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The extent of binding of glycolytic enzymes to the particulate fraction of homogenates was measured in sheep hind muscles after electrical stimulation. As compared to the control muscles, stimulation led to significant increases in the amount of phosphofructokinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase bound to the particulate fraction. The binding of other glycolytic enzymes was not significantly altered. A servey of different hind limb muscles at variable rates of stimulation revealed that each muscle exhibited its own characteristic response pattern in terms of the level of increased enzyme binding. Generally, an increased stimulation rate led to greater enzyme adsorption. The increase in enzyme binding was rapidly reversible for it was shown that the amount of enzyme bound quickly returned to control values when the muscles were allowed to recover in the live anaesthetised animal following cessation of stimulation. Those muscles which exhibited increased enzyme binding were characterised by a marked loss of glycogen and accumulation of lactate suggesting that accelerated glycolytic flux was a necessary condition for the observation of increased enzyme binding. In support of this, enzyme adsorption was observed to the greatest on stimulation of ischemic muscles, whereas in trained muscles, or muscles with depleted glycogen stores induced by prior adrenalin treatment, the increased enzyme binding response was greatly diminished. It is concluded that the variable binding of key glycolytic enzymes has a role to play in the regulation of glycolytic behaviour in skeletal muscle.

The ontogenic characteristics of lactate dehydrogenase (LDH) isozymes in mammalian pre-implantation ova have been reviewed. Evidence has been provided that the ova of mice and other mammalian species contain enzyme activity in a masked form, and display turnover processes which possess distinctive characteristics by comparison with those in adult tissues. Also, the extraordinary high levels of LDH in the extracellular secretion of the mammalian oviduct have been commented on, along with the influence of reproductive hormones on the activity and type of this enzyme. In addition, attention has been drawn to the unique characteristics of the oval micro-environment, and the influence which such factors may exert on the realization of enzyme phenotype during early mammalian development.

1. 1. The effect of the proteolysis of aldolase on both the substrate specificity of the enzyme and binding capacity for actin have been studied. 2. 2. Carboxypeptidase A, trypsin, chymotrypsin and pepsin, all acted to cleave peptides from the C-terminal portion of the enzyme, resulting initially in a marked loss of activity towards fructose-1:6-bisphosphate (FBP), without impairment of activity towards fructose-1-phosphate (F1P). In some cases, however, further proteolysis caused reductions in activity with F1P as well. 3. 3. By correlating the size of the peptide fragments released by these enzymes with the known sequence of aldolase, evidence has been provided that cleavage of His-359 and/or Tyr-361 lead to the loss of FBP activity, while further cleavage of up to six amino acids begin to affect activity against F1P, as well. 4. 4. In regard to the ability of the proteolysed aldolase to bind to F-actin, it was evident from these studies that binding ability was not impaired in the initial stages of proteolysis referred to above, but was retained until the enzyme was extensively degraded. This differential behaviour of the active and binding sites on aldolase clearly establish their separate topographical localization. 5. 5. These results have been discussed in relation to the positioning of these separate sites on the enzyme, the nature of the interaction between aldolase and actin and the phenomenon of enzyme ambiquity in cells and tissues.

1. 1. The inhibition of the proteolysis of a number of purified enzymes by a beef liver cytosol fraction has been investigated. 2. 2. The tryptic and chymotryptic digestion of catalase, aldolase and pyruvate kinase was inhibited to various degrees by beef liver cytosol. with the most marked protection occurring for aldolase. 3. 3. Malaie dehydrogenase and lactate dehydrogenase were offered no additional protection from trypsin or chymotrypsin above that exhibited by bovine serum albumin. 4. 4. With respect to pronase inactivation of these enzymes, the cytosol fraction offered little significant protection. 5. 5. The significance of these data has been discussed in terms of the regulation of the degradative processes for specific proteins in mammalian tissues.

