Lung surfactant protein SP-C from human, bovine, and canine sources contains palmityl cysteine thioester linkages.

Lung surfactant is a complex mixture of lipids and proteins that coats the alveoli to reduce surface tension and prevent airspace collapse. One of the principal protein constituents, surfactant protein C (SP-C), has been characterized following isolation from human, canine, and bovine sources. In each species, this highly hydrophobic protein is composed of 33-35 amino acids, the differences being due to NH2-terminal heterogeneity. A COOH-terminal leucine is conserved throughout. The cysteines in each species were found by fast atom bombardment mass spectrometry to be present as thioesters of palmitic acid. Acylation of recombinant SP-C with palmityl coenzyme A, followed by characterization before and after release of the acyl group with 1,4-dithiothreitol, provided corroborating evidence for the native structure.

Regulation of glycogen synthase in muscle and adipose tissue during fasting and refeeding.

Using immunoblot analysis, we examined the electrophoretic mobility of glycogen synthase from rat skeletal muscle and adipose tissue. Extracts from muscle freeze clamped in situ contained at least three forms of synthase with different electrophoretic mobilities. Extracts from adipose tissue also contained multiple forms but lacked the form with greatest mobility found in the muscle extracts. Phosphorylation at multiple sites of glycogen synthase is known to deactivate the enzyme and retard its electrophoretic mobility in sodium dodecyl sulfate gels. These results suggest that there is very little or no dephosphorylated glycogen synthase in adipose tissue and that phosphorylated forms of glycogen synthase synthesize adipose tissue glycogen. Relative to control, it is known that fasting decreases and refeeding increases glucose incorporation into glycogen in rat epididymal adipose tissue but not skeletal muscle incubated in vitro in the presence of insulin. Fasting did not change the electrophoretic pattern of muscle synthase but decreased the relative amount of adipose tissue forms with greater mobility. Refeeding increased above control the relative amount of adipose tissue synthase with greater mobility. These results indicate that changes in the phosphorylations that retard mobility contribute to the effects of fasting and refeeding on adipose tissue glycogen metabolism.

Sulfhydryl-alkylating reagents inactivate the NAD glycohydrolase activity of pertussis toxin.

The combination of ATP, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate), and DTT (dithiothreitol) is known to promote the expression of the NAD glycohydrolase activity of pertussis toxin, which resides in the toxin's S1 subunit. By monitoring changes in electrophoretic mobility, we have found that ATP and CHAPS act by promoting the reduction of the disulfide bond of the S1 subunit. In addition, ATP, CHAPS, and DTT allowed sulfhydryl-alkylating reagents to inactivate the NAD glycohydrolase activity. In the presence of iodo[14C]acetate, the combination of ATP, CHAPS, and DTT increased 14C incorporation into only the S1 subunit of the toxin, indicating that alkylation of this subunit was responsible for the loss of activity. If iodoacetate is used as the alkylating reagent, alkylation can be monitored by an acidic shift in the isoelectric point of the S1 peptide. Including NAD in alkylation reactions promoted the accumulation of a form of the S1 peptide with an isoelectric point intermediate between that of native S1 and that of S1 alkylated in the absence of NAD. This result suggests that NAD interacts with one of the two cysteines of the S1 subunit. In addition, we found the pH optimum for the NAD glycohydrolase activity of pertussis toxin is 8, which may reflect the participation of a cysteine in the catalytic mechanism of the toxin.

Structure-activity analysis of the activation of pertussis toxin.

Bordetella pertussis, the causative agent of whooping cough, releases pertussis toxin in an inactive form. The toxin consists of an A protomer containing one S1 peptide subunit and a B oligomer containing several other peptide subunits. The toxin binds to cells via the B oligomer, and the S1 subunit is activated and expresses ADP-ribosyltransferase and NAD glycohydrolase activities. Treatment of purified toxin with dithiothreitol (DTT) in vitro increases both activities. ATP and the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) synergistically reduce the A0.5 (activation constant) for DTT from greater than 100 mM to 200 microM. We studied the structure-activity relationships of activators of the toxin. In the presence of CHAPS (1%) and DTT (10 mM) the following compounds increased the NAD glycohydrolase activity of the toxin with the following A0.5's in microM and fraction of the ATP effect in parentheses: ATP, 0.2 (1.0); ADP, 6 (0.8); UTP, 15 (0.7); GTP, 35 (0.6); pyrophosphate, 45 (0.7); triphosphate, 60 (0.6); tetraphosphate, greater than or equal to 170 (greater than or equal to 0.4). Thus, the polyphosphate moiety is sufficient to stimulate the toxin, and the adenosine moiety confers upon ATP its extraordinary affinity for the toxin. Phospholipid and detergents could substitute for CHAPS in the activation of the toxin. Glutathione substituted for DTT with an A0.5 of 2 mM, a concentration within the range found in eucaryotic cells. Thus, membrane lipids and cellular concentrations of glutathione and ATP are sufficient to activate pertussis toxin without the need for a eucaryotic enzymatic process.

L-type glycogen synthase. Tissue distribution and electrophoretic mobility.

We previously reported (Kaslow, H.R., and Lesikar, D.D.FEBS Lett. (1984) 172, 294-298) the generation of antisera against rat skeletal muscle glycogen synthase. Using immunoblot analysis, the antisera recognized the enzyme in crude extracts from rat skeletal muscle, heart, fat, kidney, and brain, but not liver. These results suggested that there are at least two isozymes of glycogen synthase, and that most tissues contain a form similar or identical to the skeletal muscle type, referred to as "M-type" glycogen synthase. We have now used an antiserum specific for the enzyme from liver, termed "L-type" glycogen synthase, to study its distribution and electrophoretic mobility. Immunoblot analysis using this antiserum indicates that L-type glycogen synthase is found in liver, but not skeletal muscle, heart, fat, kidney, or brain. In sodium dodecyl sulfate-polyacrylamide gels of crude liver extracts prepared with protease inhibitors, rat L-type synthase was detected with electrophoretic mobility Mapp = 85,000. In contrast, the M-type enzyme in crude skeletal muscle extracts with protease inhibitors was detected with Mapp = 86,000 and 89,000. During purification of L-type synthase, apparent proteolysis can generate forms with increased electrophoretic mobility (Mapp = 75,000), still recognized by the antiserum. These M-type and L-type antisera did not recognize a protein with Mapp greater than phosphorylase. The anti-rat L-type antisera recognized glycogen synthase in blots of crude extracts of rabbit liver, but with Mapp = 88,000, a value 3,000 greater than that found for the rat liver enzyme. The anti-rat M-type antisera failed to recognize the enzyme in blots of crude extracts of rabbit muscle. Thus, in both muscle and liver, the corresponding rat and rabbit enzymes are structurally different. Because the differences described above persist after resolving these proteins by denaturing sodium dodecyl sulfate electrophoresis, these differences reside in the structure of the proteins themselves, not in some factor bound to the protein in crude extracts.

Isozymes of glycogen synthase.

Antisera to rat skeletal muscle glycogen synthase failed to recognize liver glycogen synthase by electroblot analysis. The antisera recognized the enzyme in skeletal muscle, heart, fat, kidney, and brain. The results support the hypothesis that there are at least two isozymes of glycogen synthase, and that most tissues contain a form similar or identical to the skeletal muscle type. There is a virtual absence of the muscle-type enzyme in adult rat liver.