G.Larry Cottam

Isozymes of pyruvate kinase in vertebrates: their physical, chemical, kinetic and immunological properties☆

Abstract The present paper tabulates the physical, chemical, immunologie and kinetic properties of the four isozymes of pyruvate kinase. The M 1 -isozymes display Michaelis-Menten kinetics, are not activated by fructose-1.6-diphosphate, and have very similar amino acid compositions. The L-isozymes are allosteric enzymes that are activated by fructose-1.6-diphosphate and have amino acid compositions that are different from the m 1 - and similar to the R-isozymes. In addition, the L- and R-isozymes are kinetically similar and immunologically cross-reactive. The M 2 -isozymes are a diverse group of enzymes. They are allosteric enzymes that are immunologically cross-reactive with the M 1 -isozyme.

Measurement of gluconeogenesis and pyruvate recycling in the rat liver: a simple analysis of glucose and glutamate isotopomers during metabolism of [1,2,3‐13C3]propionate

Simple equations that relate glucose and glutamate 13C‐NMR multiplet areas to gluconeogenesis and pyruvate recycling during metabolism of [1,2,3‐13C3]propionate are presented. In isolated rat livers, gluconeogenic flux was 1.2 times TCA cycle flux and about 40% of the oxaloacetate pool underwent recycling to pyruvate prior to formation of glucose. The 13C spectra of glucose collected from rats after gastric versus intravenous administration of [1,2,3‐13C3]propionate indicated that pyruvate recycling was slightly higher in vivo (49%) while glucose production was unchanged. This indicates that a direct measure of gluconeogenesis and pyruvate recycling may be obtained from a single 13C‐NMR spectrum of blood collected after oral administration of enriched propionate.

Evaluation of the water environments in deoxygenated sickle cells by longitudinal and transverse water proton relaxation rates

Abstract The longitudinal and transverse water proton relaxation rates of oxygenated and deoxygenated erythrocytes from both normal adults and individuals with sickle cell disease were measured as a function of temperature at two different frequencies. The simplest model which fits all of the data consists of three different environments for water molecules. The majority of the water (98%) has a correlation time indistinguishable from bulk water (3 × 10−11 sec). Secondly, there is a small amount of water (1.3–1.5%) present which has a correlation time of 2–4 × 10 −9 sec and is apparently independent of the erythrocyte sample studied. Presumably this water is the hydration sphere around the hemoglobin molecules and its correlation time is significantly slower than bulk water. The third environment contains approximately 0.2% of the water present and has a correlation time≥ 10−7 sec. This third environment is considered tightly bound to the hemoglobin because the water proton correlation time is very similar to the expected rotational correlation time for the hemoglobin molecules. The value of the transverse relaxation rate, fb(T2b)−1, for the tightly bound water fraction decreases in oxy ( S S ), deoxy ( A A ), and oxy ( A A ) erythrocyte samples as the temperature is increased as expected for a rotational correlation time process. In dramatic contrast,fb (T2b)−1 increases almost linearly as the temperature is increased over the whole 4 ° to 37 °C temperature range in samples of deoxy ( S S ) erythrocytes. The observation suggests a continual increase in the formation of deoxyhemoglobulin S polymers rather than a sudden transition from a homogeneous solution of deoxyhemoglobin S molecules to a solid gel.

Alteration in liver pyruvate kinase protein and catalytic activity upon starvation and refeeding

