Thermolabile Form Research Articles

Human platelet thermostable phenol sulfotransferase: Assay of frozen samples and correlation between frozen and fresh activities

Sulfate conjugation catalyzed by phenol sulfotransferase (PST, EC 2.8.2.1) is an important metabolic pathway for catecholamines, endogenous phenolic compounds and catechol drugs [l--4]. Thermolabile (TL) and thermostable (TS) forms of PST are present in human tissues [S-6]. The platelet is an easily obtained tissue that is useful for the study of human PST regulation [3-4,7-lo]. Only TS PST in fresh platelets prepared and assayed immediately differs between American blacks and whites in levels of activity and thermal stability [ll-121. The ability to collect, prepare and freeze multiple samples prior to assay would simplify family studies of platelet PST from American blacks. We had three objectives in this project. First, we modified the method of platelet preparation to allow frozen storage of samples and we investigated biochemical properties of the frozen enzyme. Second, we compared levels of TS PST activities and thermal stabilities from frozen and fresh samples. Third, we used the modified assay to measure TS PST activity and thermal stability in platelet samples from randomly selected subjects. The biochemical properties and activities of TS PST measured with p-nitrophenol by the modified assay compared well with the results obtained with fresh samples. The levels of TS PST activity and distributions of thermal stabilities detected with the frozen samples were essentially identical to results we reported with fresh samples [12].

Comparative genetics of albinism

Albinism in laboratory mammals is equivalent to human tyrosinase-negative oculocutaneous albinism, and thus the result of recessive mutation in the structural locus for tyrosinase (TYR), which prevents melanin biosynthesis. In the mouse, eight mutant alleles are now known at this locus, with differing effects on eye colour and on the degree of reduction in eumelanin and phaeomelanin pigmentation. Three of these alleles, namely chinchilla, himalayan (acromelanistic) and albino (c) itself, have also been recognized in a number of other species but only albino has been identified in man so far. The himalayan allele (equivalent to Siamese in the cat) is of particular interest because it converts tyrosinase into a thermolabile form, with greater production of melanin in colder areas of the body. The optic track misrouting found in human albinos also occurs in albino alleles in other mammals, which may also show reduced activity and stress responses. The TYR locus is on human chromosome 11, which now has at least 11 loci with homologues on mouse 7. However, their order is markedly different in the two species. For instance, c and Hbb (beta-globin), which are closely linked in mouse, rabbit, cat etc., are far apart on human 11q and 11p respectively. Moreover, some loci (e.g., Fes and Mod-2) which are close to c in the mouse appear to be on human chromosomes other than 11. This extensive chromosomal restructuring in mammalian evolution means that the effects of human albino deletions may differ greatly from those studied in the mouse, which are associated with defects of kidney, liver and thymus. Tyrosinase-positive albinos or near-albinos are known at a number of loci in mice and other mammals. They are the result of the absence or inhibition of melanocytes in the affected areas, so that no melanin is produced. In general they are associated with pathological pleiotropisms which may lead to anaemia, inner ear defects, megacolon, neurological effects, skeletal defects, microphthalmia, osteopetrosis, spina bifida, sterility and so on. Homologies between these and human loci affecting pigmentation are now being discovered.

Metal ion-catalyzed oxidation of proteins: Biochemical mechanism and biological consequences

