Sterol biosynthesis in the free-living nematode Panagrellus redivivus

The fate of the known sterol precursor squalene 2,3-oxide was investigated in the free-living nematode Panagrellus redivivus. The nematodes were cultured axenically in the presence of [4-(3)H]squalene 2,3-oxide. Radioactivity was found in the total lipids of the isolated nematodes. Essentially all of the radioactivity encountered in the total lipids was found in the non-saponifiable fraction. The components present in the non-saponifiable fraction were separated and isolated by t.l.c. Three labelled components were identified by a combination of t.l.c., g.l.c. and mass spectroscopy. It is established that P. redivivus has the capacity for biosynthesis of lanosterol. No labelled C(27) sterols could be detected.     

Organic Acids of Ditylenchus triformis and Turbatrix aceti

Pyruvic acid, lactic acid and several tricarboxylic acid cycle acids were extracted from Ditylenchus triformis and Turbatrix aceti and identified. Fumaric acid was predominant in both nematodes. Small amounts o f malic and α-ketoglutaric acids and intermediate quantities o f lactic, citric, succinic, and pyruvic acids occurred in D. triformis. In T. aceti citric, lactic, and α-ketoglutaric acids were less abundant than succinic, malic and pyruvic acids. Only traces of aconitic and oxalacetic acids occurred in both nematodes. All the organic acids detected accounted for only about one per cent of the dry weight of nematodes o f the two species.     

Triosephosphate isomerase from young and old Turbatrix aceti.

Triosephosphate isomerase (EC 5.3.1.1) has been purified from young and old Tubatrix aceti. The enzyme shows a sharply lower specific activity in homogenates from old nematodes compared to similar preparations from young animals. However, when the enzyme activity of the respective homogenates was adjusted to the same level, equal amounts of antiserum (prepared to pure “young” triosephosphate isomerase) were required to remove the activity. Therefore, the lower specific activity observed in “old” homogenates was due to the presence of less enzyme and not to “altered” enzyme. The same results were obtained by immunotitrations of pure preparations of “young” and “old” enzyme. Moreover, in contrast to results reported for other enzymes, the specific activity of “old” triosephosphate isomerase, during purification, rose to the same value as that of pure “young” enzyme. The evidence indicates that altered triosephosphate isomerase does not exist in old T. aceti. The above results contradict the idea of an “error theory” in which all proteins would develop altered sequences. Pure triosephosphate isomerase (old or young) from T. aceti consists of two subunits, each of molecular weight 26,500. No isozymes could be detected.     

Protein degradation in aged nematodes (Turbatrix aceti).

The rates of degradation of total soluble proteins in the free-living nematode, $\text{Turbatrix aceti}$, have been estimated by following the loss of acid-insoluble insoluble radioactivity from protein during a nonradioactive chase period after initial labeling with [³⁵S]methionine. These proteins appear to lose label kinetically as a homogeneous class in age-synchronized nematode populations. However, proteins are degraded more slowly in senescent cultures than in young cultures. Protein degradation rates decline progressively during nematode aging. These findings suggest that the protein degradative system in T. aceti may become partially defective with advancing age which may result in the accumulation of aberrant protein molecules in senescent organisms.     

Glutamine and asparagine synthesis in the nematodes Heligmosomoides polygyrus and Panagrellus redivivus

Low levels of glutamine synthetase were demonstrated in Panagrellus redivivus and Heligmosomoides polygyrus. Asparagine synthetase was detected in P. redivivus but activity was too low to be detected in H. polygyrus.     

Studies on Turbatrix aceti beta-N-acetylglucosaminidase. 1. Purification and physicochemical characterization

N-Acetyl-beta-D-glucosaminidase was purified, from the culture medium of the nematode Turbatrix aceti, to homogeneity, as judged by electrophoresis in polyacrylamide gel and ultracentrifugation. The purification scheme involved the following steps: (i) concentration of the culture medium by ultra-filtration by an Amicon PM-30 membrane; (ii) ammonium sulfate precipitation; (iii) DEAE-Sephadex and (iv) Sephadex G-200 chromatography; and (v) affinity chromatography on succinyldiaminopropyl amino-Sepharose bearing the ligand p-aminophenyl 2-acetamido-2-deoxy-1-thio-beta-D-glucopyranoside. The molecular weight of the enzyme was 112,000 +/- 4800 and 124,000 as determined by polyacrylamide gel electrophoresis and by gel filtration through Sephacryl S-200, respectively. The enzyme showed a pH optimum of 4.8 for N-acetylglucosaminidase and 5.4 for N-acetylgalactosaminidase. The detailed substrate specificity studies were carried out on both synthetic and natural oligosaccharides and glycopeptides. The chitin oligosaccharides and asialo-agalacto complex type as well as high mannose-type glycoproteins such as fetuin and ovalbumin, respectively, were good substrates for the enzyme. Substrate analogs in which the oxygen atom of the acetamido group was replaced by sulfur atom proved to be poor substrates.     