1. 1. The degradation of tritiated catalase. which had been purified from mouse liver, was studied under in vitro conditions by incubating the enzyme with subcellular fractions from the same tissue. 2. 2. The degradative process was monitored by following both the loss of catalase enzymatic activity and the formation of soluble radioactivity. 3. 3. Only the large granule fraction caused appreciable degradation of catalase at neutral pH and 37°C, but under conditions of lower pH the degradative rate due to this fraction increased markedly. 4. 4. Additionally, it was noticed that the cytosol fraction of liver slowed the rate of heat inactivation of the purified enzyme, and that a similar inhibition of inactivation by cytosol also extended to the tryptic inactivation of the enzyme. 5. 5. These results have been discussed in relation to the established characteristics of the degradation of mouse liver catalase in vivo , and possible means of regulation of this degradation

The role of catalase in lipid metabolism has been studied by means of a comparison of the turnover characteristics of the major lipid classes in the normal mouse with those of animals in which the catalase activity had been inhibited and blocked by aminotriazole and allylisopropylacetamide. Double isotope ratios were determined in the lipid fractions of several tissues following the injection of labeled glycerol, and a number of significant differences were identified between these treatments. Since catalase is recognized as an integral component of the peroxisomal pathway of fatty acid oxidation, these results may be taken as indicating that interruption of the process of peroxisomal beta-oxidation in this manner cause extensive perturbations of lipid metabolism in the living animal, and these perturbations extend well beyond those tissues where the predominant localization of these organelles occurs. The concept which derives from these data--that of a significant regulatory role of peroxisomes in relation to the overall balance of lipid metabolism in the animal body--is described and discussed.

1. 1. An affinity chromatography technique for the determination of turnover parameters has been utilized for the derivation of new data for lactate dehydrogenase and its isozymes in a wide variety of tissues from female rats. 2. 2. This methodology allowed the establishment of relative turnover characteristics in all tissues examined, and the evaluation of rate constants for synthesis and degradation and half-life values in the majority of these enzyme sources. 3. 3. Considerable variation was evident in the turnover characteristics of total protein and lactate dehydrogenase in the different tissues, with the synthesis of the enzyme being highest in reproductive tissues, heart and liver, and degradation most prominent in reproductive tissues. 4. 4. With regard to the isozymes, marked differences in half-lives were evident not only between the same isozyme in different tissues, but also between the various isozymes in any one tissue. 5. 5. Bi-directional trends in the half-life values for the isozyme sequences in several tissues, indicated a distinctive contribution to turnover from the different cell types present. 6. 6. These data have been discussed in relation to the available comparisons in the literature, and the known physiological correlations of lactate dehydrogenase in these tissues.

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.

1. 1.The suitability of a combined application of the techniques of affinity chromatography, double labelling and gel electrophoresis in the determination of the turnover characteristics of the lactate dehydrogenases isoenzymes (l-lactate:NAD+ oxidoreductase EC 1.1.1.27) in rat tissues has been studied. 2. 2.Affinity chromatography was established as affording the advantages of rapidity, high yield and purity to such studies, and the double-labelling procedure was modified to encompass the differential decay kinetics in the separate rat tissues. In addition, a convenient method for the resolution and separate collection of radioactively labelled isoenzymes has been described. 3. 3.Using this methodology, comparative turnover values for the isoenzymes of lactate dehydrogenase and for total soluble protein have been determined. 4. 4.The comparability of these results with other methodologies, and the advantages of this approach in facilitating broad comparative studies on turnover are discussed.

Recent studies of lipid turnover in living mammals support the concept that peroxisomes have a significant physiological role in the metabolism of neutral lipids. These findings also raise intriguing questions about the influence of peroxisomes on phospholipid catabolism, the biosynthesis of complex lipids, and inter-tissue and inter-compartmental communication.

1. 1. As an extension of a previous line of research, and in order to provide detailed information on the developmental characteristics of lactate dehydrogenase (EC 1.1.1.27), the properties of this enzyme have been studied over a representative range of intervals during the gestation and post-natal maturation of the cat and guinea pig. 2. 2. These animals occupy significant positions in relation to the available data in other mammalian species, and the results have enabled a number of informative comparisons to be drawn in regard to the morphogenetic processes and the epigenetic control of lactate dehydrogenase biosynthesis in mammals. 3. 3. In particular, the relative ratios of monomer types (A and B) of this enzyme prior to the initiation of funtional differentiation, have been shown to bear a limited phylogenetic inter-relationship. Species within the same sub-order possessed values of a similar magnitude, and a broader correspondence existed between the ratios in related sub-orders. The significance of this phenomenon is discussed in relation to the control of isoenzyme synthesis. 4. 4. Additional information has been provided on the rolel of oxygen and other factors in determining the properties of tissue lactate dehydrogenase in the pre- and post-natal situations.