In the presence of O 2, Fe(III) or Cu(II), and an appropriate electron donor, a number of enzymic and nonenzymic oxygen free radical-generating systems are able to catalyze the oxidative modification of proteins. Whereas random, global modification of many different amino acid residues and extensive fragmentation occurs when proteins are exposed to oxygen radicals produced by high energy radiation, only one or a few amino acid residues are modified and relatively little peptide bond cleavage occurs when proteins are exposed to metal-catalyzed oxidation (MCO) systems. The available evidence indicates that the MCO systems catalyze the reduction of Fe(III) to Fe(II) and of O 2 to H 2O 2 and that these products react at metal-binding sites on the protein to produce active oxygen (free radical?) species (viz; OH, ferryl ion) which attack the side chains of amino acid residues at the metal-binding site. Among other modifications, carbonyl derivatives of some amino acid residues are formed; prolyl and arginyl residues are converted to glutamylsemialdehyde residues, lysyl residues are likely converted to 2-amino-adipylsemialdehyde residues; histidyl residues are converted to asparagine and/or aspartyl residues; prolyl residues are converted to glutamyl or pyroglutamyl residues; methionyl residues are converted to methionylsulfoxide residues; and cysteinyl residues to mixed-disulfide derivatives. The biological significance of these metal ion-catalyzed reactions is highlighted by the demonstration: (i) that oxidative modification of proteins “marks” them for degradation by most common proteases and especially by the cytosolic multicatalytic proteinase from mammalian cells; (ii) protein oxidation contributes substantially to the intracellular pool of catalytically inactive and less active, thermolabile forms of enzymes which accumulate in cells during aging, oxidative stress, and in various pathological states, including premature aging diseases (progeria, Werner's syndrome), muscular dystrophy, rheumatoid arthritis, cataractogenesis, chronic alcohol toxicity, pulmonary emphysema, and during tissue injury provoked by ischemia-reperfusion. Furthermore, the metal ion-catalyzed protein oxidation is the basis of biological mechanisms for regulating changes in enzyme levels in response to shifts from anaerobic to aerobic metabolism, and probably from one nutritional state to another. It is involved in the killing of bacteria by neutrophils and in the loss of neutrophil function following repeated cycles of respiratory burst activity.

Sulfation pharmacogenetics: correlation of human platelet and small intestinal phenol sulfotransferase.

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic drugs. All human tissues studied contain a thermostable (TS) form of PST, which catalyzes the sulfate conjugation of "simple" phenols such as p-nitrophenol, and a thermolabile (TL) form, which catalyzes the sulfation of dopamine and other monoamines. In the present study we tested the hypothesis that genetically controlled levels of TS and TL PST activity in the platelet, as well as inherited variations in the thermal stability of platelet TS PST, might reflect those same characteristics of the enzyme in the less accessible tissue, human small intestinal mucosa. Platelet TS and TL PST activities and TS PST thermal stability were measured in blood samples from 45 randomly selected healthy subjects, and 14 of those subjects were selected to have intestinal biopsies performed. There was a significant correlation between levels of platelet and jejunal mucosal TS PST activity (rs = 0.574, p less than 0.030), but there was not a significant correlation between levels of TL PST activity in the two tissues (rs = 0.265, p = 0.368). There was also a significant correlation between the trait of TS PST thermal stability in the two tissues (rs = 0.828, p less than 0.0001). These observations suggest that inherited variations in TS PST activity and thermal stability in an easily obtained tissue, the platelet, might be used to predict individual differences of those properties of the enzyme in the human small intestine, an organ that plays an important role in drug metabolism.

Triiodothyronine: A Substrate for the Thermostable and Thermolabile Forms of Human Phenol Sulfotransferase*

The enzyme(s) responsible for the sulfate conjugation of L-T3 in man has not been characterized. T3 sulfotransferase (T3-ST) activity was characterized in normal human liver tissue obtained during clinically indicated surgical resection. Subcellular distribution studies showed that the T3-ST activity was localized to the cytoplasmic fraction. This finding raised the possibility that T3-ST activity might be similar to the 2 previously identified forms of cytoplasmic phenol sulfotransferase (PST) found in human tissue. A thermostable (TS) form of PST catalyzes the sulfate conjugation of micromolar concentrations of p-nitrophenol, and a thermolabile (TL) form catalyzes the sulfate conjugation of dopamine and other monoamines. Thermal stability and enzyme inhibitor experiments showed that T3-ST activity in pooled liver homogenates was very similar to the TS form of PST. The apparent similarity of T3-ST to TS PST was studied further by measuring T3-ST, TS PST, and TL PST activities in 20 individual liver samples. T3-ST activities correlated significantly with TS PST activities (r = 0.939; P less than 0.001) measured with p-nitrophenol, but not with TL PST activities (r = -0.118; P greater than 0.6) measured with dopamine. However, sulfation of T3 by the TL form of the enzyme might have been masked by the 18-fold higher specific activity of TS than TL PST in human liver homogenates. When the two forms of PST were separated by ion exchange chromatography, T3 was found to be a substrate for both the TS and TL forms of PST. "True" Km values for T3 were similar for TS and TL PST (81 and 127 microM, respectively).

Phenol sulfotransferase inheritance.

1. Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of many phenolic and catechol neurotransmitters. Human tissues contain both thermostable (TS) and thermolabile (TL) forms of PST that differ in their substrate specificities, inhibitor sensitivities, physical properties, and regulation. 2. Individual variations in the levels of activity of both TS and TL PST in the human platelet are strongly influenced by inheritance. 3. Individual differences in the level of platelet TS PST activity are correlated with individual variations in the activity of this form of the enzyme in human cerebral cortex, liver, and intestinal mucosa. 4. There are also individual familial differences in the thermal stability of TS PST in the platelet. These differences are correlated with individual variations in the thermal stability of TS PST in cerebral cortex, liver, and intestinal mucosa. 5. Individual variations in the thermal stability of TS PST in hepatic tissue are associated with the presence of one or both of a pair of TS PST isozymes that can be separated by ion-exchange chromatography and that differ in their thermal stabilities. 6. This series of observations suggests that a structural gene polymorphism may be one mechanism by which inheritance controls TS PST in humans. The isozymes of TS PST in liver may represent the products of alternative alleles for this polymorphism, alleles that might control the structure of TS PST in many human tissues.

Inheritance of human platelet thermolabile phenol sulfotransferase (TL PST) activity.

Sulfate conjugation is an important pathway in the biotransformation of drugs and neurotransmitters. The thermolabile (TL) form of the enzyme phenol sulfotransferase (PST) catalyzes the sulfation of catecholamine neurotransmitters and drugs such as methyldopa and acetaminophen. Platelet TL PST activity was measured in blood samples from 232 individuals in 49 nuclear families. Correlations ranged from 0.43 to 0.45 for parent-offspring pairs and from 0.44 to 0.47 for siblings. Mother-father correlations were not significantly different from zero. Although evidence was not unequivocal, both segregation and commingling analyses provided some support for a major gene influence on TL PST activity, with other variation due to polygenic background. In both sets of analyses, however, support for a major gene hypothesis depended upon skewness in the TL PST activity distribution. A polygenic model with high heritability (0.77) was most strongly supported with the log transformed data. These results confirm and extend a previous report of high heritability of TL PST based on a study of twins. In addition, our results raise the possibility of a major gene effect on this important catecholamine- and drug-metabolizing enzyme--a possibility that can now be evaluated using biochemical techniques.

Human pheochromocytoma phenol sulfotransferase: biochemical properties and activities of thermolabile and thermostable forms

To determine whether pheochromocytoma phenol sulfotransferase (PST) activities were similar to blood platelet PST activities, we established assay conditions and biochemical properties for the human pheochromocytoma enzymes. At least two forms of PST were present in high speed supernatant (HSS) preparations of the tumors. A thermolabile form (TL) and a thermostable form (TS) were similar to those of human platelet PST with regard to pH optima, apparent K m values, responses to 2,6-dichloro-4-nitrophenol and thermal stability. PST activities were measured in 74 tumors of neuroectodermal origin that had been stored at −80° C for a mean of 37.9 months. Levels of TL and TS PST activities decreased in a nonlinear fashion with time of sample storage. TL and TS PST activities of 4 samples assayed after 1.08 ± 1.95 (mean ± SD) month of storage were 167 ± 73 and 3110 ± 1817 U/mg protein, respectively (mean ± Sem). Our results indicated that the TL and TS forms of PST in pheochromocytoma HSS preparations were biochemically similar to platelet PST activities.

β-Hexosaminidase From Colon and Sera of Dukes-Classified Colorectal Cancer Patients: Activity Levels, Isozyme Patterns, and Kinetic Properties<xref ref-type="fn" rid="FN1">2</xref>

