Hans W. Behrisch

Regulation of enzyme activity at low temperature: Ionic influences on fructose-1,6-diphosphatase from muscle of the arctic tanner crab Chionocetes Bairdi

Abstract 1. 1. The possible roles of pH and potassium ion in the regulation of tanner crab muscle fructose-1,6-diphosphatase (FDPase) were examined over the physiological temperature range of the animal. 2. 2. A rise in temperature causes a shifting of the normally observed alkaline pH optimum towards the neutral range. The addition of K+ ion appears to counter this by inhibiting enzyme activity more at the neutral pH range than at alkaline pH. 3. 3. Tanner crab muscle FDPase is inhibited powerfully by 5′AMP. A decrease in temperature from 15 to 0°C results in an overall increase in affinity for the inhibitor. However, at the acclimatization temperature, sensitivity for AMP is markedly reduced. 4. 4. A rise in pH diminishes the effectiveness of AMP as an inhibitor of activity of tanner crab muscle FDPase. On the other hand, K+ causes a large increase in FDPase-AMP interaction. 5. 5. At high (approaching physiological) concentrations of AMP, activity of FDPase is markedly insensitive to the thermal range. 6. 6. These findings suggest mechanisms for the maintenance and regulation of muscle FDPase from the tanner crab over the temperature range of the animal.

On the subunit structure of seasonal isoenzymes of fructose 1,6-diphosphatase from muscle of the Alaskan king crab Paralithodes camtschatica

1. 1. Fructose 1,6-diphosphatase (FDPase) from skeletal muscle of the Alaskan king crab (Paralithodes camtschatica) occurs as a single form with an isoelectric point (pI) of about 5·2 in the cold-acclimatized animal, and as a single form with a pI value of 5·8 in the warm-acclimatized animal. 2. 2. The enzyme has a molecular weight of around 135,000, as judged by ultracentrifugation through a sucrose density gradient. 3. 3. The enzyme can be dissociated into two 66,000 mol. wt “halves” by exposure to 4 M urea. These halves have values of pI that are identical to those of the native enzyme. 4. 4. Exposure of the “halves” of FDPase to 2% sodium dodecyl sulfate results in a complete loss of activity, which appears to be founded on a further dissociation into small subunits of unequal molecular weight. The weights of these subunits were around 25,000 and 40,000, respectively. 5. 5. The pI values of the 40,000 mol. wt subunits from both the cold and warm FDPases are identical, while the pIs of the 25,000 mol. wt subunits differed markedly. 6. 6. These results suggest that the physical differences that presumably underlie functional differences between these thermal isoenzymes are symmetrically arranged, and that perhaps only a small portion of the enzyme molecule is altered to yield a new isoenzyme during thermal acclimatization.

Temperature and the regulation of enzyme activity in heterothermic tissues of an alpine mammal. Lactate dehydrogenase from skeletal muscle of the chamois

Abstract 1. 1.|The kinetic behaviour of lactate dehydrogenases (LDHs) from skeletal muscle from the extremities and deep parts of the body of the chamois ( Rupicapra rupicapra L. ) was examined over a wide range of temperatures. 2. 2.|One species of LDH is observed in the heterothermic leg muscle and two in the homeothermic tissue. 3. 3.|When pH of the assay medium is “corrected” for temperature to conform to temperature-caused changes observed in the pH of intra and extracellular fluids, the homeothermic and heterothermic LDHs may be differentiated kinetically. 4. 4.|Strikingly, under such conditions a decrease in temperature does not bring about an increase in apparent affinity of the heterothermic LDH for substrate. Rather, values of the apparent Km for pyruvate remain constant, with the result that the Q 10 of the reaction remains unchanged over the entire range of temperature and substrate concentrations tested. 5. 5.|The significance of this behaviour under conditions that we believe approach the physiological one is discussed.

