On the variation in enzyme activities during the growth of burkitts lymphoma cells in suspension culture

• 1.1. The growth characteristics of Burkitt's lymphoma cells in suspension culture have been studied, and a mean population doubling time of 20–21 hr established for this cell line under a range of nutritional and physical conditions; data which have provided a basis for the assessment of the reproducibility of the culture techniques and conditions which were employed in the subsequent studies.
• 2.2. Activities of lactate dehydrogenase (LDH), aldolase and esterase, as well as the cellular content of total soluble protein, and the isoenzyme pattern of LDH, were monitored in randomly growing Raji cells for the duration of a complete growth cycle.
• 3.3. In this period, the temporal pattern of variation in the levels of total soluble protein were seen to reflect alterations in LDH activity during a single growth cycle.
• 4.4. The fluctuations observed in LDH activity were greater than those observed for either aldolase or esterase activity, and, from the data considered, the maximum degree of variation appeared to be confined to the initial stages of growth.
• 5.5. Extracellular levels of LDH activity remained relatively constant throughout the growth cycle. so that the large fluctuations in intracellular LDH activity could not be attributed to either secretion or leakage of the enzyme into the culture medium.
• 6.6. No gross changes in the pattern of LDH isoenzymes in these Raji cells were detected during the course of a single growth cycle.

     

Temperature acclimation in amphibians: Changes in lactate dehydrogenase activities and isoenzyme patterns in several tissues from adult Discoglossus pictus pictus (Otth.)

• 1.1. The effect of cold (8 ± 2°C) acclimation on the lactate dehydrogenase activities and isoenzyme patterns from sartorius muscle, liver, heart and brain of adult Discoglossus pictus pictus (Otth.) was studied.
• 2.2. Two groups of animals were studied: one set of animals was trapped in October and another set in December. In both cases some of the animals were sacrificed upon collection and some others subjected to 5 months of acclimation at 8 ± 2°C before being sacrificed for analysis.
• 3.3. A general trend towards a decrease in LDH specific activity was observed during cold acclimation. The magnitude of change, but not the direction, depends on both the tissue examined and the season at which the experiment was initiated.
• 4.4. A complex LDH isoenzyme reorganization was also found in liver, heart and brain. In liver from Experiment 1 and in heart from both experiments, a relative maintenance in M-type LDH activity during cold acclimation was observed. However, in brain there was a relative maintenance of LDH₃ activity in both experiments.
• 5.5. The low behavioral activity (and its metabolic consequences) and the existence of an intrinsic annual rhythm in D. pictus metabolism are suggested as responsible for the observed enzymatic changes.

     

Separation of β-N-acetylglucosaminidase isoenzymes from liver and plasma of six mammalian species by DEAE-cellulose chromatography

• 1.1. The β-N-acetylglucosaminidase (EC 3.2.1.30) activities of human, pig, calf, lamb, rat and rabbit liver and plasma have been investigated.
• 2.2. All preparations had maximum activity between pH 4.0 and 4.5 and K_m values with the substrate 4-methylumbelliferyl-2-acetamido-2-deoxy-β-d-glucopyranoside ranged from 0.54 to 2.54 mM.
• 3.3. The isoenzyme profiles of liver and plasma β-N-acetylglucosaminidase activity were compared using DEAE-cellulose chromatography. In all species the major anionic component of liver (β-N-acetylglucosaminidase A) was eluted at a higher salt concentration than the most anionic plasma isoenzyme.
• 4.4. The plasma β-N-acetylglucosaminidase A isoenzyme of all species contained sialic acid residues whereas only the rabbit, pig and calf liver isoenzymes were sialylated.

     

Purification,isolation and characterization of a phosphoglycolate phosphatase isoenzyme from human erythrocytes

• 1.1. Preparation, purification and characterization of a phosphoglycolate phosphatase (PGP)3 isoenzyme from human erythrocytes was achieved by DEAE-Sepharose CL.-6B chromatography and isoelectric focusing using carrier ampholytes. pH 4–6.
• 2.2. The isoenzyme has an isoelectric point of 5.00 ± 0.05 and could be purified 33.000 fold to a specific activity of 32.7 U/mg of protein. It represents the PGP phenotype 1 consisting of a single isoenzyme.
• 3.3. The enzyme is composed of two subunits (mol. wt 35,000) which are identical and not connected by SS-bridges.
• 4.4. At 4°C the isoenzyme is more stable in the pH range of 7–9 than at acid pH values.
• 5.5. Incubation at 30 and 40°C for 4 hr does not affect the activity of the isoenzyme.
• 6.6. It has a K_m-value of 0.28 mM for phosphoglycolate (PG) as substrate.

