DHPR activity

Screening for a BH4 deficiency should be done in all newborns with plasma phenylalanine levels higher than 120 pmol/1, as well as in older children with neurologic signs and symptoms [10]. The following tests are recommended (1) analysis of pterins in urine, (2) measurement of DHPR activity in blood from a Guthrie card,... 

DHPR activity may be determined from whole blood, RBC, dried blood from Guthrie cards, cultivated primary dermal fibroblasts, or from lysates of tissue samples (e.g., liver or brain). 

DHPR activity measurements are not available in external control programs and can only be compared by exchanging results from other laboratories (for examples see www.bh4.org). For internal control, we recommend using dilution II (40 pi, with an activity of 0.75 mU/ml see below), and blood from normal control subjects. 

DHPR activity is assayed by measuring the linear increase of ferrocytochrome c at 550 nm (AEssonm = 21) upon enzymatic incubation at 25°C for 5 min in a volume of 1 ml. The incubation mixture contains 40 tl lysate, 875 tl of 50 mM Tris-HCl pH 7.6, 50 pi of the 1 mM ferricytochrome solution, and 25 pi of the 2 mM NADH solution. The blank control contains 40 pi of Tris-HCl pH 7.6 instead of the lysate. These mixtures are preincubated for 5 min at 25°C, before the reaction is started by addition of 10 pi of the 1 mM 6-MPH4 substrate. The reaction is monitored by measuring the AE550nm/min over a period of 5 min. The starting lysate of 40 pi was diluted 25-fold. 

DHPR activity decreases with increasing Hb concentrations (unpublished observation). 

A small subset of patients with hyperphenylalaninemia show an appropriate reduction in plasma phenylalanine levels with dietary restriction of this amino acid however, these patients still develop progressive neurologic symptoms and seizures and usually die within the first 2 years of life ("malignant" hyperphenylalaninemia). These infants exhibit normal phenylalanine hydroxylase (PAH) activity but have a deficiency in dihy-dropteridine reductase (DHPR), an enzyme required for the regeneration of tetrahydro-biopterin (BH4), a cofactor of PAH (see Fig. 39.18). Less frequently, DHPR activity is normal but a defect in the biosynthesis of BH4 exists. In either case, dietary therapy corrects the hyperphenylalaninemia. However, BH4 is also a cofactor for two other hydroxy-lations required in the synthesis of neurotransmitters in the brain the hydroxylation of tryptophan to 5-hydroxytryptophan and of tyrosine to L-dopa (see Chapter 48). It has been suggested that the resulting deficit in central nervous system neurotransmitter activity is, at least in part, responsible for the neurologic manifestations and eventual death of these patients. 

Measurement of DHPR activity in blood from a Guthrie card... 

Patients sustain convulsions and neurological deterioration. The urine contains low levels of the metabolites of serotonin, norepinephrine and dopamine. The reductase also plays a role in the maintenance of tetrahydrofolate levels in brain, and some patients have had low folate levels in the serum and CNS. Treatment has been attempted with tryptophan and carbidopa to improve serotonin homeostasis and with folinic acid to replete diminished stores of reduced folic acid. This therapy is sometimes effective. Diagnosis involves assay of DHPR in skin fibroblasts or amniotic cells. Phenylalanine hydroxylase activity is normal. 

Additional freeze-thawing of tissue or fibroblast lysates may result in reduced GTPCH activity, and it is thus recommended to assay activity always from freshly lysed material. Alternatively, lysates may be kept for 1-2 days at -80°C (this may not be the case for PTPS, SR, and DHPR assays, as extracts can be kept at -80°C for a longer period without loosing activity). 

Besides the enzymatic incubation in the reaction mixture, all procedures are carried out at 4°C. PTPS activity is assayed by measuring the biopterin produced upon enzymatic incubation at 37°C for 120 min in a final volume of 110 pi in the dark (due to the light sensitivity of pterins), followed by chemical oxidation. To stabilize the produced BH4, DHPR and NADH are present in the enzyme assay. Two separate blanks are prepared, a blank reaction with cell lysate that is immediately oxidized to detect the biopterin that was present in the lysate, and a blank reaction without cell lysate to detect the biopterin that is generated from the incubation (substrate) buffer. 

In RBC, the activity for DHPR is calculated in U/g Hb. The Hb concentration is determined in g Hb/ml by standard laboratory methods. The results for tissues or other cells like fibroblasts or amniocytes are given in mg protein/ml. To determine the protein concentration, use filtrates from Sephadex G-25 (MicroSpin) columns and proceed according to the protein assay method (BioRad). Supernatants are kept at 4°C, or can be shock frozen and stored at -80°C. 

Additional information <2, 3> (<2> the concentration of glutamate which yields half-maximal activity is 33 mM for y-glutamyl kinase DHPr, and 37mM for y-glutamyl kinase w-l-, no typical Michealis-menten kinetics [2] <3> plots of the enzyme activity as a function of ATP concentration are non-hyperbolic [5]) [2, 5]... 

