Lysosomal enzyme activities in cultured lymphoid cell lines

Several lysosomal enzyme activities in cultured lymphoid cell lines were studied during 3 phases of cell culture; logarithmic growth phase, stationary phase and decline phase.Enzyme induction during cell growth was found in N-acetyl-hexosaminidase, β-galactosidase and α-l-fucosidase, but no induction in a-d-mannosidase, α-glucosidase and β-glucuronidase. The latter two enzymes were unchanged during all cell culture phases.A drop in α-l-fucosidase and α-d-mannosidase activity was found during the stationary and decline phases of cell culture.     

Lysosomal enzyme variations in thirteen cell strains cultured from one amniotic fluid

Fifteen primary amniotic fluid cultures were established from a single sample of amniotic fluid. Three different methods were used to set up these cultures which yielded 13 cell strains. Nine lysosomal enzymes (acid phosphatase, β-glucuronidase, β-galactosidase, α-galactosidase, α-glucosidase, α-mannosidase, α-arabinosidase, $\text{N-}\text{acetyl}\text{-ß-}\text{d}\text{-}\text{glucosaminidase}$ and arylsulphatase A) were assayed in these 13 cell strains. The coefficients of variation of these enzyme levels were less than the coefficients for enzyme levels in cell strains grown from different samples of amniotic fluid but greater than those for the combined culture and assay system used. No assay values were found which could have suggested a possible enzyme deficiency disease.     

Acid hydrolases in amniotic fluid and chorionic villi at various stages of pregnancy

A study was made at various stages of pregnancy of five acid hydrolases which occur in amniotic fluid and chorionic villi and which are relevant to serious storage disorders.In amniotic fluid β-galactosidase and α-mannosidase decreased moderately towards term, while β-glucosidase decreased markedly. N-Acetyl-β-glucosaminidase and β-glucuronidase were relatively unchanged.In chorionic villi N-acetyl-β-glucosaminidase, β-galactosidase, and α-mannosidase were substantially decreased towards term, while β-glucosidase was unchanged and β-glucuronidase markedly increased.In both amniotic fluid and chorionic villi the enzyme pattern was approximately the same as that found in liver in a previous study.The findings suggest that these enzyme assays might be useful in the diagnosis of inborn errors prenatally by using amniotic fluid, and early postnatally by using chorionic villi.     

Activity of beta-galactosidase, beta-glucuronidase and N-acetyl-beta-glucosaminidase in human rectal mucosa

Activity of β-galactosidase, β-glucuronidase and N-acetl-β-glucosaminidase was determined by biopsy specimens of rectal mucosa of control subjects, cystinotic children and their parents.The studied enzymes exhibited maximal activity at pH 5.0, 4.0 and 4.5, respectively. Apparent K_m values using P-nitrophenyl-β-galactoside, p-nitrophenyl-β-glucuronide and p-nitropheny-β-glucosaminide were found to be 0.52 mM, 0.70 mM, and 0.67 mM.The activity of all three enzymes was found to be closely correlated in the 11 subjects of the control group. The values found in parents and their cystinotic children fit into these correlations, but show higher scatter of data caused by the fact that values of β-galactosidase were found to be higher and of β-glucuronidase and N-acetyl-glucosaminidase lower in the group of parents than in the other two groups.     

Dynamics of the binding capacity of plasma sex hormone binding globulin (SHBG) for testosterone and dihydrotestosterone during puberty

Plasma sex hormone binding globulin binding capacity (SHBG-b.c.) has been evaluated in 203 normal subjects (114 males and 89 females) aged 3 to 51 years. The subjects were divided into groups: prepubertal, early pubertal (Tanner's Stages 1 and 2), late pubertal (Tanner's Stages 4 and 5) and adult.In both sexes, plasma mean values of SHBG binding capacity both for dihydrotestosterone (DHT) and testosterone (T) were significantly higher in prepubertal subjects, falling during puberty to adult levels.During pubertal development DHT-BG binding capacity and T-BG binding capacity showed different plasma values with respect to sex and phase of puberty.Our data do not support an absolute relationship between sex hormones and SHBG binding capacity, but suggest other mechanisms as well: (a) SHBG modifies its physicochemical properties during puberty, or (b) the binding capacity is the result of a pool of proteins which modifies its composition during pubertal evolution.     