     

Purification and characterization of camel liver glutathione S-transferase

• 1.1. Glutathione S-transferases have been purified (18-fold) in 65–70% yield from the liver of one humped camel using affinity chromatography on glutathione-linked agarose.
• 2.2. Chromatofocusing technique resolves the glutathione S-transferases into seven distinct isoenzymes with apparent pI of 8.7, 8.4, 8.0, 7.8, 7.3 and 6.5.
• 3.3. The major isoenzyme (pI 8.7) which accounted for over 95% of the total activity was composed of two identical subunits of molecular mass 24,000 and was immunologically similar to the other six isoenzymes.
• 4.4. The substrate specificities and the effect of various inhibitors on the activity of the abundant camel liver isoenzyme were also examined.

     

Preparation of two neurotoxic esterases from the chick central nervous system

• 1.1. A standard procedure for lipid-extraction of lyophilized hen brain material is decribed.
• 2.2. Nine carboxylesterase isoenzymes (EC 3.1.1.1) are identified in lipid-extracted lyophilized material (LELM) using kinetic analysis of organophosphate inhibition. Total phenyl valerate (PV) hydrolysing carboxylesterase activity in LELM is 43.3U.g⁻¹
• 3.3. Two carboxylesterase isoenzymes of LELM are classified as neurotoxic esterases (NTE_A and NTEg_B).
• 4.4. Using n-octylglucoside 51% of the water-insoluble neurotoxic esterase activity from LELM are solubilized.
• 5.5. Six carboxylesterase isoenzymes including NTE_A (6.5 U-l⁻¹) and NTE_B (4.2 U-l⁻¹) are present in the solubilized preparation.
• 6.6. Throughout purification and separation steps carboxylesterase isoenzymes are identified by their rate constants for the reaction with organophosphorus inhibitors.

     

NAD(P)H dehydrogenase from rabbit and rat liver: Purification and some properties

• 1.1. NAD(P)H dehydrogenase from rabbit liver was purified to electrophoretic homogeneity using a procedure also found applicable for the rat liver enzyme.
• 2.2. Rabbit and rat liver enzymes showed different behaviour in isoelectric focusing and different K_m values and turnover numbers.
• 3.3. Both enzymes were inhibited to similar extents by warfarin.
• 4.4. The rabbit enzyme is composed of two subunits of mol. wt 27,000 and contained 1 FAD group per subunit.
• 5.5. Some absorption and circular dichroism properties of the rat enzyme are shown.

     

Lactate dehydrogenase (LDH) in 27 species of amazon fish: Adaptive and evolutive aspects

• 1.1. Among the 27 species of Amazon fish belonging to the orders Rajiformes, Clupeiformes, Osteoglossiformes, Characiformes, Siluriformes and Perciformes here analyzed, 56% showed an electrophoretic pattern of five, 7% of four, 30% of three, and 7% of two LDH isozymes, suggesting the presence of both LDH-A¹ and LDH-B¹ loci. In addition to these loci, the third gene LDH-C¹ was detected only in the Osteoglossiform species O. bicirrhosum and in the perciform species P. squamosissimus, with a generalized expression in the first and a restricted in the second.
• 2.2. Only P. squamosissimus (Perciformes) showed a LDH reversed pattern, in which the A₄ is more anodic than the B₄.
• 3.3. Like other vertebrates, in most (93%) of the species here analyzed, a direct correlation between electrophoretic mobility and thermostability was observed. The inactivation temperatures varied from 55°C in the Rajiformes species of 70°C in the Perciformes species.
• 4.4. Polymorphism in at least one of the LDH loci was detected in 22% of the species studied here: P. castelnaena (Clupeiformes) and B.cf. cephalus (Characiformes) at the LDH-A¹ locus, R. myersi and H. unitaeniatus (both Characiformes) at the LDH-B¹ and L. agassizi (Characiformes) at both loci.
• 5.5. No modifications of the classic LDH pattern found by other authors in organisms routinely subjected to hypoxic stress were observed in these Amazon species. In 93% of the species screened here, subjected to considerable hypoxic stress, large daily oscillations in temperature, O₂ and CO₂ levels, pH, low ionic content, and seasonal drought, a bidirectional pattern of expression of the LDH loci was observed.