Abstract Liver pyruvate kinase (L-type isozyme) was purified from the livers of rats fed a high carbohydrate, low protein diet for 4 days. The protein was homogeneous as judged by polyacrylamide-gel electrophoresis with and without added sodium dodecyl sulfate and as judged by high speed sedimentation and low speed equilibrium centrifugation. The specific activity of the purified protein was 190–220 international units (IU)/mg. A precipitating antiserum directed specifically against liver pyruvate kinase was obtained from rabbits and was used to determine the amount of liver pyruvate kinase protein present in the 80,000g supernatant fraction of rat liver homogenates in response to the dietary status of the animal. Rats maintained on a high carbohydrate, low protein diet for 4 days prior to sacrifice have at least 20 mg of precipitable liver pyruvate kinase protein per liver. Starvation of the animal results in a marked reduction in liver pyruvate kinase so that by 3 days of starvation less than 7 mg of liver pyruvate kinase protein per liver remains. Refeeding the animal a high carbohydrate, low protein diet results in a return of the liver pyruvate kinase protein to the prestarvation level of 20 mg per liver. The liver pyruvate kinase activity per liver varies in the same direction as does the liver pyruvate kinase protein but does not parallel the change in protein. Animals fed a high carbohydrate, low protein diet for 4 days have 60–70 IU/mg of liver pyruvate kinase protein whereas animals starved for periods exceeding 30 h have greater than 100 IU/mg of liver pyruvate kinase protein. Refeeding starved animals with a high carbohydrate, low protein diet initially causes a large increase in activity per milligram of liver pyruvate kinase protein followed by a return of this value to the prestarvation level. The observed rise in the ratio of activity per milligram of liver pyruvate kinase protein during starvation suggests a modification in the enzyme protein resulting either in an increase in the specific activity of the enzyme or in a decrease in the affinity of the enzyme for the antibody.

The gelation of deoxyhemoglobin S in erythrocytes as detected by transverse water proton relaxation measurements

Abstract At 37 °C, when samples of blood, washed erythrocytes, or isolated hemoglobin from individuals with sickle cell disease are deoxygenated, the transverse water proton relaxation time is sharply decreased. In similar samples from normal adults homozygous for hemoglobin A, only a slight decrease in t 2 is observed upon deoxygenation at 37 °C. In samples containing deoxyhemoglobin S the value of t 2 increases as the temperature is decreased from 37 °C to 4 °C, in contrast to samples containing oxyhemoglobin S, oxyhemoglobin A, or deoxyhemoglobin A where t 2 decreases as the temperature decreases. It is suggested that this decrease in t 2 observed in samples of deoxyhemoglobin S at 37 °C is the result of an increase in the amount of preferentially oriented water at macromolecular interfaces which occurs under conditions known to produce deoxyhemoglobin S gelation. Conditions which reverse deoxyhemoglobin S gelation such as lowering the temperature to 4 °C decrease the amount of preferentially oriented water which results in an increase in the value of t 2 . Thus, measurement of the transverse water proton relaxation time can be used to monitor the gelation of deoxyhemoglobin S inside the erythrocyte.

Essential fatty acid deficiency in critically III surgical patients

Abstract Plasma fatty acid profiles of 33 critically ill surgical patients receiving fat-free parenteral nutrition were examined at weekly intervals up to 28 days. While plasma total fatty acid concentration remained relatively constant and within the normal range, marked compositional alterations were apparent. Levels of linoleate (18:2ω6), the major essential fatty acid in man, fell below normal values (754 ± 259 μg/ml) in 67 percent of patients within 1 week after cessation of oral intake. Decreases in other ω6 unsaturated fatty acids, derived from linoleate, were also apparent. In contrast, gradual increases were observed in levels of endogenously synthesized fatty acids, palmitate (16:0), palmitoleate (16:1) and oleate (18:1ω9). A fatty acid unique to essential fatty acid deficiency, 5,8,11 eicosatrienoate (20:3ω9), appeared in 25 percent of the patients during the first week and in all patients by the third week of study. Considering the rapid appearance and progression of these bio-chemical changes, early initiation of linoleate supplementation appears justified to forestall the development of related clinical sequelae.