Biochemical activities of lysosomal acid hydrolases in leukemic cells

The biochemical activities of 8 lysosomal acid hydrolases in leukemic cells from 48 patients were examined. Characteristic alterations were found in α-mannosidase, β-galactosidase and N-acetyl-β-glucosaminidase activities of leukemic cells. The level of α-mannosidase activity was much higher in myelo(mono)genous leukemias (AML, AMoL, AMMoL, CML and CMMoL) than in lymphogenous ones (ALL, T-cell leukemia, hairy cell leukemia and CLL) without exception. The β-galactosidase activity also differed as a result of α-mannosidase, except in T-cell leukemia. In T-cell leukemia it was within the range of normal lymphocytes, but in the other lymphogenous leukemias it was significantly below normal. N-acetyl-β-glucosaminidase activity in myelo(mono)genous leukemic cells was above the range of normal granulocytes. The changes in these enzyme levels were consistent. The lymphocytic or myelocytic nature of three cases of acute undifferentiated leukemia could be determined by enzyme studies. In two cases it was lymphocytic and in one it was myelocytic. The enzymatic abnormalities were also found in morphologically mature neutrophils from patients with not only chronic types (CML, CMMoL) but also acute types (AMoL, AMMoL) of leukemias, and were similar to those of their respective leukemic cells. Analysis of lysosomal enzymes (at least three of those mentioned above), can elucidate one of the biochemical properties of leukemic cells and may be valuable in the differentiation of leukemias.     

Enzymes of lysosomal origin in human plasma and serum: assay conditions and parameters influencing the assay

The condition for maximal activity (pH, buffer, saturating substrate concentration, range of linear relationships between enzyme activity versus incubation time, and versus enzyme concentration) in the fluorimetric assay of several glycohydrolases of lysosomal origin in human plasma and serum have been established. The following enzymes were studied: alpha-galactosidase, beta-galactosidase, beta-N-acetylglucosaminidase, beta-glucosidase, beta-glucuronidase, alpha-mannosidase, alpha-fucosidase. All examined enzymes turned out to be more or less unstable upon storage at 37 degrees C, 4 degrees c, and -20 degrees C in both serum and plasma. The only exceptions were beta-glucuronidase, which was stable in plasma and serum, and alpha-fucosidase which was stable only in plasma. Generally the degree of instability was greater in serum than in plasma. The levels of some enzymes (alpha-galactosidase, beta-galactosidase, beta-N-acetyl glucosaminidase, beta=glucuronidase) were markedly higher in serum than in plasma; conversely the levels of the same enzymes in "platelet free" serum equalled those in plasma. This stresses the necessity to use freshly prepared plasma for lysosomal glycohydrolase assay. Under the procedural conditions recommended for the assay the methods for the determination of lysosomal glycohydrolases in plasma appeared to be simple, sensitive and reproducible.     

Isoenzymes of four acid hydrolases in human kidney and urine

The occurrence of different isoenzymes of β-glucosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, and α-mannosidase in human urine and kidney tissue was studied by isoelectric focusing. Artificial substrates were used for the enzymatic assays. There was a predominance of isoenzymes with a low isoelectric point in the urine. In the kidney tissue isoenzymes with higher isoelectric point predominated. This difference may be due to a higher proportion of N-acetylneuraminic acid-containing enzymes in the urine than in the kidney tissue.     

Hypothermia and Infection I. Influence of Hypothermia on Antibody Formation in Mice in the Secondary Response to Typhoid-H-Antigen

Öckerman, P. A. Lysosomal Acid Hydrolases in the Liver in Gargoylism. Deficiency of 4-methylumbelliferyl-β-galactosidase. Scand. J. clin. Lab. Invest. 22, 142-146, 1968.

The activites of six lysosomal acid hydrolases were measured in liver biopsy specimens from six patients with gargoylism and in post-mortem tissue from two patients. A marked diminution was noted for the activity of 4-methylumbelliferyl-β-glactosidase in all patients with gargoylism. The deficiency of β-galactosidase did not seem to be due to the presence of an inhibitor in the liver.