Molecular adaptations to the diving habit: on the possible role of glycerol in the ischemic seal kidney

Abstract 1. 1. Isolated perfused kidneys of the spotted seal (Phoca vitulina richardsi) are able to withstand long periods of ischemia and resultant anoxia. In this state the major source of energy of the kidney cortex appears to be glycogen, wich is converted stoichiometrically to lactate. 2. 2. After about 45 min of ischemia glycogen stores in kidney cortex are effectively depleted: yet lactate concentrations continue to rise, albeit at a lower rate. 3. 3. On the basis of an observed rapid increase in concentrations of the free fatty acids in the tissue, the source of this additional lactate is glycerol from triglycerides within the tissue. 4. 4. When seal kidney, which is ischemic during the dive, is compared to seal heart, which is perfused during the dive, the activities of the enzymes glycerokinase, α-glycerophosphate dehydrogenase and triosephosphate isomerase are substantially higher in seal kidney cortex than in seal heart, or kidney of the domestic dog. 5. 5. This suggests the existence of a short metabolic sequence that can augment the finite stores of carbohydrate in this usually active tissue during a long and arduous dive. 6. 6. This sequence does not become active until glycogen stores are virtually depleted and the kidney cortex cells energy charge is much reduced (from 0.84 to 0.71). 7. 7. These results suggest the existence of a late metabolic “reserve” for an ischemic tissue of the diver during a particularly strenuous or long dive.

Unusual regulatory characteristics of fructose 1,6-diphosphatase from muscle. Studies on the kinetic behaviour of the enzyme from muscle of the arctic tanner crab Chionocetes bairdi

Abstract 1. 1. The regulatory behaviour of fructose 1,6-diphosphatase (FDPase) from skeletal muscle of the tanner crab ( Chionocetes bairdi ) was examined over the physiological temperature range of the animal. 2. 2. In addition to being inhibited by 5′AMP (commonly accepted as being the major modulator) tanner FDPase is inhibited by ribulose-5-phosphate (Ru-5-P), uridine diphospho-N-acetylglucosamine (UDPAG) and mannose-6-phosphate. These results are discussed in light of the crabs moulting cycle. 3. 3. Tanner FDPase displays negative co-operativity in the binding of substrate (FDP), but only at the temperature of acclimatization. 4. 4. Phosphoenolpyruvate (PEP), a positive modulator of crustacean FDPase enhances activity of FDPase by increasing enzyme-substrate affinity, and also abolishing the negative co-operativity observed in substrate saturation curves at low (acclimatization) temperatures. 5. 5. The role of negative co-operativity in the regulation of enzyme activity is discussed.

Molecular adaptations to the diving habit: Fructose 1,6-bisphosphatase of a diver, the harbor seal

Abstract 1. 1. The regulatory properties of -1,6-bisphosphatase (FbPase) from seal liver were examined and compared with those of the dog FbPase. 2. 2. The liver enzyme appears to be identical with that from kidney cortex and occurs as one form with a pI of 5.85. 3. 3. This FbPase has a pH optimum near 7.0 and is relatively insensitive to pH in affinity for substrate over the range 6.8 to 7.4. 4. 4. An activator of FbPase, phosphoenolpyruvate (PEP), markedly reduces the S 0.5 for FbP over a wide pH range and also enhances the degree of interaction between the substrate binding sites. 5. 5. Seal FbPase is inhibited by 5′-AMP but affinity for the inhibitor is relatively unaffected by a wide pH range. PEP reduces FbPase-AMP affinity and also maintains the S 0.5 for AMP independent of pH. 6. 6. These observations, particularly the marked insensitivity of the enzyme to large pH changes, which contrasts strongly with that of the non-diving mammal, suggest a mechanism for extended control of carbohydrate metabolism by this key enzyme in the diver.

Differentiation between isoproteins by derivative spectroscopy

Abstract 1. 1. A method extending the application of derivative spectroscopy by utilizing the second derivative spectra is presented. It is independent of protein concentration and may be used under denaturing or non-denaturing conditions. 2. 2. By using mixtures of the carboxyl- and amino-blocked derivatives of phenylalanine, tyrosine and tryptophan as model proteins, it may be shown that the method is sufficiently sensitive to distinguish between proteins differing by one or two aromatic amino acid residues. 3. 3. The procedure may be employed reliably in the differentiation between isoenzymes or isoproteins. and is simple, rapid and sensitive.