Evidence for a proteolytic modification of liver pyruvate kinase in fasted rats

Abstract Liver pyruvate kinase was purified to homogeneity from rats fed a high carbohydrate, low protein diet (LPK-C) and from rats fasted for 84 h (LPK-F). Although the enzymes have similar electrophoretic mobilities in 7% polyacrylamide disc gels, the specific activity of LPK-C was two to three times the value of the specific activity of LPK-F. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of LPK-C yields a single protein band of 56,000 daltons. In contrast, LPK-F yields two bands of protein. Approximately one-third of the LPK-F has an electrophoretic mobility similar to the 56,000-dalton LPK-C peptide. The remaining two-thirds of the LPK-F protein migrates as a 51,000-dalton peptide. Cyanogen bromide was used to cleave LPK-C and LPK-F. Similar peptide patterns were obtained from LPK-C and LPK-F when the cyanogen bromide fragments were resolved by 12% polyacrylamide gel electrophoresis in 7.5 m urea containing 6 m m Triton X-100 and 5% acetic acid. Separation of the two peptides from LPK-F was accomplished by selective immunologie absorption of the 56,000-dalton peptide with anti-LPK-C gammaglobulin immobilized on Sepharose 4B. Tryptic digests of LPK-C, LPK-F and the 51,000-dalton peptide yield similar peptide patterns when analyzed via sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. These results suggest that the 51,000-dalton peptide could be derived by a proteolytic cleavage or limited digestion of the 56,000-dalton subunit. Phosphorylation of LPK-C and LPK-F by [γ-32P]ATP in vitro with cyclic AMP-activated protein kinase results in covalent incorporation of 32 P into only the 56,000-dalton subunit. These results suggest that anin vivo proteolytic modification that yields the 51,000-dalton subunit.

Anti-sickling nature of dimethyl adipimidate

Dimethyl adipimidate (DMA), a bifunctional cross-linking agent, is known to be a powerful anti-sickling agent in vitro . The hemoglobin isolated from DMA-treated erythrocytes is heterogeneous with respect to charge and size as shown by starch gel electrophoresis and sedimentation velocity studies; some hemoglobin tetramers being cross-linked together resulting in high molecular weight species. Measurement of transverse water proton relaxation times of DMA-treated sickle erythrocytes shows that while some aggregation may take place within deoxygenated erythrocytes, it is not of sufficient extent to cause the cells to change shape. Therefore, it is suggested that the anti-sickling effect of DMA is due to its ability to react with hemoglobin S inside sickle erythrocytes and inhibit the aggregation process.

Effect of pH, carbamylation and other hemoglobins on deoxyhemoglobin S aggregation inside intact erythrocytes as detected by proton relaxation rate measurements.

Abstract Measurement of the transverse water proton relaxation rate has been used to study the effect of pH, carbamylation, and other hemoglobins on the aggregation of deoxyhemoglobin S inside intact erythrocytes. Upon complete deoxygenation, cyanate-treated ( S S ) erythrocytes and erythrocytes heterozygous with respect to hemoglobin S ( A S , C S , and S D ) have high transverse water proton relaxation rates very similar to the values obtained with homozygous ( S S ) erythrocytes. These results suggest extensive intermolecular interactions between deoxyhemoglobin S molecules and a resultant increase in the correlation time for the small fraction of “irrotationally bound” water. When the transverse relaxation rate in deoxygenated ( S S ) erythrocytes was measured as a function of pH, the maximum rate was observed between pH 7.0 and 7.5. Upon increasing the pH beyond this range the observed relaxation rate decreases as does the number of sickled cells. Upon decreasing the pH, the observed transverse relaxation rate also decreases but the ratio of values from deoxy oxy ( S S ) erythrocytes remains in the normal range of 4–6 and the number of sickled cells does not change. Therefore, the deoxyhemoglobin S aggregate inside sickled erythrocytes, as observed by water proton relaxation rates, is not altered by carbamylation or by the presence of nongelling hemoglobins. In addition, the enhancement of the relaxation rates as a function of pH is consistent with the number of sickled forms observed.

Turnover of liver pyruvate kinase

Abstract The relative rates of synthesis and degradation for liver pyruvate kinase have been determined in rats fed standard lab chow, fasted, and refed a high-carbohydrate-low-protein diet. Relative rates of synthesis and apparent rates of degradation were determined by pulse-labeling the enzyme in vivo with l -[4,5- 3 H]leucine and by measuring the incorporation of radioactivity into liver pyruvate kinase after quantitative precipitation of the enzyme with anti-liver pyruvate kinase immunoglobulin. The relative rate of synthesis decreased approximately 75% upon fasting and then increased 20- to 30-fold upon refeeding the high-carbohydrate diet. The apparent half-lives for liver pyruvate kinase in fasted, control, and refed animals are very similar (55, 59, and 47 h, respectively). Thus, the nutritional alterations in the levels of liver pyruvate kinase seem to result primarily from alterations in the rate of enzyme synthesis.