1. 1. The relative incorporation of 14 C-labelled fatty acids by ovine liver and adipose tissue has been studied under a variety of conditions. 2. 2. In liver incorporation was mainly in the form of free fatty acid, whereas adipose tissue showed greatest incorporation into glycerides. 3. 3. Desaturation of stearate and endogenous synthesis of oleate were both markedly increased by comparison with the available data for monogaslric animals, and would seem to provide an explanation for the occurrence of high concentrations of oleate in ovine tissues

1. 1. The multiple forms of esterase activity have been studied in the tissues of eight representative invertebrate species: the sea anemone, the liver fluke, the round worm, the segmented worm, the king prawn, the garden snail, the chiton and the sea cucumber. 2. 2. These esterases have been characterized in terms of their relative mobilities on polyacrylamide gel electrophoresis, substrate and inhibitor specificity, molecular weights and physicochemical properties. 3. 3. In the representative of the phylum Coelenterata, the sea anemone, eleven esterase multiple forms were characterized and included carboxylesterases and cholinesterases. In the liver fluke (phylum Platyhelminthes) eight esterases were distinguishable. All of these were again carboxylesterases and cholinesterases. 4. 4. Thirteen multiple forms of esterases were found in the round worm of the phylum Nematoda, while only ten were present in the representative of the phylum Annelida, the earth worm. Both these species contained carboxylesterase, cholinesterase and arylesterase forms. 5. 5. The most complex system was found in the tissues of the king prawn (phylum Arthropoda) with twenty-nine isoenzymes. In the garden snail, eighteen isoenzymes were found, but in the marine mollusc, the chiton, only eleven forms were distinguishable. All these species contain the three types of esterases classified in the round worm and the earth worm. 6. 6. In the sea cucumber, from the phylum Echinodermata, only five carboxylesterases were found. 7. 7. These results have been discussed in relation to the phylogenetic variability of the esterases, and the complex structural and genetic inter-relationships of these multiple enzyme forms.

The applicability of the methodology of disc electrophoresis to the determination of the component molecular weights of complex protein mixtures has been investigated. To this end, variable factors in the formation of polyacrylamide gel columns have been studied, along with their influence on the determination of relative mobilities. The effect of gelling time was demonstrated as a major factor in this regard, and the influence of catalyst and monomer concentration on this parameter defined. Migration of proteins of established amino acid composition at different pH's has been shown to vary in a parallel fashion to the alteration in charge of the protein. As a result of these investigations, the adoption of a standardized set of conditions which allow for the accurate estimation of the molecular weights of the protein components of biological systems has been made possible, a method that seems capable of wide application in assessing protein interrelationships.

The incorporation of 14C-labelled myristic, palmitic, stearic, oleic and linoleic acids in vitro into the lipids of bovine spermatozoa was measured at intervals from 2min to 2h. All acids were rapidly incorporated into diglycerides, myristic acid being metabolized to the greatest extent. Whereas the low incorporation of acids into total phospholipids reflected the relative stability of the major phospholipid fractions in sperm, the minor phospholipids, particularly phosphatidylinositol, showed comparatively high metabolic activity. Although, in general, saturated acids were incorporated more actively than unsaturated substrates, stearic acid was poorly incorporated into all lipids except phosphatidylinositol. In regard to fatty acid composition of sperm lipids it was notable that diglycerides contained myristic acid as the major component, and this acid was also a prominent moiety of phosphatidylinositol. Docosahexaenoic acid was the principal fatty acid of the major phospholipid classes. These findings have been discussed in relation to the role of lipids in the metabolism of spermatozoa.