Regulation of enzyme activity in the hibernator: Effect of cations on liver pyruvate kinase in the arctic ground squirrel

Abstract 1. 1.|The kinetic behavior of pyruvate kinase (PyK) from liver of the Arctic ground squirrel (Citellus undulatus) was examined over a wide temperature (5–37°C) range in the presence of its substatres and modulators, and cations that are known to influence activity of this enzyme. 2. 2.|The substrates adenosine diphosphate (ADP) and phosphoenolpyruvate (PEP) as well as the modulators fructose 1,6-bisphosphate (FBP) and adenosine triphosphate (ATP) appear to be bound in such a manner that a decrease in the S0.5 value is accompanied by an increase in the Hill constant (h), indicating an enhanced degree of site-site interaction in the binding of these metabolites. 3. 3.|In marked contrast, the cations K+, Mg2+ and Mn2+, are bound in such a manner that falling S0.5 values are accompanied by decreases in the attendant h values. 4. 4.|The significance of these results is discussed and it is suggested that only the intermediates play a role in regulating activity of liver pyruvate kinase while the ions appear to enhance the Q10 effect on the PyK reaction, and function as an integral part of the intracellular milieu that maintains the efficiency of enzyme regulation over a wide temperature range.

Regulation of enzyme activity in a hibernator: Hepatic 6-phosphogluconate dehydrogenase from the arctic ground squirrel☆

Abstract 1. 1. Kinetic parameters of hepatic 6-phosphogluconate dehydrogenase (6PGD) from the Arctic ground squirrel were examined over the range of seasonal variation in the body temperature of the animal. 2. 2. Electrofocusing of the enzyme shows a single form of the enzyme in both hibernating and nonhibernating animals, with an isoelectric point of 5.15. 3. 3. Specific activity is significantly higher in the nonhibernator than in the hibernator, but substrate affinities for 6-phosphogluconate (6PG) and NADP + are not significantly different between the two states, or over the temperature range 5–37°C. 4. 4. ADP, ATP, Mg 2+ and fructose-1,6-bisphosphate (FBP) have no effect on enzyme activity, while AMP is mildly inhibitory ( K i = 2.18 mM). NADPH is a potent inhibitor ( K i = 0.02 mM), and may be a major regulator of 6PGD activity. 5. 5. These results suggest that the major changes in the activity of 6PGD accompanying the onset of hibernation are brought about by a combination of seasonal changes in enzyme concentration, and by the effects of a reduction in thermal energy accompanying the lowering of body temperature.

Temperature and the regulation of enzyme activity in homeo- and heterothermic tissues of arctic marine mammals: Some regulatory properties of 6-phosphogluconate dehydrogenase from adipose tissue of the spotted

Abstract 1. 1. Electrofocusing of 6-phosphogluconate dehydrogenase (6-PG deh.) from flipper (heterothermic) and deep (homeothermic) adipose tissues of the spotted seal (Phoca vitulina) shows two enzyme variants to be present in each tissue with isoelectric points of 5·91–5·96 and 5·34–5·40. However, the relative activities of both components are markedly different in each tissue. 2. 2. A decrease in temperature causes a large decrease in the apparent Km for substrate (6-PG) in the homeothermic (deep) 6-phosphogluconate dehydrogenase. In contrast, in the heterothermic (flipper) enzyme Km for substrate is relatively independent of thermal change. 3. 3. Affinity of the homeothermic 6-phosphogluconate dehydrogenase for its inhibitor TPNH is powerfully enhanced by a decrease in temperature, a finding which would make this enzyme unsuited for heterothermic function. On the otherhand, affinity of the flipper 6-PG deh. for TPNH is conspicuously insensitive to temperature. 4. 4. These results suggest formal mechanisms for continued function and thermal independence of regulation of 6-phosphogluconate dehydrogenase in heterothermic tissues from an Arctic marine mammal.