Total activity levels, isozyme patterns, and kinetic properties of beta-hexosaminidase were studied in crude supernatants of malignant and adjacent, uninvolved normal colon tissues and in sera from 28 Dukes-classified colorectal cancer patients. A significant increase (P less than .001) in both beta-hexosaminidase activity and beta-hexosaminidase specific activity and a significant increase (P less than .01) in the relative percentage of activity comprised by the basic thermostable form of beta-hexosaminidase (Hex B) isozyme were found in malignant tissue compared to the activities seen in uninvolved normal colon tissue. No apparent correlations were found between either beta-hexosaminidase activity levels or relative percentage of Hex B and Dukes category. Kinetic analysis indicated that the thermolabile form of beta-hexosaminidase and Hex B from malignant colon were comparable to the corresponding isozymes from normal colon with regard to thermostability after preincubation at 50 degrees C and pH activity curves (optimum between pH 4.0 and 5.0). Significantly decreased (P less than .05) beta-hexosaminidase activity was found in sera of the 28 colorectal cancer patients (17.3 +/- 5.2 U/ml, mean +/- SD) when compared to the activity in 19 controls with nonmalignant diseases (21.4 +/- 8.2 U/ml) and 17 normal controls (21.3 +/- 6.4 U/ml). Isoelectric focusing indicated that a peak of beta-hexosaminidase activity with an isoelectric point value (9.5) comparable to that of the peak found in increased amounts in malignant colon was detectable in the sera of 36% of the colorectal cancer patients and 11% of the controls.

Lung phenol sulfotransferases: Thermal stability of human and bovine enzymes

Phenol sulfotransferase (PST) from human and bovine lung was purified about 1000-fold and the activity was measured with different acceptor substrates. Human lung PST catalyzes sulfation of both exo- and endogenous phenols, but the bovine lung enzyme only exogenous phenols. This difference in substrate specificity seems to be related to the presence of at least two different molecular forms of the enzyme: a thermostable and a thermolabile form in human lung, and two thermostable forms in bovine lung. Like the human platelet PST, the thermostable form from human lung is active with low concentrations of phenol ( K m = 45 μM) and the thermolabile form with dopamine and high concentrations of phenol ( K m = 909 μM). Bovine lung, which shows no catecholamine sulfating activity, contains two thermostable phenol sulfotransferases, both active with phenol but differing in their affinity to this substrate ( K m = 40 μM and 335 μM, respectively).

Human pituitary phenol sulfotransferase: biochemical properties and activities of the thermostable and thermolabile forms.

Pituitary tissue contains phenol sulfotransferase (PST), the enzyme that catalyzes the sulfate conjugation of monoamine neurotransmitters. We carried out these studies with pituitaries obtained 21.3 +/- 3.0 h postmortem (mean +/- SEM; n = 21) to determine whether the biochemical properties and variations in levels of human pituitary PST activities were similar to those of PST in platelets from control subjects. PST in the human platelet has been studied thoroughly because of the possibility that platelet PST might reflect levels of PST activity in other tissues such as the pituitary and brain. Our results demonstrated 2 forms of the pituitary enzyme that were similar to the thermostable (TS) and thermolabile (TL) forms of platelet PST with regard to assay conditions, pH optima, Km values for multiple substrates, responses to 2,6-dichloro-4-nitrophenol (DCNP), and thermal stability properties. Pituitary samples also were obtained at autopsy 6.3 +/- 0.33 h (mean +/- SEM; n = 3) after death to determine the effects of storage at 4 degrees C on PST activities. After storage for 6-18 h, 83-99.6% of the TS PST activity remained and 44-66.9% of the TL PST activity remained. Pituitary TS PST activity in samples obtained within 12.1 +/- 3.25 h after death was 121.0 +/- 49.1 units/mg protein (mean +/- SEM; n = 7) with a range from 9.7 to 367.6. TL PST activity was 35.6 +/- 11.6 units/mg protein (mean +/- SEM; n = 6) with a range from 6.1 to 80.7. Wide variations of both enzyme activities were also present in 3 pituitary tumor samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermolabile and thermostable human platelet phenol sulfotransferase. Substrate specificity and physical separation.

Human platelets contain at least two forms of phenol sulfotransferase (PST), a thermolabile (TL) form for which dopamine is a substrate and a thermostable (TS) form for which micromolar concentrations of phenol can serve as substrate. At higher concentrations phenol is also a substrate for the TL form. Studies of the regulation and the possible clinical value of measurements of platelet PST have been hampered because there is no specific substrate for the TS form of the enzyme. The purposes of these experiments were to determine whether there might be a better substrate than phenol for use in measurement of the activity of the TS form of platelet PST, and to attempt to physically separate the two forms of the platelet enzyme. The results of substrate kinetic, thermal stability, and inhibitor studies performed with platelet homogenates were all compatible with the conclusion that p-nitrophenol and 6-OH-melatonin were substrates for both the TS and TL forms of platelet PST. Norepinephrine, epinephrine and 5-OH-tryptamine were substrates for only the TL form. The apparent Km constants of the two forms of PST for p-nitrophenol differed by 7,100-fold when measured in platelet homogenates. This difference was 200 times greater than that which has been reported for phenol. Therefore, p-nitrophenol is the preferred substrate for measurement of the TS PST activity if interference by the TL activity is to be avoided. This information made it possible to use p-nitrophenol as a substrate in experiments designed to separate the two forms of platelet PST.(ABSTRACT TRUNCATED AT 250 WORDS)

Human platelet phenol sulfotransferase: partial purification and detection of two forms of the enzyme.

