Margaret L. Smith

Cystine: Binding protein assay

Publisher Summary The cystine-binding protein is utilized for cystine transport through the osmotic shock-sensitive transport system of Escherichia coli. This protein is located in the periplasmic space and can be isolated following osmotic shock of this bacterium. It reversibly binds cystine with a dissociation constant of 10 n M . When purified, the cystine-binding protein may be used as described to measure cystine levels in physiological fluids. The chapter also discusses the use of cystine-binding protein for quantitative cystine assay in biological samples. Nanomolar concentrations of the disulfide, cystine, are assayed based on the competitive binding between [ 14 C] cystine and the nonradioactive cystine present in a sulfosalicylic acid extract of the biological material. The cystine-binding protein does not bind reduced glutathione or other thiols that are present in much larger amounts than cystine in most biological samples, eliminating the need for purification procedures prior to assay. The specificity of the cystine binding is discussed.

Renal cell culture using autopsy material from children with cystinosis

SummaryRenal cell cultures were initiated using fresh autopsy material from two individuals with cystinosis, ages 5 and 8 yr. Cells obtained from collagenase treated autopsy material were grown in a selective kidney medium containing Coons modified F12, 2.5% fetal bovine serum, transferrin, insulin, selenium, hydrocortisone, PGE1, and fibronectin. These cells had an epithelial appearance, formed domes, and were periodic acid-Schiff positive. Both tight junctions and microvilli were seen by electron microscopy. Fibroblasts had a cloning efficiency of zero in the selective medium and grew poorly compared to their growth in Coons F12 with 10% fetal bovine serum. The lysosomal cystine content of the renal cells was greatly elevated and comparable to that of fibroblasts from cystinotic patients. Renal cell lysosomal cystine levels were only partially reduced by exposure to either pantethine or the aminothiol, cysteamine. However, exposure to either compound effectively depleted cystinotic cultured fibroblasts of their lysosomal cystine. Study of cultured renal material may have practical significance in pharmacologic considerations.

Early occurrence of end-stage renal disease in a patient with infantile nephropathic cystinosis

We report the case of a patient with infantile nephropathic cystinosis who required renal transplantation at age 30 months. Exhaustive evaluation did not identify a cause of progressive renal failure other than cystinosis. The patients genetic lesion was allelic with those of other patients with cystinosis; fusion of this patients fibroblasts with fibroblasts from another patient with infantile nephropathic cystinosis did not demonstrate complementation of the biochemical defect.

Lysosomal cystine storage in cystinosis and mucolipidosis type II.

ABSTRACT: Cultured fibroblasts from mucolipidosis II (ML-II) patients demonstrated an elevated cystine content which increased with time in culture compared to fibroblasts from cystinotic patients or normal controls under the same conditions. In both cystinotic and ML-II cells the increased levels of cystine could be derived either from endogenous proteolysis or from in vitro supplementation of the cultured cells with cysteine-glutathione mixed disulfide. Cystine was depleted from both cell types by cysteamine. When cysteamine was replaced with complete medium, the cystine reaccumulated in both cystinotic and ML-II cells within 24 h, although a lag of 4 h was seen with ML-II cells. The intracellular location of the increased cystine in cultured fibroblasts was examined utilizing free-flow electrophoresis and found to be in the purified population of secondary lysosomes of both cystinotic and ML-II cells. White blood cell and hepatic cystine, which was greatly increased in cystinotic patients, was not elevated in ML-II patients. Compared to normal control fibroblasts the efflux of cystine from isolated granular fractions was virtually absent in cystinotic fibroblasts and considerably reduced in ML-II fibroblasts. The examination of such similarities and differences in cystine accumulation and transport in tissues from cystinotic and ML-II patients has provided some insight into the defects in these diseases.

Cystine exodus from lysosomes: cystinosis.

Publisher Summary This chapter discusses the cystine exodus from lysosomes. The concept of an intracellular transport process for the movement of cystine from the lysosomal space to the cystosol owes its existence to the search for the metabolic defect in the inherited disease cystinosis. Because the cytoplasmic catabolic pathways for cystine appeared normal in cystinosis, investigators suspected that the defect in this disease involved the escape of cystine from the lysosome to the cytosol. Using the techniques of amino acid loading and exodus as well as that of amino acid uptake in the presence of analogs, lysosomal transport systems in human fibroblasts have been characterized for small neutral amino acids, for anionic acids, and for branched and aromatic dipolar amino acids. Exodus is determined from the amount of cystine remaining in the lysosomes at the various time points. This chapter discusses the procedures for measuring exodus for both radiolabeled and unlabeled methyl ester loaded lysosomes.

