. Acid alpha-glucosidase could hydrolyze maltose about 10 times faster than isomaltose, and maltotetraose about 5 times faster than tetrasaccharide isolated from urine. In leucocytes of the patient with late onset glycogenosis type II, acid alpha-glucosidase activities towards maltose, isomaltose, maltotetraose and tetrasaccharide isolated from urine showed 75.3%, 67.4%, 76.5% and 41.4% of normal control values, respectively. Neutral alpha-glucosidase activities towards these four oligosaccharides were normal. Tetrasaccharide with alpha-1,6-glucosidic linkage might be accumulated by the impaired hydrolysis in the circulation as well as the leakage of undegraded glycogen to the circulation from the affected muscle.