1. 1. The effect of rumen development in the lamb on the in vivo metabolism of stearic-1- 14 C, oleic-1- 14 C, and linoleic-1- 14 C acids has been investigated. 2. 2. Calculations of the total incorporation of these fatty acids into individual tissues in the whole animal showed vastly increased incorporation after rumen development was completed, even though isotope concentration in the tissue lipid, in general, decreased. In the very young lamb, much of the free fatty acid was apparently oxidized. 3. 3. There appeared to be an increased uptake of stearate into triglyceride during rumen development, but the bulk of the incorporated fatty acid was present as phospholipid at all ages. 4. 4. The individual fatty acids were incorporated to different extents into the different tissues. In addition, incorporation into the principal lipid fractions varied with the acid. 5. 5. The metabolism of injected [ 14 C] tristearin and triolein was compared with the results for free fatty acids. It was found that incorporation into liver, heart, lung, spleen, and adipose tissue was greater for triglycerides. However, adipose tissue incorporation at 10 weeks of age was still low.

The multiplicity and subcellular distribution of liver catalase activity have been studied in 10 species of mammals, in order to clarify comparative aspects of the properties of this enzyme. All mammals studied, except mouse, displayed the bulk of liver catalase activity in the cytosol, but the relative proportions of soluble and particulate activity varied quite markedly between species. Sheep, pig, and guinea pig provided the richest sources of supernatant liver catalase; while mouse, rat and pig contained the highest peroxisomal catalase activity. Rabbit, mouse, and rat liver supernatants exhibited 3–4 multiple forms of catalase activity, and displayed an electrophoretic pattern which was distinctly different to that of the aqueous peroxisomal extract. This latter fraction contained a single form of activity of higher anodal migration; a pattern of which was similar to that observed in all fractions of the other species examined. These results may be reconciled with current knowlledge of catalase genetics, subunit structure, and turnover by means of a postulated epigenetic mechanism, involving modifications of the enzyme in rabbit, rat, and mouse liver cytosol.

1. 1. The incorporation of [I-14C]myristic, -palmitic, -stearic, -oleic and -linoleic acids in vitro into the lipids of bovine testicular tissues was measured after incubation for 2 hours. Tissue slices from the testes of adult, 4-month and 2-week-old animals were used. 2. 2. The mature testis incorporated less of each substrate than immature testes, but each tissue incorporated linoleic acid to the greatest extent. 3. 3. Phosphatidylinositol and diglycerides were metabolically more active than other major phospholipid and neutral lipid fractions in the mature testis, and the metabolism of cholesteryl esters was also significant at this stage. 4. 4. With the immature testes a more general metabolism was apparent, reflecting the lack of functional specialization in these tissues. 5. 5. Incorporated linoleic acid was readily desaturated, with maximal desaturase activity occurring in the testis tissue from the 4-month-old calf. Fatty acid incorporations were not related to the endogenous fatty acid concentrations of testis lipids. 6. 6. The lipid fractions of all tissues contained very little docosapentaenoic acid, but considerable amounts of docosahexaenoic acid, particularly in the ethanolamine phosphoglycerides. Within the phospholipids, docosahexaenoic acid content increased with maturation, whereas arachidonic acid content decreased. PIasmalogenic aldehydes were more saturated in character in the mature testis.

1. 1. As part of a comparative study of lipid metabolism, the fatty acid uptake of bovine spermatozoa has been investigated. 2. 2. A very active incorporation of labelled fatty acids into the individual lipids of bovine spermatozoa was evident, in general, but the extent of this uptake was shown to vary with the different individual acids. 3. 3. Phospholipid shows greater incorporation than diglycerides initially, but within minutes diglyceride incorporation predominates. 4. 4. Uptake into phospholipids is usually directed towards phosphatidylcholine principally or the corresponding plasmalogen. 5. 5. Fatty acid analysis of the lipids of bovine spermatozoa reveals that myristic acid is the major component in the diglycerides.

1. 1Multiple forms of soluble esterase activity have been resolved in horse, sheep, ox and possum tissue extracts and sera. 2. 2.By comparing esterase zymograms from different tissues and from different species, it is apparent that the distribution and multiplicity of esterase activity is tissue and species specific. 3. 3.By means of substrate and inhibitor studies, the esterase multiple forms have been characterized into four main classes: carboxylesterases, arylesterases, acetylesterases, and cholinesterases. Each of these can be considered as an isoenzymic group. 4. 4.Evidence is presented for further differentiation of activity within these isoenzymic divisions giving 2 groups of arylesterases, 3 groups of cholinesterases, and 5 groups of carboxylesterases.