There are number of unanswered questions about MH. These include 1. Is the enhanced sensitivity of RyRl to MH-producing triggers due to an inherent change in the sensitivity of RyRl to these agents, or does altered Ca2+ homeostasis in the muscle drive other changes that enhance RyRl sensitivity to activation 2. Why are MH episodes more prevalent in children 3. Why are males more likely to have an MH response 4. Do mutations in proteins other than RyRl and the DHPR produce MH ... 

The close alignment of cytoplasmic and ER membranes is, in fact, cmcially important for the workings of excitation-contraction coupling in the skeletal muscle. In these cells, we have a unique mechanism of activation of one channel by another The RyR is directly hooked up to a cytosolic loop of the dihydropyridine receptor (DHPR Figure 6.6a, b). Membrane depolarization will cause a conformational change to the DHPR, which in turn is directly and mechanically transmitted to the RyR, so that both channels open synchronously. This even works in the absence of any calcium flux across the cytoplasmic membrane - experimentally, skeletal muscle cells can be induced to contract in calcium-free buffers. 

It is now widely believed that in skeletal muscle a depolarization-induced change in the structure of the DHPR a 1 subunit directly influences the RyR in such a way as to markedly increase its conductance for Ca +. Since the resulting increase in [Ca +]i can open other RyR channels, this produces a surge of Ca + release. In contrast, cardiac muscle expresses a different a 1 DHPR subunit than skeletal muscle. The cardiac subunit has much higher channel conductance and faster kinetics than the skeletal muscle type, and so admits much more extracellular calcium during the action potential. Thus, CICR does play an important role in cardiac muscle. In both cases, high [Ca +]i reduces RyR conductance, by direct binding of Ca + (or Ca +-calmodulin) to RyR, by activation of a kinase that phosphorylates RyR, and probably both. At the same time, Ca-calmodulin (Ca-CaM) activates a protein kinase that phosphorylates the SR Ca-ATPase, which increases its activity 10- to 100-fold. These two mechanisms combine to terminate the Ca " " spike. 

A. The patient, despite being put on a low-Phe diet, exhibits neurologic problems resulting from an inability to synthesize catecholamine and indoleamine neurotransmitters. This is caused by a deficiency in dihydropteridine reductase (DHPR). DHPR regenerates tetrahydro-biopterin (BH ), which is oxidized to dihydrobiopterin by phenylalanine hydroxylase, as well as tyrosine hydroxylase and tryptophan hydroxylase (tryptophan 5-monooxygenase). If phenylalanine hydroxylase were deficient, a diet low in Phe would alleviate the effects. Since the urinary biopterin concentration is elevated, a deficiency in GTP cyclohydrolase I is eliminated because that is an enzyme in the biosynthetic pathway of BH. Phe hydroxylase, Tyr hydroxylase, and Tip hydroxylase activities are low because of a lack of BH. ... 

D. Because of the DHPR deficiency, the activities of Phe hydroxylase and Tyr hydroxylase are low, hence the synthesis of catecholamine neu-rotransmitters are depressed. The synthesis of the indoleamine neurotransmitter serotonin is also depressed because BH is required for the hydroxylation of tryptophan. The best treatment is to decrease the Phe load by a low-Phe diet and provide the precursors for the catecholamine and indoleamine neurotransmitters that occur after the enzymes affected by the deficiency of BH, which would be L-dopa and 5-hydroxytryptophan. 

Fig. 47.3. Events leading to sarcoplasmic reticulum calcium release in skeletal muscle. 1. Acetylcholine, released at the synaptic cleft, binds to acetylcholine receptors on the sar-colemma, leading to a change of conformation of the receptors such that they now act as an ion pore. This allows sodium to enter the cell and potassium to leave. 2. The membrane polarization that results from these ion movements is transmitted throughout the muscle fiber by the T-tubule system. 3. A receptor in the T-tubules (the dihydropyridine receptor, DHPR) is activated by membrane polarization (a voltage-gated activation) such that activated DHPR physically binds to and activates the ryanodine receptor in the sarcoplasmic reticulum (depolarization-induced calcium release). 4. The activation of the ryanodine receptor, which is a calcium channel, leads to calcium release from the SR into the sarcoplasm. In cardiac muscle, activation of DHPR leads to calcium release from the T-tubules, and this small calcium release is responsible for the activation of the cardiac ryanodine receptor (calcium-induced calcium release) to release large amounts of calcium into the sarcoplasm.

Hg. 4. Many Ca sparks, activated in a synchronized manner by Ca influx during an action potential, sum to a global Ca transient to cause contraction. Ca " influx through dihydropyridine receptors (DHPR) triggers the release of Ca from SR through ryanodine (RYR2 ) channels, which cause muscle contraction (Berridge 1997). 

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