To begin to study the usefulness of platelet phenol sulfotransferase (PST) as a possible measure of the enzyme activity in other organs such as the brain, we purified human platelet PST 36-120-fold. Activity toward 3-methoxy-4-hydroxyphenylglycol (MHPG), dopamine, 5-hydroxytryptamine (5-HT), and phenol eluted in the same Sephadex G-100 and Affi-Gel Blue column fractions. Specific activities of the enzyme with MHPG, dopamine, 5-HT, and phenol as substrates were 1198, 1068, 401, and 408 units/mg protein, respectively. Optimal assay conditions were established for each substrate. Apparent Km values were 598 microM, 21 microM, 19 microM, and 500 microM for MHPG, dopamine, phenol, and 5-HT, respectively. Apparent Km values for 3'-phosphoadenosine-5'-phosphosulfate (PAPS) with the same four substrates ranged from 0.11 to 0.25 microM. The pH optima were 6.3 for phenol, 6.8 for dopamine, and 7.0 for MHPG and 5-HT. An additional pH optimum at 8.6 was present for 5-HT. A thermolabile form of the enzyme measured with dopamine and 5-HT, as well as a thermostable form measured with phenol, were present. Dichloronitrophenol (10(-5) M) noncompetitively inhibited the thermostable enzyme activity by 96% but decreased the thermolabile activity by only 36%. These studies provide the basis for a more accurate comparison of human platelet PST with the enzyme in the human brain and in other tissues.

Sulphate conjugation of p-hydroxytriamterene by platelet phenol sulphotransferase: assay conditions and correlation with metabolism in man.

1 Sulphate conjugation catalyzed by phenol sulphotransferase (PST) is an important pathway in the metabolism of many drugs including triamterene. Variations in PST activity in an easily obtained tissue such as the platelet might reflect individual differences in the sulphate conjugation in other organs and tissues. Human platelets contain at least two forms of PST, a thermolabile (TL) form for which dopamine is a substrate and a thermostable (TS) form for which low concentrations of p-nitrophenol serve as a substrate. 2 p-OH-triamterene, the major metabolite of triamterene, is conjugated with sulphate in vivo. p-OH-triamterene was a substrate for platelet PST with an apparent Michaelis-Menten value of 26 microM. Thermal stability studies indicated that p-OH-triamterene was a substrate for only the TS form of platelet PST. 3 When platelet homogenates from 29 individual subjects were tested, there was a significant correlation between PST activities measured with 4 microM p-nitrophenol and with p-OH-triamterene (r = 0.985, P less than 0.0001) but not between activities measured with dopamine and with p-OH-triamterene (r = 0.023, P greater than 0.2). These results confirmed that p-OH-triamterene was a substrate for only the TS form of human platelet PST. 4 The same 29 subjects were treated with 1 mg/kg of triamterene orally. 24-h urinary excretions of triamterene, p-OH-triamterene and p-OH-triamterene sulphate averaged 15.3%, 6.3% and 78.4%, respectively, of the total of triamterene plus measured metabolites excreted. The excretion of triamterene plus the two metabolites averaged 43.1 +/- 2.6% (mean +/- s.e. mean) of the ingested dose. There was not a significant correlation between the proportion of p-OH-triamterene excreted as sulphate conjugate and the activities of either the TS or TL forms of platelet PST activity.

Alpha-Methyldopa, alpha-methyldopamine an alpha-methylnoradrenaline: substrates for the thermolabile form of human platelet phenol sulphotransferase.