THE ROLE OF ATP IN LYSOSOMAL CYSTINE EFFLUX

In cystinosis there is a defect in cystine efflux from the lysosomes which is expressed in both cultured fibroblasts and E.B. virus transformed lymphoblasts. Cystine efflux from lysosomes isolated in Hepes/Sucrose from control lymphoblasts is stimulated by the addition of MgATP, while efflux from lysosomes isolated from control fibroblasts is not. To clarify the role of ATP and the lysosomal ATPase in cystine efflux, we determined the pH of lysosomes isolated in simultaneous experiments from both cell types by measuring the fluorescence of intralysosomal FITC-dextran. The pH of lymphoblast lysosomes is 6.5±.05. This is lowered to 6.2±.03 by the addition of MgATP to the incubation buffer. Fibroblast lysosomes have a pH of 5.5±.07 which is not altered by the addition of MgATP. However, if the pH of fibroblast lysosomes is raised by preincubation with KCl, ATP causes acidification to near-initial values. In both cell types, there is no significant difference between cystinotic and control lysosomes in initial pH, ability to use ATP to produce an acidic pH, or the amount of proton-translocating ATPase activity. Our interpretation of these data is that cystine efflux from lysosomes requires a highly acidic intralysosomal pH. Since fibroblast lysosomes isolated in Hepes/Sucrose have a pH of 5.5 independent of ATP addition, cystine efflux is not stimulated by ATP in this system. In contrast, lymphoblast lysosomes are acidified in the presence of ATP, explaining the requirement of cystine efflux for ATP.

CYSTINE EXODUS FROM LEUKOCYTE GRANULAR FRACTIONS IS STIMULATED BY ATP AT SUB-SATURATING CYSTINE LEVELS

Cystine exodus from the granular fractions of fibroblasts, lymphoblasts, and rat liver is stimulated by ATP. We observe a similar ATP effect in leukocytes. N-ethyl maleimide (NEM), an inhibitor of the lysosomal H+-translocating ATPase (H+ATPase), eliminated the stimulation by ATP, but had no effect on basal exodus. In a series of three experiments on different days the % cystine loss in 30 min. at 37° was 54.5±1.9 (mean±SD) in pH 7.0 Hepes/sucrose buffer, 51.6±3.7 in buffer with 1 mM NEM, 76.6±4.8 in buffer with 2 mM MgATP, and 57.3±8.1 in buffer with NEM and MgATP. The initial concentration of cystine in these experiments ranged from 19 to 52 nmol cystine/unit β-hexosaminidase (cys/hex). The ATP effect disappeared when the load was increased to 1200 nmol cys/hex. The cystine loss for the same four conditions as above was 25.2, 29.8, 23.6, and 20.1%, respectively. At initial loads from 22-117 cys/hex, ATP stimulated exodus 2.2±0.6 (mean±SD, n=5) fold, whereas at loads from 800-1300 cys/hex, ATP stimulated exodus 1.1±0.2 (n=3) fold. The increased rate in the presence of ATP may represent improved binding of the cystine to the partially saturated inner transporter resulting from conformational or charge optimization brought about by the H+ATPase. The effect disappears either in the presence of the H+ATPase inhibitor NEM or when the transporter becomes saturated by high load levels.

860 CYSTINOSIS: DEFECTIVE FIBROBLAST LYSOSOMAL CYSTINE TRANSPORT

Defective cystine efflux has been demonstrated in lysosomes from both peripheral leukocytes and cultured EBV-lymphocytes from cystinotic patients. However, this abnormality has been difficult to demonstrate in fibroblast lysosomes, raising the question of whether lysosomal transport is the only defect in cystinosis. We have substantiated that some previous methods of loading fibroblast lysosomes with cystine, using cystine dimethyl ester (CDME), lead to cystine accumulation in a non-lysosomal as well as the lysosomal compartment (E. Harms, personal communication). If fibroblasts are removed from their anchorage substrate and incubated with 0.25 mM CDME for 15 min., only lysosomes are loaded with cystine as shown on Percoll gradients. In cystine-loaded lysosomes from two normal control fibroblast cultures the 20 min. cystine efflux was 19% and 20% without ATP and 56% and 74% with 2 mM MgATP. Lysosomes from two cystinotic fibroblast cultures had no cystine efflux over 20 min. both with and without ATP. We have used the dye, dipropylthiodicarbocyanine iodide to measure lysosomal membrane potential. MgATP causes a positive shift in membrane potential, in both cystinotic and normal lysosomes. In both cases, inhibitor and anion effects are consistent with an electrogenic proton pump. All of these findings support the concept of an abnormal lysosomal cystine transport protein as the primary defect in this disease.