To explore how enzyme affinities and enzyme activities regulate hydrolysis of water-insoluble substrates, we compared hydrolysis of phospholipid-stabilized emulsions of medium-chain (MCT) versus long-chain triacylglycerols (LCT). Because substrate solubility at the emulsion surface might modulate rates of hydrolysis, the ability of egg yolk phosphatidylcholine to solubilize MCT was examined by NMR spectroscopy. Chemical shift measurements showed that 11 mol % of [13C]carbonyl enriched trioctanoin was incorporated into phospholipid vesicles as a surface component. Similar methods with [13C]triolein showed a maximum solubility in phospholipid bilayers of 3 mol % (Hamilton & Small, 1981). Line widths of trioctanoin surface peaks were half that of LCT, and relaxation times, T1, were also shorter for trioctanoin, showing greater mobility for MCT in phospholipid. In assessing the effects of these differences in solubility on lipolysis, we found that both purified bovine milk lipoprotein lipase and human hepatic lipase hydrolyzed MCT at rates at least 2-fold higher than for LCT. With increasing concentrations of MCT, saturation was not reached, indicating low affinities of lipase for MCT emulsions, but with LCT emulsion incubated with lipoprotein lipase, saturation was reached at relatively low concentration, demonstrating higher affinity of lipase for LCT emulsions. Differences in affinity were also demonstrated in mixed incubations where increasing amounts of LCT emulsion resulted in decreased hydrolysis of MCT emulsions. Increasing MCT emulsion amounts had little or no effect on LCT emulsion hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

The

Purpose: Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation resulting from defective carnitine transport. This disease is caused by mutations in the carnitine transporter gene SLC22A5. The objective of this study was to extend mutational analysis to four additional families with this disorder and determine whether recurrent mutations could be found. Methods: The SLC22A5 gene encoding the OCTN2 carnitine transporter was sequenced, and the missense mutations identified were expressed in Chinese hamster ovary (CHO) cells. Results: DNA sequencing revealed four novel mutations (Y4X; dup 254 -264, 133X; R19P; R399Q).Alleles introducing premature STOP codons reduced the levels of OCTN2 mRNA. Carnitine transport in CHO cells expressing the R19P and R399Q mutations was reduced to Ͻ 5% of normal. The 133X mutation was found in two unrelated European families. Two patients within the same family, both homozygous for the same mutation

Sand rats (Psammomys obesus) maintained on a diet providing a free choice between laboratory chow and salt bush (Atriplex halimus) were classified into four groups differing in extent of the diabetic syndrome: A, normoglycemic-normoinsulinemic; B, normoglycemic-hyperinsulinemic; C, hyperglycemic-hyperinsulinemic; or D, hyperglycemic with reduced insulin levels. The metabolic pattern of these groups was characterized by measuring the uptake of fatty acid-labeled, very-low-density lipoprotein-borne triglycerides (VLDL-TG) and [3H]-2-deoxyglucose (2-DOG) into muscle and adipose tissues; incorporation of [14C]alanine into glycogen in vivo; gluconeogenesis from lactate, pyruvate, and alanine in hepatocytes; the effect of insulin on glycogen synthesis from glucose; the oxidation of albumin-bound [1-14C]palmitate and [14C]glucose in strips of soleus muscle; activities of muscle and adipose tissue lipoprotein lipase; and activities of rate-limiting enzymes of glycolysis, gluconeogenesis, and fatty acid synthesis in liver. In group A, uptake of VLDL-TG and activity of lipoprotein lipase were higher in adipose tissue and lower in muscle than in albino rats. In the liver, gluconeogenesis and the activity of phosphoenolpyruvate carboxykinase, as well as lipid synthesis and the activity of NADP-malate dehydrogenase, were higher than in albino rats, whereas activity of pyruvate kinase was lower. In group B, uptake of VLDL-TG by adipose tissue and muscle and lipoprotein lipase activity were similar or higher than in group A. Uptake of 2-DOG by muscle and adipose tissue and activity of liver phosphoenolpyruvate carboxykinase were lower than in group A. In groups C and D, uptake of VLDL-TG and lipoprotein lipase activity in muscle were further increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Three of four nonketotic hyperglycinemia patients homozygous for a novel GLDC mutation (A802V) were treated by assisted respiration and/or sodium benzoate with or without ketamine and had transient neonatal or absent symptoms and normal developmental outcome, despite persisting biochemical evidence of nonketotic hyperglycinemia. This exceptional outcome may be related to the high residual activity of the mutant protein (32% of wild type) and therapeutic intervention during a critical period of heightened brain exposure and sensitivity to glycine.