1. 1. The properties of the multiple enzyme forms of soluble esterase activity have been studied in three non-mammalian species-lizard, frog and catfish. 2. 2. The resultant data has enabled a review and classification of esterase heterogeneity over a representative range of vertebrate phylogeny. 3. 3. Four main classes of esterase activity have been characterized (carboxyl-esterases, arylesterases, acetylesterases and cholinesterases), but evidence for further differentiation of activity-types within these groups is also presented. 4. 4. Considerable molecular variation is evident between species and a late evolutionary development of some esterase types (e.g. acetylesterases, group I carboxylesterases and cholinesterases) seems indicated.

Summary 1. In this review, representative data on the nature of enzyme multiplicity and the developmental progressions of multiple enzyme forms have been collected, and the significance of this material has been discussed in relation to gene involvement during tissue differentiation. 2. In addition, emphasis has been directed towards the relevance of this onto-genetic information to a wide variety of biological phenomena, such as the possibilities of metabolic advantage conferred by the distinctive properties and physiological locations of these multiple enzyme forms, the insight into the subunit structure and compositional interrelationships of proteins, the aetiology of disease states, the relationship to hormone action, the extra nuclear specification of enzymes, and the transformations between particulate and soluble enzymes. 3. Three isoenzyme systems in particular have been chosen for the special development of these themes and treated in some detail; lactate dehydrogenase (LDH), aldolase and the esterases. Briefer accounts of the ontogeny of other less-studied series of multiple enzyme forms have also been included. 4. In relation to LDH, the major findings and conclusions of the pioneering investigators have been outlined, together with the reassessments which have been necessitated by recent studies of LDH ontogeny. It is evident, for example, that the early embryonic form of this enzyme is not necessarily parental as previously thought, and the realization appears to possess wide-ranging implications in regard to the control of synthesis of the enzymes. An indirect role of oxygen in LDH biosynthesis is indicated together with the independent regulation of the separate genes. 5. Recent developments in the enzymology of preimplantation ova have been discussed. It is noteworthy that the extraordinary levels of LDH activity which have been reported to be associated with these early developmental stages, may arise by extracellular adsorption rather than by an intrinsic synthesis of enzyme. Hence contemporary interest in this area has shifted towards a comprehension of the biochemical significance of the extremely unusual, selective concentrations of extracellular lactate dehydrogenase in the oviduct, the relation of this enzyme to the specialized growth requirements of the ova, and the influences of reproductive hormones on these oviducal isoenzymes. 6. The role of aldolase ontogeny as well as the composition, gene control, and native state of these lysases is also reviewed. This enzyme provides a particular example of the manner in which tissue modification of the catalytic and structural properties of enzymes may influence interpretations of the native state of purified proteins, and in this case, also, the observable microheterogeneity satisfies previous disquietude in regard to the existence of different subunits in the muscle enzyme. 7. The basic developmental importance of aldolase-C is stressed by the occurrence of this form of the enzyme in the early embryos of many different species. Furthermore, a significant addition to our knowledge of the possible biological purposes of isoenzymes and the rationale of their ontogenetic development during the establishment of tissue identity would appear to follow from the recent studies on reversible adsorption of aldolase to cell membranes. Aldolase-C. for example, is differentially inhibited in this way by the end product of anaerobic glycolysis, and other such metabolite-isoenzyme-tissue interactions allow for a wide range of tissue-specific responses regulating energy derivation and other important functions of the cell. 8. The remarkable extent of heterogeneity among the esterases, the distinctive species-specific nature of their multiplicity, and the overlapping substrate specificities have long presented a formidable barrier to the unravelling of the isoenzyme status of these enzymes. The advantages of systematic approaches utilizing differential substrate and inhibitor specificities have been emphasized, and the most recently available information summarized. 9. The carboxylesterases may be divided into five distinct groups, and recognizable subdivisions also occur within the other classes of esterases. Most of the different possibilities of isoenzyme causation are exemplified within the esterases, and the ontogenetic specificity of their heteromorphs seems to be greater than for any other recorded isoenzymic system. 10. In addition to the generative features displayed by the ‘soluble’ enzyme forms, the potential utility of latent isoenzyme activities have been illustrated by reference to catalase. The properties of such systems provide a useful means of studying such problems as the subcellular specification of enzyme synthesis and the nature of the mechanism governing the distribution of specific proteins between organelles and cytosol. 11. Catalase also provides a situation of special interest in that the existence of isoenzymes and even of activity in the cytosol have been disputed until very recently. Evidence establishing the reality of these occurrences, and the epigenetic nature of catalase multiplicity in some species, has been cited. 12. Recently available ontogenetic information of a number of oxidoreductases, transferases and hydrolases has also been collected together and discussed.