     

Lactate dehydrogenase in amoeba,Acanthamoeba sp. in different phases and conditions of culture

• 1.1. The activity and kinetic changes of amoeba LDH in different phases and conditions of culture were investigated.
• 2.2. LDH of the amoeba is specific against d(−)LDH irrespective of the hypoxic conditions created.
• 3.3. In hypoxic conditions it was not possible to visualize the presence of another LDH isozyme of muscle type by kinetic or electrophoretic analysis.
• 4.4. However, the changes in the K_m value and the L:H ratio as well as the decrease of electrophoretic mobility of LDH band indicate the change in kinetic properties of the enzyme from an obviously heart type in oxygenated culture in the direction of a muscle type LDH in strongly hypoxic culture conditions.
• 5.5. The influence of factors producing either environmental or metabolic hypoxia on possible repression or induction of LDH in amoeba is discussed.

     

The influence of ehrlich ascites tumour growth on the turnover of lactate dehydrogenase in tissues of the mouse

• 1.1. The influence of Ehrlich ascites tumour growth on the turnover of total soluble protein and lactate dehydrogenase (LDH) in mouse tissues has been studied.
• 2.2. Turnover parameters were determined by means of double-labelling technique, with the enzyme (LDH) being isolated by affinity chromatography.
• 3.3. Tumour growth was accompanied by a decreased rate of synthesis of total protein in all tissues.
• 4.4. Lactate dehydrogenase by contrast snowed an increased rate of synthesis in all tissues but kidney.
• 5.5. These directions of change, in combination with the lesser response of degradation constants, resulted in a consequent conservation of enzyme activity in all tissues except kidney.
• 6.6. A generalized shift in the LDH isozyme pattern of these tissues was also observed during tumour growth with an increased contribution of A-type subunit.
• 7.7. These results have been discussed in relation to the redirection of protein synthesis and degradation, the occurrence of foetal isozymes, and possible mechanisms involved in the redistribution of protein resources in the animal during tumour development.

     

Calcutta-1 LDH: Kinetic and thermodynamic properties of an electrophoretic variant of human LDH

• 1.1. Kinetic constants determined for the purified heterozygous variant LD₁ were closely similar to those of normal human LD₁.
• 2.2. Calcutta-1 homozygote LDH differed from normal LDH in K_m NADH and in Arrhenius activation energy.
• 3.3. The normal B subunits confer stability on the mutant subunits in the heterotetramers of Calcutta-1 LD₁.

     

Tissue specific isoenzymes of d-lactate dehydrogenase from the foot,mantle and hepatopancreas of Patella caerulea (L). purification and properties

• 1.1. Isoenzymes of d-lactate specific dehydrogenase from foot, mantle and hepatopancreas of Patella caerulea have been purified by Chromatographic techniques. d-lactate dehydrogenase (d-Ldh) from P. caerulea tissues was found to be tetrameric with a M_r of ca 140,000 as judged by gel filtration; subunit M_r of ca 37,000 was obtained from SDS-electrophoresis.
• 2.2. Kinetic studies suggest that P. caerulea foot and mantle d-Ldh is similar to vertebrate muscle-type l-Ldh; furthermore hepatopancreas d-LDH resembles vertebrate heart-type l-LDH since it has a higher affinity for d-lactate and is inhibited by pyruvate.
• 3.3. The results imply that the P. caerulead-Ldh isoenzymes may have distinct metabolic functions.

     

Purification and characterization of arylsulfatase from sea urchin (Hemicentrotus pulcherrimus) embryos

• 1.1. Arylsulfatase was extracted from sea urchin (Hemicentrotus pulcherrimus) plutei and purified to electrophoretical homogeneity by means of DEAE-cellulose, acetone fractionation and Sepharose CL-6B, successively.
• 2.2. The molecular weight of this enzyme was approx, 670,000. The molecular weight of a single subunit was approx. 63,000. The K_m value for p-nitrophenyl sulfate was 0.59 mM.
• 3.3. This enzyme was competitively inhibited by the sulfate ion and was classified as the type II arylsulfatase. The pH optimum was between 5.0 and 6.0.

     

Respiration in the eastern water dragon,Physignathus lesueurii (agamidae)

• 1.1. Some aspects of the gas exchange system of a diving lizard, Physignathus lesuewii were studied.
• 2.2. Breathing patterns were analysed.
• 3.3. Breathing rate increases logarithmically with temperature and Q₁₀ = 1.8. LogBR = −0.237 + 0.0256 T.
• 4.4. Gas tensions in lung air and arterial and venous blood were measured. Arterial pH declines with increasing temperature.
• 5.5. Temperature has a marked effect on oxygen affinity of the blood (ΔH = −10.1 kcal mol⁻). A Bohr effect was also noted.
• 6.6. CO₂ equilibrium curves were drawn.
• 7.7. The results are considered with a view to anticipating the efficiency of the gas exchange system of this species under conditions of variable temperature and during diving.

