Replacement of C305 in Heart/Muscle-Type Isozyme of Human Carnitine Palmitoyltransferase I with Aspartic Acid and Other Amino Acids

Abstract

Liver- and heart/muscle-type isozymes of human carnitine palmitoyltransferase I (L- and M-CPTI, respectively) show a certain similarity in their amino acid sequences, and mutation studies on the conserved amino acids between these two isozymes often show essentially the same effects on their enzymatic properties. Earlier mutation studies on C305 in human M-CPTI and its counterpart residue, C304, in human L-CPTI showed distinct effects of the mutations, especially in the aspect of enzyme stability; however, simple comparison of these effects on the conserved Cys residue between L- and M-CPTI was difficult, because these studies were carried out using different expression systems and distinct amino acids as replacements. In the present study, we carried out mutation studies on the C305 in human M-CPTI using COS cells for the expression system. Our results showed that C305 was replaceable with aspartic acid but that substitution with other amino acids caused both loss of function and reduced expression.

Molecular Pathways: Tumor Cells Co-opt the Brain-Specific Metabolism GeneCPT1Cto Promote Survival

“…for fulfilling their metabolic needs, particularly when more broadly expressed family members, namely CPT1A and CPT1B, are also available but apparently not activated in cancer cells (44). Presumably, both CPT1A and CPT1B can also increase FAO (45,46), so it stands to reason that CPT1C upregulation must provide a special activity that spurs tumor growth. The simplest explanation would be that CPT1C acts on different substrates than CPT1A and CPT1B.…”

Unique Behavior of Bacterially Expressed Rat Carnitine Palmitoyltransferase 2 and Its Catalytic Activity

“…Finally, we asked whether bacterially expressed full-length CPT2, partially purified by simple centrifugation as described above, shows catalytic activity. As a comparison, we also expressed rat CPT1b in COS7 cells as previously described, [14][15][16] and used lysates of these cells to represent CPT1b. As shown in Fig.…”

“…L-[methyl-3 H] carnitine hydrochloride as a tracer, essentially as described previously for measurement of CPT1 activity. [14][15][16] Briefly, the reaction was initiated by addition of 50 µL sample solution containing 30 µg protein sample of crudely purified CPT2 to the prewarmed reaction solution (450 µL), to make final concentrations of 150 mM KCl, 1 mM EDTA, 0.25 mM reduced glutathione, 1.3 mg/mL fatty acid-free bovine serum albumin (BSA), 0.5 mM L-carnitine, 50 µM palmitoyl-CoA, 2 mM NaCN, 9.25 kBq L-[methyl-3 H] carnitine and 50 mM N-(2-hydroxyethyl) piperazine-N′-2-ethanesulfonic acid (Hepes) buffer, pH 7.4. After incubation at 30 °C for 20 min, the reaction was terminated by the addition of 200 µL 3 M HC1.…”