Age dependent changes in enzymes involved in macromolecular synthesis in Turbatrix aceti.

The specific activities of elongation factor 1, RNA and DNA polymerase, as well as fructose-1,6-diphosphate aldolase were determined as a function of age in the nematode Turbatrix aceti. It was found that the specific activities of both DNA polymerase and aldolase declined constantly as the animal aged. In contrast, both elongation factor 1 and RNA polymerase showed sharp increases in their specific activities at 5 and 15 days, respectively, before ultimately declining. It was also shown that elongation factor 1, which exists mainly as a high molecular weight species in the young animal, undergoes a conversion to a lower molecular weight species as the organism ages. In addition there is a progressive accumulation of inactive or partially active elongation factor 1 molecules in older animals. The addition of α-tocopherol to the growth medium of these nematodes resulted in an increased life-span as well as alterations in the patterns of the enzyme activities.     

Two FMRFamide-like peptides from the free-living nematode Panagrellus redivivus.

Peptides of the FXRFamide family, where X = M, I or L, are broadly distributed among invertebrates. Two such peptides were purified and sequenced from the free-living nematode, Panagrellus redivivus. Immunohistochemical techniques localized FMRFamide-like material in several regions of these organisms, including the nerve cords and, most prominently, in paired groups of cells located caudally to the base of the pharynx. RIA determinations gave an estimate of 2.8 nmol immunoreactive peptide/g of an acetone extract of P. redivivus. Four sequential HPLC purification steps, followed by sequencing by automated Edman degradation and FAB-MS, led to the identification of Ser-Asp-Pro-Asn-Phe-Leu-Arg-Phe-amide (SDPNFLRFamide) and Ser-Ala-Asp-Pro-Asn-Phe-Leu-Arg-Phe-amide (SADPNFLRFamide) as members of the FXRFamide family in this nematode.     

Multiple forms of isocitrate lyase in the matrix of Turbatrix aceti mitochondria

The localization of five separate forms of isocitrate lyase (EC 4.1.3.1) in the free-living nematode Turbatrix aceti was determined by analyzing their distribution among subcellular fractions with continuous (Tris-acetate, pH 7.5) polyacrylamide gel electrophoresis. Enzyme activity on gels was detected either by substrate-dependent Schiff's-aldehyde staining or absorbance of phenylhydrazone at 324 nm. Following rate sedimentation of worm homogenates, the highest specific activity for isocitrate lyase was recovered in the pellet containing intact mitochondria. Glyoxysomes (microbodies) were not observed by electron microscopy in this or any other fraction. Selective removal of mitochondrial outer membranes (and hence components in the intermembrane space) was accomplished by two different procedures: (1) passage of mitochondria in a hypersomotic medium through a French pressure cell at 1500 psi, or (2) treatment with 0.6 mg digitonin mg protein. Electron microscopy revealed essentially complete removal for the former procedure, but only partial removal following digitonin treatment. Equilibrium centrifugation on sucrose gradients was necessary to strip the residual outer membranes from the digitonin-treated mitochondria. Mitoplasts resulting from the two procedures were subfractionated into matrix and inner membrane components by high-pressure disruption (14,000 psi) and subsequent rate sedimentation (144,000g, 60 min). Identical electrophoretic patterns were found using both slab and disc gels, whether stained with Schiff's reagent or scanned at 324 nm, in samples taken from clarified homogenates, intact mitochondria, mitoplasts, or matrix fractions. The results indicate that all five forms of the enzyme occur together in the mitochondrial matrices. Their individual functions are not yet known, but they may be involved in the regulation of isocitrate metabolism common to the tricarboxylic acid and glyoxylate cycles occurring within the same mitochondria.     

Mineral content of the free-living nematode Turbatrix aceti and ruminant parasite Trichostrongylus colubriformis

1. The parasitic nematode Trichostrongylus colubriformis and free-living nematode Turbatrix aceti were examined for element content. 2. T. colubriformis contained significantly less phosphorus, potassium, manganese and boron than T. aceti. 3. T. colubriformis contained significantly more nitrogen and copper than T. aceti. 4. Comparative study of the chemical composition of parasitic vs free-living nematodes may facilitate in vitro cultivation of the parasitic forms.