1 Sulphate conjugation catalyzed by phenol sulphotransferase (PST) is an important pathway in the catabolism of alpha-methyldopa (MD). Variations in PST activity in an easily obtained tissue such as the human platelet might reflect individual differences in the sulphate conjugation of MD in other organs and tissues. There are at least two forms of human platelet PST, a thermolabile form for which dopamine is a substrate and a thermostable form for which low concentrations of phenol can serve as a substrate. 2 MD, alpha-methyldopamine (MDA) and alpha-methylnoradrenaline (MNA) were tested as substrates for human platelet PST. All three were substrates for the thermolabile form of the enzyme and none were substrates for the thermostable form of PST. Apparent Michaelis-Menten (Km) values for MD, MDA and MNA were 5.5, 0.014 and 0.28 mM, respectively. Apparent Km values for 3'-phosphoadenosine-5'-phosphosulphate, the sulphate donor for the reaction, were 0.08, 0.13 and 0.10 microM, respectively, for the three catechol substrates. The pH optima for the reaction were 7.5 for MD and 6.5 for both MDA and MNA. 3 When platelet homogenates from 20 individual subjects were tested, there were significant correlations between PST activities measured with dopamine and those measured with MD, MDA and MNA (r = 0.54, 0.98 and 0.93, P less than 0.02, less than 0.001, and less than 0.001, respectively), but not between activities measured with low concentrations of phenol and those measured with MD, MDA and MNA (r = 0.021, 0.045 and 0.046, respectively). There results were also compatible with the conclusion that MD, MDA and MNA were substrates for the thermolabile form of platelet PST. 4 These observations will make it possible to test the hypothesis that variations in the activity of the thermolabile form of platelet PST may reflect individual differences in the sulphate conjugation of MD, MDA and MNA.

Comparison of amino acid sequence and thermostability of tyrosinase from three wild type strains of Neurospora crassa.

The thermostability of tyrosinase from three wild type strains of Neurospora crassa has been investigated. For this purpose a sequence comparison of two thermostable and one thermolabile tyrosinase isoenzyme was carried out. It revealed that at position 201 the thermostable enzyme forms share an aspartate residue in contrast to an asparagine residue in the thermolabile form. In addition, one of the thermostable isoenzymes displays five other substitutions. Since the relative stability of the thermostable forms as compared to the thermolabile one decreases with increasing ionic strength, the common aspartate residue is thought to bring about the additional stability of the thermostable isoenzymes by forming a salt bridge between aspartate 201 and a positively charged group of the protein. The strong pH-dependency of the thermostability with an apparent pKA of 6.6 indicates a histidinium side chain as the most likely ionic group to be involved in the salt bridge. This conjecture is also supported by measurements of the stability towards the chaotropic agent guanidinium chloride. The difference of the free energy change of denaturation delta GDH2O between the apoenzymes of a thermostable and a thermolabile isoenzyme was calculated as 2.5 kcal mol-1. Furthermore, it was shown that the copper ions of the native and the cobalt ions of Co(II)-substituted tyrosinase strongly enhance the stability of the protein as compared to its apoform.

Rapid cessation of phospholipid synthesis in fructose-1,6-diphosphate aldolase mutants of Escherichia coli.

Escherichia coli GH352, which was originally described as a temperature-sensitive strain containing a thermolabile acyl coenzyme A:monoacylglycerol 3-phosphate acyltransferase, does not now contain a thermolabile form of this enzyme. It has a defect in fructose-1,6-diphosphate aldolase and at least one additional temperature-sensitive lesion. Both strains GH352 and NP315, a temperature-sensitive aldolase mutant, show rapid cessation of 32-P1 incorporation into nucleic acids and phospholipids at 42 C. These characteristics of strain GH352 are therefore no longer attributed to thermolabile phospholipid synthesis, but can be attributed to the fructose-1,6-diphophate aldolase lesion.

Thermoadaptation of enzymes in thermophilic and mesophilic cultures of Bacillus stearothermophilus and Bacillus caldotenax