     

Direct adaptation of cells to temperature: Similar changes of LDH isozyme patterns by in vitro and in situ adaptations in Xenopus laevis

• 1.1. Cells of Xenopus laevis were cultured in vitro chronically at different temperatures and their isozyme patterns of lactate dehydrogenase (LDH) were compared with those of liver of temperature-acclimated toads.
• 2.2. Relative increase of cathodic isozyme by cold adaptation was observed both in two independent cell lines and in toad tissue.
• 3.3. This supports the concept that adaptation of cells to local body temperature would be responsible to the change in isozyme pattern by temperature adaptation of organisms.
• 4.4. Such changes would not be a direct result of elevated pO₂, judging from tissue distribution of isozymes.

     

Esterase heterogeneity in the oyster drill,Urosalpinx cinerea say (Prosobranchia: Muricidae)

• 1.1. Four regions of Urosalpinx cinerea foot muscle esterases were differentiated electrophoretically and partially characterized with the use of inhibitors and substrates.
• 2.2. The isozymes of Esterase II are inferred to be that of a dimeric molecule, each subunit encoded at a different locus, on the basis of their electrophoretic variants, tissue distributions and selective inhibition by PMSF.
• 3.3. Esterase IV, probably a diallelic polypeptide, is significantly heterogeneous for allele frequencies among the Connecticut and Virginia populations examined; this is probably associated with the limited larval dispersal pattern in this species.

     

Lactate dehydrogenase isozymes of the leopard danio,Brachydanio nigrofasciatus: their characterization and ontogeny

• 1.1. The tissue specific patterns and ontogeny of lactate dehydrogenase (LDH) are reported.
• 2.2. While all tissues (eye, brain, heart, intestine, liver, ovary and skeletal muscle) show isozymes of A and B subunit composition, only liver extracts possess isozymes resulting from C subunit synthesis.
• 3.3. The A₄ homopolymer appears simultaneously with initial muscle contractility and is correlated with the physiological function of muscular contraction.
• 4.4. The activation of the Ldh-C locus is correlated with the first functioning of liver. It is suggested that the state of differentiation of liver cells may be the stimulus required for C locus expression.

     

Shift in vivo in the lactate dehydrogenase isoenzyme pattern in experimental models conditioning the metabolic adaptation of rat brain

• 1.1. In order to analyse the possible shift in the lactate dehydrogenase isoenzyme pattern, experimental models affecting the glycolytic pathway are developed, including phenylalanine, thiamine and epinephrine administration, and a vitamin free diet.
• 2.2. The results obtained show that a shift in the lactate dehydrogenase isoenzyme pattern occurs in every change in physiological condition.
• 3.3. These shifts are in accordance with the kinetic properties of each isoenzyme and account for a metabolic adaptation to each physiological state.

     

The effects of temperature and eyestalk extracts on oxygen consumption of the crayfish Cambarus acuminatus (Faxon)

• 1.1. The pattern of VO₂ of 4 and 25°C acclimated Cambarus acuminatus was one of normal compensation with cold acclimation resulting in translation and clockwise rotation of the rate-temperature (R-T) curves.
• 2.2. Acclimation patterns were significantly influenced by the sequence in which crayfish experienced experimental temperatures.
• 3.3. Eyestalk extracts prepared from warm acclimated crayfish significantly decreased VO₂ in eyestalkless cold acclimated recipients.

     

Comparative dehydrogenase activity during the ontogenesis of the yellow fever mosquito Aedes (Stegomyia) aegypti L. (diptera:culicidae)

• 1.1. α-GPDH in most active in adults, LDH in third instar larvae, and MDH in third instar larvac.
• 2.2. During pre-pupal growth, LDH is the most active enzyme, followed by MDH and α-GPDH; while during post-pupal growth, MDH is most active followed by α-GPDH and LDH.
• 3.3. Increased enzyme activity is in response to changing physiological and physical environments, and is due to temporal activation of new gene loci indicated by the production of new α-GPDH and MDH isozymes. LDH activity is probably controlled by the temporal action of regulatory gene(s).
• 4.4. Dehydrogenase activity profiles during ontogenesis are probably species-specific.