Glycine Transport by Human Diploid Fibroblasts—Absence of a Defect in Cells from Patients with Nonketotic Hyperglycinemia

Kelly, John; Otto, Elaine F; Hillman, Richard E.

doi:10.1203/00006450-197902000-00008

Cited by 7 publications

(2 citation statements)

References 8 publications

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“…The K,' s for glycine transport in the controls and NKH cell lines for laboratory (1.4 to 2.0 mM) are comparable with those obtained elsewhere (1.1 to 2.4 and 0.65 to 4.2 mM) (5,12 (12) because the latter authors used 20-min incubation times while the former incubated for only one min. However.…”

Section: Discussionsupporting

confidence: 70%

Studies of Glycine Metabolism and Transport in Fibroblasts from Patients with Nonketotic Hyperglycinemia

Halton

Krieger

1980

Pediatr Res

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SummaryGlycine transport in both normal and nonketotic hyperglycinemia fibroblasts was shown to occur by a sodium-dependent system. No significant difference could be detected in either the Km's (1.4 to 2.0 mM) or the Vm,' s (6.2 to 16 nmole per mg protein per min) of the three control and three patient cell lines. Valine was a weak competitive inhibitor of glycine uptake. Ki's from both groups fell into the 5.6 to 5.8 mM range. Plasma levels of valine of one patient reached a maximum of 0.6 mM following a valine load. Glycine cleavage activity could not be detected in either control or nonketotichypergl~cinemia fibroblast lines. Serine utilization was the same in both nonketotic hyperglycinemia and control lines. SpeculationThe fibroblast lines of nonketotic hyperglycinemia patients used in our study indicate that a glycine transport defect is not the cause of the elevated cerebrospinal fluid glycine levels observed in nonketotic hyperglycinemia. The clinical valine effect is unlikely to be related to the inhibition of glycine transport by valine.Nonketotic hyperglycinemia (NKH) is a metabolic disease characterized by the early onset of hypotonia, lethargy, and myoclonic convulsions. No treatment yet devised has avoided the profound mental retardation in surviving patients. A disproportionate elevation of glycine is seen in the cerebrospinal fluid and a decrease in the activity of the glycine cleavage enzyme in brain and liver (10. 12) has been reported. However. this abnormality has also been found in the biochemically distinct disorder, ketotic hyperglycinemia (2, 12) which raises the possibility that it may not be the primary genetic defect in NKH.In some patients with NKH, valine has been shown to induce coma and markedly exacerbate the hypotonia (7. 8). This striking effect has led to the suggestion that NKH represents a defect in valine metabolism (8).More recently. Revsin and Morrow ( I 1 ) found V, . , differences in fibroblast glycine transport between NKH and control cell lines. An inhibition of glycine transport by valine was briefly noted by these authors.In our study, we investigated the influence of valine on glycine transport as a possible cause of the clinical valine effect. Glycine oxidation and serine utilization in fibroblasts were also examined. MATERIALS AND METHODSHuman diploid fibroblasts (24) from three normal and three NKH patients were grown in Eagle's minimal essential medium (13) containing 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 pg/ml).For transport studies, cells were grown on washed sterile coverslips ( 1 l x 22 mm) (14). With the fibroblasts at confluency, the 93 coverslips were rinsed twice with phosphate-buffered saline glucose (PBSG) [I30 mM NaCI, 5 mM KCI, 1.2 mM MgS04, 1 mM CaC12, 5 mM glucose. and 10 mM Na2HP04 (pH 7.4)]. In a final wash, the coverslips were left for one hr in PBSG at 37OC to minimize endogenous glycine levels.The incubation procedure was similar to that of Foster and Pardee (3). Four yl of [2-L4C]glycine (15) were added per ml of...

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Section: Discussionsupporting

confidence: 70%

Studies of Glycine Metabolism and Transport in Fibroblasts from Patients with Nonketotic Hyperglycinemia

Halton

Krieger

1980

Pediatr Res

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“…Glycine transport in human fibroblasts has not yet been completely defned (Revsin and Morrow, 1976: Kelly et al, 1979) 9 A single system was also found by Kelly et al (1979). The range of Km values of these authors (1.1-1.3 mmol/l) were nearly the same as those reported here ( 1.73-2.17 mmol/1).…”

Section: Discussionsupporting

confidence: 65%

Glycine transport by cultured skin fibroblasts from a patient with isolated hyperglycinuria

Feneant

Moatti

Lemonnier

et al. 1980

J of Inher Metab Disea

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Glycine transport is studied in cultured skin fibroblasts from a patient with isolated hyperglycinuria and from five normal subjects. Fibroblasts from the patient take up glycine less well than do cell lines from controls. Kinetic studies are consistent with a single transport system in the patient's and controls' cell lines. Vmax value in the hyperglycinuric lines is normal, but the apparent affinity is always reduced as opposed to those of four different control lines separately tested. Statistical analysis shows significant difference between Km values.

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Transport of L‐alanine in cultured human fibroblasts: Evidence for two kinetically distinguishable systems

Feneant

Moatti

Lemonnier³

et al. 1981

Journal Cellular Physiology

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The transport of L-alanine in human diploid fibroblasts was investigated. Transport measurements were performed on subcultures between the third and eighth passages with subconfluent cells growing on glass coverslips. Kinetic analysis of approximate initial rates of transport at substrate concentrations from 0.05 to 10 mmole/liter indicate the presence of two distinguishable systems. The high affinity system has a Km of 0.24 mmole/liter and a Vmax of 6.4 nmole/100 micrograms protein/2 min. For the low affinity system, the contribution of the high affinity system to the uptake must absolutely be taken into account. The Km and Vmax values, obtained by using a computer program, are a Km of 15.0 mmole/liter and a Vmax of 14.7 nmole/100 micrograms protein/2 min. For alanine concentrations below 1 mmole/liter, the contribution of the Na+-independent uptake is less than 10%, and the kinetic constants of the high affinity system are in the same range if this contribution is taken into account. On the contrary the influence of a diffusion-like process is more significant on the low affinity system whose Km is about 49 mmole/liter after subtraction of the Na+-independent uptake from the experimental velocities. Inhibition studies were performed with NCH3-alanine. They permitted us first to confirm the existence of system A in cultured human fibroblasts in agreement with two recent works and second to show how this system contributes to L-alanine uptake. This contribution seems very small in low concentrations but it rises as the concentrations increase.

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Glycine Transport by Human Diploid Fibroblasts—Absence of a Defect in Cells from Patients with Nonketotic Hyperglycinemia

Cited by 7 publications

References 8 publications

Studies of Glycine Metabolism and Transport in Fibroblasts from Patients with Nonketotic Hyperglycinemia

Studies of Glycine Metabolism and Transport in Fibroblasts from Patients with Nonketotic Hyperglycinemia

Glycine transport by cultured skin fibroblasts from a patient with isolated hyperglycinuria

Transport of L‐alanine in cultured human fibroblasts: Evidence for two kinetically distinguishable systems

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