A purified arylesterase preparation from bovine plasma was characterized to the extent that it has a partial specific volume of 0.91ml/g and an apparent z-average molecular weight of 440000. The relatively large magnitude of the former reflects the presence of phospholipids, cholesterol, triglycerides and beta-carotene, the last-named being responsible for the pronounced yellow colour of the preparation. Removal of the lipid material is accompanied by a decrease in the apparent z-average molecular weight to 120000, the size of the smallest species detected by high-speed sedimentation equilibrium being in the vicinity of 70000 daltons: denaturation of the lipid-free preparation with 6m-guanidine hydrochloride caused essentially complete breakdown into subunits of this size. In kinetic studies on the enzyme the maximal velocity for the hydrolysis of phenyl acetate was found to increase by 60% on addition of 1 mm-Ca(2+), with the K(m) showing a concomitant decrease from 6.6 to 2.1 mm. Removal of lipid had no detectable effect on V(max.) or K(m) in either the presence or the absence of Ca(2+). It is concluded that the bovine plasma arylesterase preparation is either a lipoprotein or an enzyme-lipoprotein complex with properties very similar to those of the alpha(1)-lipoprotein or high-density lipoprotein (HDL(2)) fraction of serum.

1. 1. The latency, multiplicity, and subcellular distribution of catalase activity have been studied in a variety of mammalian tissues, in order to clarify comparative aspects of the localization and isoenzyme status of this enzyme. 2. 2. Much of the catalase activity was found to be of a soluble nature, but the proportion of soluble contribution to the total catalase activity varied between species and between different tissues. Sheep liver displayed an especially high proportion of activity in the soluble form. 3. 3. The comparative efficiency of a number of solubilization procedures was studied, and blending with 0.05 per cent Triton X-100 was demonstrated as the most efficient means of releasing paniculate catalase activity. 4. 4. Representative enzymes, resulting from this solubilization procedure, were resolved into multiple forms by high-voltage starch-gel electrophoresis. In this way, evidence was provided for a complex heterogeneity of the enzyme, and for a differential distribution of the multiple enzyme forms.

Changes in the properties and distribution of LDH isoenzymes have been studied in the tissues and fluids of the reproductive tract during the oestrous cycle and during postnatal development in the rat. The specific activity values of the tissue LDH over this period indicate a sequential response along the length of the tract. In the extracellular secretions of the tract, peak activities coincide with, or closely follow, peak values in adjacent tissues and isoenzyme patterns tend to bear a reciprocal relationship between individual sections of the tract tissues and the enclosed secretions. The high levels of extracellular activity occurring within the oviducal secretion appear to be the result of contributions from the circumjacent tissues along the length of the tract, the Fallopian tube being indicated as an especially rich source. Hormonal and developmental influences on the synthesis of LDH isoenzymes are discussed in relation to the changes observed.

1. 1. As an extension of previous comparative studies, the ontogeny of amphibian aldolase has been studied. 2. 2. Unusual features of aldolase multiplicity in the frog include the broad tissue distribution of aldolase C, the atypical mobility of aldolase B, and the low substrate analogue ratio of aldolase A. 3. 3. The results also comment on the relative phyletic divergence and ontogenetic significance of the aldolase activity types.

1. 1. The incorporation of saturated and unsaturated 14 C-labelled fatty acids into the erythrocytes of sheep and cattle has been studied under a variety of in vitro conditions. 2. 2. Total incorporation was unaffected by factors such as incubation period, concentrations of glucose, adenosine triphosphate, coenzyme A, l -α-glycero-phosphate and oxygen; but uptake was dependent on the concentration of carrier acid and albumin. 3. 3. Preferential esterification of unsaturated fatty acids was observed with phospholipid being the main initial fate. 4. 4. From these data, it is concluded that fatty acid uptake in these species involved a physical binding predominantly and a secondary esterification process.

