Morton Rothstein

Heme requirement for reproduction of a free-living nematode.

The free-living, hermaphroditic nematode Caenorhabditis briggsae has a nutritional requirement for sterols. It will reproduce indefinitely in a liquid medium containing only bacterial cells (Escherichia coli) and salts if various sterols are present. Several other lipid-soluble materials are ineffective in supporting reproduction.

Recent developments in the age-related alteration of enzymes: A review

Recent developments dealing with the age-related alteration of enzymes have been examined. Since the last review of this field, three enzymes have been purified to homogeneity from young and old nematodes (enolase, phosphoglycerate kinase, triosephosphate isomerase) and one from the liver of young and old rats (superoxide dismutase). In all cases except for triosephosphate isomerase, the enzymes from old animals show a reduced catalytic ability compared to those from young animals. In addition, new reports of increases in the amount of altered enzymes in late-passage cells in tissue culture have appeared, though contrary evidence has also been published. The data from these and other papers are compared and discussed. Possible explanations for the alteration of enzymes include error theory, substitution of individual amino acids and conformational change without sequence change. Though final conclusions cannot be made, the evidence favors the latter idea to explain the presence of altered enzymes in old animals.

The formation of altered enzymes in aging animals

Evidence is accumulating which supports the idea that the alteration of enzymes is due to post-synthetic modifications rather than to sequence changes. Although unequivocal proof is still lacking, the idea that the changes in old enzymes are due to a subtle denaturation rather than to covalent changes is strengthened by recent work. That altered enzymes may result from a slowed protein turnover in old organisms appears to be a viable hypothesis.

Age-related alteration of enolase in the free-living nematode, Turbatrix aceti.

Abstract Enolase has been purified approximately 60–70-fold from both young and old Turbatrix aceti (nematodes). Both preparations are homogeneous as shown by gel electrophoresis, immunodiffusion and other criteria. The “old” enolase has a sharply reduced specific activity both in crude homogenates and in the pure preparation, compared to the “young” enzyme. The “old” enzyme is also much more sensitive to heat as shown by heat-stability determinations. At 52.5 °C, a biphasic pattern is obtained with a rapidly deteriorating component amounting to 40% of the enzyme. The remaining activity depreciates more slowly but more rapidly than the rate of loss of “young” enzyme activity. The results support the idea that “old” enzyme consists of active and partially active molecules, or perhaps entirely of the latter, rather than a mixture of active and inactive molecules. Molecular weight (82,000), subunit size (41,000), absence of isozymes, K m , K i (phosphate), index of fluoride inhibition and net charge (gel electrophoresis) are the same for both preparations. Enolase from T. aceti has been compared with enolase from other sources. The nematode enzyme is similar to the rabbit enzyme with regard to molecular weight, subunits and index of fluoride inhibition but differs in charge and pH optimum.

The effect of age on enolase turnover in the free-living nematode, Turbatrix aceti

Abstract The rates of synthesis and degradation of enolase and total soluble proteins slow with age in the free-living nematode, Turbatrix aceti . The half-lives are 73 and 58 h for soluble protein and enolase, respectively, in young organisms (5 days old). The respective figures are 163 and 161 h for old organisms (22–30 days old). Similar slowing of protein turnover occurs when the organisms are aged by a repeated screening procedure which avoids the use of fluorodeoxyuridine, an inhibitor of DNA synthesis normally added to aging cultures to obtain synchrony. The results support the idea that slowed protein turnover may be responsible for the formation of altered enzymes in old organisms.

Heat-labile isozymes of isocitrate lyase from aging Turbatrix aceti

Abstract The temperature stabilities of pure isocitrate lyase, isolated from young and old Turbatrix aceti , have been compared. The “old” enzyme shows the presence of a heat-labile component lacking in the enzyme derived from young organisms. This component has been shown to be associated with two of the five isozymes comprising the isocitrate lyase. A mechanism is proposed which might account for the presence of altered enzymes in aged organisms.

Biosynthesis of fatty acids in the free-living nematode, Turbatrix aceti.

Abstract The free-living nematode, Turbatrix aceti , in axenic culture, has the ability to synthesize polyunsaturated fatty acids de novo. Experiments with labeled fatty acids have provided some insight into the metabolic pathways involved. Of particular interest is the synthesis of 8, 11, 14, 17-eicosatetraenoic acid in greater quantities than the more common 5, 8, 11, 14 isomer (arachidonic acid).

Nematode biochemistry—IX. Lack of sterol biosynthesis in free-living nematodes

Abstract 1. 1. Under axenic conditions, the free-living nematodes, Caenorhabditis briggsae, Turbatrix aceti and Panagrellus redivivus , are unable to synthesize cholesterol from acetate-2-C 14 or DL -mevalonate-2-C 14 . 2. 2. No evidence could be found that sterols other than cholesterol are synthetized by any of the organisms.

Triosephosphate isomerase from young and old Turbatrix aceti.

Abstract 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.

NEMATODE BIOCHEMISTRY. III. EXCRETION PRODUCTS.

Abstract 1. 1. The free-living nematode, Caenorhabditis briggsae, after incubation with various radioactive substrates, excretes nitrogen chiefly in the form of ammonia and amino acids. No significant amounts of urea, uric acid, allantoin or creatinine are produced. 2. 2. Highly radioactive acidic and neutral components are excreted into the incubation media. 3. 3. The results obtained are in general agreement with the known excretion products of a number of parasitic nematodes.