1. Bacillus stearothermophilus was adapted to 37° C (mesophilic culture) and to 55° C (thermophilic culture) by cultivation via an intermediate temperature of 46° C. In the crude extract of the thermophilic bacterial cells the glucokinase, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and isocitrate dehydrogenase are more thermostable than the corresponding enzymes in the crude extract of mesophilic cells. At the intermediate temperature of 46° C both types are probably formed. 2. 37° C-precultures of Bacillus caldotenax were further cultivated (in different samples) at 5° C intervals between 30° C and 70° C. It was shown that in 70° C-cells of the above mentioned enzymes more thermostable forms and in 37° C-cells more thermolabile forms are present. Furthermore, as demonstrated in the case of glucose-6-phosphate isomerase and isocitrate dehydrogenase, cells cultured in the temperature range between 30–50° C produced thermolabile enzyme variants (M-type), while cultures between 60–70° C produced thermostable variants (Th-type). At cultivation temperatures above 50° C a pronounced lag-period expressing the metabolic changes was found. In the lag-period, mesophilic enzymes are no longer present as early as 20 min after increasing the temperature (70° C), and synthesis of thermostable enzymes starts about 1 h before the beginning of growth. 3. Similar results were obtained with Bacillus caldotenax precultivated at 70° C and cultivated between 30° C and 70° C.

Separation and comparison of isoenzymes of [formula omitted] of pregnancy serum by polyacrylamide gel electrofocusing

Sera from normal and pregnant subjects as well as those from subjects with Tay-Sachs disease, hyperlipidemia and cancer were subjected to electrofocusing in the pH range of 3–10 in polyacrylamide gel (7.5%). The separated isoenzyme bands were stained for visualization on the gel for activity with α- naphthol-AS-BI-N- acetyl-β- D-glucosaminide as substrate. Elevation of N- acetyl-β- D-glucosaminidase (hexosaminidase) activity during pregnancy was found to be due to a progressive increase in number of isoenzymes: 1–2 bands in the early stage of pregnancy (5 weeks) to 6–8 bands in the last trimester. By comparison with the known isoenzymes of human aortic hexosaminidase, at least one of these bands corresponds to the A form, 4–5 bands to the P form and 1–2 bands to intermediate between the A and P forms. In the B form region only a single band with weak enzyme activity was observed, and its intensity of staining remained almost unchanged during pregnancy. Heat treatment (50° for 3 h at pH 4.4) resulted in inactivation of isoenzymes in the area on the gel of the A form and of the intermediates between the A and P forms but had no effect on the P and B forms. However, some bands found in the A area, between the A and P areas and in the P regions, transformed into forms having more alkaline isoelectric points (pIs) by this treatment. The new forms were found predominantly in the B area. The proportion of the thermostable (P and B) and the thermolabile (A) forms of the enzyme by such treatment was 32% and 68%, respectively, for normal serum, and 70% and 30%, respectively, for pregnancy serum. The value for pregnancy serum remained fairly constant throughout pregnancy (5 weeks to the last trimester). Tay-Sachs serum (one case) possessed three very minor bands corresponding to the P region and two major bands in the B region. When heat-treated, a partial transformation of minor bands and a complete transformation of one of the major bands in the B region occurred. One B band was transformed into the other B band having the most alkaline pI. Total hexosaminidase activity, meanwhile, did not show any change. Increase in number of isoenzymes of hexosaminidase in the P region was also found in sera of hyperlipidemic and cancer patients, suggesting that the P form may not be specific to pregnancy.

Human lens hexokinase

Clear crystalline lenses were obtained post-mortem from human prenates, neonates, young and old adults. Senile cataracts removed surgically were also studied. The specific activity of soluble fetal lens hexokinase is equal to that of the rat lens. There is a progressive decrease in specific activity as the lens size increases. In the fetal lens cortex and nucleus there is high hexokinase activity; young adult and cataractous lens nuclei are practically devoid of hexokinase activity. The epithelium and most superficial cortex possess high hexokinase levels even in the senile cataract. There is a large amount of insoluble hexokinase in the epithelium and most superficial cortex of the senile cataract. This can be solubilized with salts and detergents. No soluble or insoluble hexokinase is found in the nucleus of the senile cataract. Starch gel electrophoresis reveals the presence of Type I and probably also Type II hexokinase in the human lens. Types I and II were definitely found in the lens of the Rhesus monkey. There is a thermolabile form (presumably Type II hexokinase) and a thermostable form (presumably Type I) of hexokinase in the human lens. Michaelis constants were measured. In the fetal lens: K M glu = 0·11 m m, K M ATP = 0·74 m m; in the senile cataract: K M glu = 0·045 m m, K M ATP = 0·385 m m. There is great similarity between the hexokinases of the rat, rabbit and human lens. Further investigation employing lenses of these experimental animals is likely to yield results relevant to the human lens.