1. 1. The developmental, physicochemical and molecular weight properties of 14 soluble esterases of green turtle tissue extracts have been studied. 2. 2. Eight arylesterases and five carboxylesterases were resolved but cholinesterase existed as a single species. At least 5 structural genes would appear to be implicated in the synthesis of these esterase forms, as three distinct groups of arylesterases were differentiated on the basis of the physicochemical and developmental parameters utilized. 3. 3. During early development, the A and B genes responsible for lactate dehydrogenase synthesis were both active. Subsequent developmental studies showed two periods of peak enzyme activity, preceding regular change to adult distribution. 4. 4. The distribution and developmental aspects of multiplicity were not as complex as those of animals higher on the evolutionary scale, and apparently reflect a simpler form of genetic control of this stage of phylogeny.

1. 1. In order to assess the immunological inter-relationships of the vertebrate esterases, antisera were produced against several purified enzymes from the different esterolytic classifications, namely sheep liver carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1.), horse serum cholinesterase (acylcholine acyl-hydrolase, EC 3.1.1.8), ox testis acetylesterase (acetic-ester hydrolase, EC 3.1.1.6) and ox serum arylesterase (acyl-ester hydrolase, EC 3.1.1.2). 2. 2. These antisera were tested for their interactions at the level of enzyme heterogeneity, by gel electrophoresis of a representative range of tissues before and after incubation with the sera, followed by observation of the alteration in the patterns of esterase heteromorphs consequent upon this treatment. 3. 3. From these results, it appears that a close immunological relationship exists between the multiple forms of each individual esterase type within a single species. In the case of the acetylesterases, this immunological relationship extends between the species as well. A close relationship was also observed between chicken liver carboxylesterase and horse serum cholinesterase, and to a lesser degree between these two latter enzymes and sheep liver carboxylesterase. 4. 4. In addition to the electrophoretic studies, complement fixation inhibition assays were carried out between each of the antisera and all the purified antigens. Also, micro-complement fixation assays between the antisera to the chicken liver carboxylesterase and horse serum cholinesterase were effected with all the antigens. These immunological tests with pure enzymes served to verify that a degree of relationship exists between the hose cholinesterase and the chicken carboxylesterase. 5. 5. The significance of these results has been discussed in relation to the phylogenetic variability of the esterases, the complex structural and genetic inter-relationships of these multiple enzyme forms, and the comparative situation in intensively studied isoenzyme systems.

Although cytosolic enzymes are known to contribute significantly to the metabolic potential of the cell, it is becoming increasingly clear that their activities are modified by interactions with structural components of the cell. Specification of these interactions has been limited in the past by the availability of suitable experimental techniques, but studies with purified muscle proteins and new methodologies have advanced an understanding of these processes, and posed some interesting questions for the future.

Previously reported results of equilibrium-partition experiments on the interaction of aldolase with actin-containing filaments [Walsh, Winzor, Clarke, Masters & Morton (1980) Biochem. J. 186, 89-98] have been subjected to a more rigorous theoretical analysis involving consideration of the consequences of cross-linking interactions between enzyme and filament. The experimental results obtained with F-actin-tropomyosin are best described by a model with one binding site per heptameric repeat unit of filament and a value of 39000 M-1 for the site binding constant, k. Similar analyses of the influence of Ca2+ on aldolase binding to F-actin--tropomyosin--troponin substantiate the existence of two equivalent binding sites (k = 14900 M-1) for the enzyme on each repeat unit of the thin filament. The Ca2+-sensitivity of this interaction reflects either a decrease in the strength of aldolase binding to these two sites (k = 8200 M-1) or the elimination of one site.

The kinetic parameters of fructose bisphosphate aldolase (EC 4.1.2.13) were shown to be modified on binding of the enzyme to the actin-containing filaments of skeletal muscle. Although binding to F-actin or F-actin-tropomyosin filaments results in relative minor changes in kinetic properties, binding to F-actin-tropomyosin-troponin filaments produces major alterations in the kinetic parameters, and, in addition, renders them Ca2+-sensitive. These observations may be relevant to an understanding of the function of this enzyme within the muscle fibre.