The activities of β-glucuronidase, β-acetylglucosaminidase, acid phosphatase and α-fucosidase were significantly increased in some of the patients. The activity of α-mannosidase was not significantly different from the values in the controls.     

Impaired degradation of keratan sulfate in GM1-gangliosidosis

We have prepared a new radiolabeled substrate (galactose-N-acetylglucosamine 6-sulfate-[1-³H]galactitol), from shark cartilage keratan sulfate, for an assay of acid β-galactosidase activity. Using this substrate, we found that there was a striking deficiency of β-galactosidase activity in the cultured skin fibroblasts of patients with GM₁-gangliosidosis. However, there seemed to be no quantitative differences in residual enzyme activity between type 1 and type 2 GM₁-gangliosidosis.     

LYSOSOMES OF THE ARTERIAL WALL : I. ISOLATION AND SUBCELLULAR FRACTIONATION OF CELLS FROM NORMAL RABBIT AORTA

Smooth muscle cells were dissociated from normal rabbit aorta by incubating the tissue in Hanks'' solution containing elastase, collagenase, and hyaluronidase. The isolated cells contained significant amounts of the following acid hydrolases: N-acetyl-β-glucosaminidase, N-acetyl-β-galactosaminidase, β-galactosidase, β-glucuronidase, α-mannosidase, β-glucosidase, acid phosphatase, and cathepsins C and D. The cells were disrupted and fractionated by isopycnic centrifugation on sucrose density gradients in the Beaufay automatic zonal rotor. Lysosomes with a modal density of 1.16 were identified by the distribution of these acid hydrolases and by the latency of N-acetyl-β-glucosaminidase and β-galactosidase. Other particulate enzymes studied in these sucrose gradients included cytochrome oxidase and monoamine oxidase (mitochondria), 5''-nucleotidase and leucyl-β-naphthylamidase (plasma membrane), and catalase (? peroxisome). This microanalytical subcellular fractionation technique is applicable to the study of milligram quantities of many other tissues, both normal and pathological.     

A new variant mucolipidosis: Biochemical investigations on two siblings

Biochemical studies are presented on two siblings with some features of Mucolipidosis III, but with distinctive clinical findings. Levels of β-galactosidase, -mannosidase, β-glucuronidase, N-acetyl-β-glucosaminidase and -fucosidase found in serum from these patients ranged from 10 to 100 times higher than normal. The ratio of heat stable to heat labile serum isoenzymes of N-acetyl-β-glucosaminidase is considerably greater than normal.

An extremely low activity of β-galactosidase was found in fibroblasts cultured from one patient. Levels of the remaining enzymes were in the low normal range. Similarly, β-galactosidase levels were low in heart, kidney, liver, spleen and lung of one patient who died during the course of the study. Activities of the remaining enzymes were close to normal.

No excessive excretion of mucopolysaccharide was noted, however, changes in distribution of several fractions were found. Mucopolysaccharide labeled with radioactive sulfate was degraded by cultured fibroblasts at a normal rate.

In addition to clinical differences, the biochemical studies further demonstrate the uniqueness of these patients.     

Substrate inhibition of lysosomal hydrolases: α-Galactosidase A and β-glucocerebrosidase

ObjectiveWe have investigated the kinetics of α-galactosidase A and β-glucocerebrosidase deficient in Fabry and Gaucher diseases, respectively.Design and methodsWe have performed spectrofluorymetric measurements of the activity of enzymes using a derivative of 4-methylumbelliferone as a substrate and a human T-cell line as a source of enzymes.ResultsWe have observed the substrate inhibition effect, which is related to temperature.ConclusionsThe diagnostic procedures for Fabry and Gaucher diseases used now in laboratory practice neglect temperature-dependent substrate inhibition, which may significantly reduce the sensitivity of enzyme activity determinations.     

Involvement of sialic acid residues in electroimmunodiffusion of α1-acid glycoprotein. A method for determining the degree of sialylation of serum glycoproteins

When the two immunological methods, radial immunodiffusion (RID) and electroimmunodiffusion (EID), were used for the determination of α₁-acid glycoprotein, a significant discrepancy in the results was encountered depending on the degree of sialylation. It appeared that with desialylated α₁-acid glycoprotein, amounts estimated by EID were much lower than those actually present as assayed by the RID method. Determination of glycoprotein samples treated with neuraminidase for varying periods of time revealed an increasing underestimation by the EID method with decreasing sialic acid content. Partial resialylation of asialo-α₁-acid glycoprotein by bovine colostrum β-galactoside α(2 → 6) sialyltransferase on the other hand resulted in less underestimation. Differential determination of α₁-acid glycoprotein by the two immunological methods thus offers a method for the estimation of the degree of sialylation of α₁-acid glycoprotein and other sialoglycoproteins in serum and body fluids.     

Molecular epidemiology of β-lactamase production in penicillin-susceptible Staphylococcus aureus under high-susceptibility conditions

Little is known about the prevalence of β-lactamase production in penicillin-susceptible Staphylococcus aureus isolates under high-susceptibility conditions. We analyzed S. aureus isolates with penicillin G minimum inhibitory concentration (MIC) ≤ 0.12 μg/ml that were recovered from in-/outpatients (n = 108) between 2016 and 2017 in Japan. β-Lactamase production was detected by nitrocefin-based and Clinical and Laboratory Standards Institute penicillin zone edge testing and blaZ PCR. All isolates were categorized as having penicillin G MIC ≤0.03 μg/ml using an automated system; MICs determined based on the microdilution method were 0.016 μg/ml (2%), 0.03 μg/ml (44%), and 0.06 μg/ml (54%). Notably, no isolates harbored the blaZ gene. The results from the nitrocefin-based and zone edge tests were consistent with those obtained by PCR. S. aureus isolates with penicillin G MIC ≤0.03 μg/ml exhibited a low frequency of β-lactamase production. Thus, screening for β-lactamase production may be unnecessary for isolates showing such high susceptibility.     

Urinary lysosomal glycosidases after renal allotransplantation: correlation of enzyme excretion with allograft rejection and ischemia

Urinary β-galactosidase, β-glucuronidase and N-acetyl-β-glucosaminidase were measured in patients with renal allotransplants and compared with normal controls. Increased excretion of all three enzymes was noted in the transplant patients resulting possibly from mild chronic rejection.A second part of the investigation correlated renal function with daily N-acetyl-β-glucosaminidase excretion by the patients. In acute rejection, enzyme levels rose sharply from a baseline then decreased following successful treatment. With cadaveric grafts and initially good urinary flow, N-acetyl-β-glucosaminidase levels were high and decreased as creatinine clearance improved; however, with initial oliguria, levels were low and rose as diuresis began then decreased to a baseline. This was attributed to a washing out of enzyme released during the unavoidable ischemic period involved in handling cadaver kidneys.Because it reflects physiological changes in the kidney, daily monitoring of urinary N-acetyl-β-glucosaminidase should be helpful in the diagnosis of renal damage caused by rejection and ischemia.     

Lysosomal enzymes in cerebral atrophy

In seven patients with cerebral atrophy due to pre-senile dementia and/or cerebrovascular disease, the activity of acid phosphatase in lumbar cerebrospinal fluid (CSF) was higher (p < 0.05) than in six controls. The activity of arylsulphatase and β-galactosidase in CSF was the same in the two groups. In the serum, the activities of acid phosphatase and arylsulphatase were the same in the two groups but the activity of β-galactosidase was lower (p < 0.02) in patients with cerebral atrophy.     

Methods for semi micro or automated determination of thrombin, antithrombin, and heparin cofactor using he substrate, H-D-Phe-Pip-Arg-p-nitroanilide-2HCl.

Methods for the measurement of thrombin and plasma antithrombin, by end point determination at a semi micro level and also by rate assay measurement in a fully automated system have been devised using the thrombin specific chromogenic substrate, H-D-Phe-Pip-Arg-p-nitroanilide. Preliminary defibrination of plasma is avoided in both methods. The semi micro method has been correlated with antitrhombin measured in plasma of postoperative patients by established clotting and immunological assays. The automated method has been found to be highly reproducible and to have less scatter than the other procedures.