Research into cellular regulation engenders a basic dilemma: although analytical accuracy may be improved by using purified components, such approaches often proceed at the cost of physiological credibility. Some of the classical concepts of control require reexamination in the light of the realities of the microenvironment.

Lactate dehydrogenase (E.C.I.l. 1.27) exists in most mammalian tissues as five molecular forms, whose subunit composition may be represented by the sequence Ad, A3B, A,B,, AB3, B, where A and B are different polypeptides. During embryonic maturation, differential gene function is reflected in the redistribution of activity amongst the multiple forms of this enzyme, and these developmental progressions have been widely utilized in studies of tissue differentiation, and the control of enzyme synthesis in vertebrate systems [l-3]. Among the most salient observations which have been made concerning the ontogeny of this enzyme in recent years, were the reports of extremely high lactate dehydrogenase activities of the B-type which were associated with mammalian ova in the preimplantation stages [4], and the subsequent abrupt decrease in this activity and change of sub-unit type which coincided with uterine attachment [S, 61. These findings held quite dramatic implications in developmental biochemistry, and provided new insight into the mode of energy utilization of these early embryonic stages. Nevertheless, there remained several puzzling features of the reported phenomenon, in terms of the relative stability of the different subunit types, and the control of synthesis of this enzyme, for instance. The present communication describes a reexamination of the early ontogenetic properties of lactate dehydrogenase in the mouse, and reveals a disposition of the enzyme which casts new light on the role of lactate dehydrogenase in early ontogeny.

A comparative study has been carried out on the micro-localization of catalase in mouse tissues subsequent to treatment with a representative range of hypolipidemic drugs. A commonality of effect was shown by clofibrate (ethyl-alpha-p-chlorophenoxyisobutyrate), Wy-14,643 (4-chloro-6-[2,3 xylidino)-2-pyrimidinylthio] acetic acid), RMI-15,414 (5-tetradecyloxy-2-furancarboxylic acid) and aspirin (acetyl salicylic acid), in that treatments with each of these drugs was associated with the release of peroxisomal catalase into the cytoplasmic compartment of liver and kidney. It was also noticeable that this increased cytosolic activity was characterized by the presence of an 'aged' form of the enzyme with different mobility and activity characteristics to that of the peroxisomal enzyme. Possible molecular bases for these effects and their relationship to peroxisomal biogenesis are discussed.

1. 1. In an extension of previous studies which demonstrated the existence of an association of glycolytic components in extracts of rat skeletal muscle, the properties of this complex have been further investigated. 2. 2. It has been shown that this association of glycolytic enzymes may also be observed in extracts of ovine muscle under physiological conditions of pH and ionic strength, and that the association is sensitive to variations of pH and the concentrations of proteins and specific metabolites such as ATP and fructose 1,6-diphosphate. Furthermore, myosin was identified as a necessary component for complex formation under the experimental conditions. 3. 3. The size of the observed complex of glycolytic enzymes increases with increasing protein concentration; at 52 mg protein/ml, s 20,w values of over 100 are indicated. 4. 4. These data have been discussed in relation to the possible physiological significance of the multi-enzyme association.

1. 1. The hydrolytic cleavage of purified mouse liver catalase by a number of proteinases has been shown to be subject to inhibition by a liver cytosol fraction. 2. 2. It has also been demonstrated that the inhibitory function afforded by the liver cytosol is a characteristic of the tissue per se, and not due to contamination by blood proteinase inhibitors. 3. 3. Of the hydrolytic enzymes tested, inhibition is most apparent against the trypsin-like proteinases; however, liver cytosol has little inhibitory capacity against tryptic hydrolysis of the synthetic trypsin substrate, benzoyl arginine—para—nitroanilide hydrochloride. 4. 4. Inhibition of proteolysis was not specific for catalase as substrate. Pyruvate kinase, for example, was protected to a similar extent against tryptic digestion in the presence of mouse liver cytosol, and other enzymes were also afforded a degree of protection. 5. 5. Investigation of the characteristics of the liver cytosol inhibitor indicated that the factor is evidently a high molecular weight protein or protein aggregate. 6. 6. The significance of these data has been discussed in terms of the regulatory process involved in the degradative processes of catalase and other enzymes.

In interpreting the developmental progressions of isozymes and the influence of cellular factors on these phenotypic expressions, increasing attention is being directed to the contribution of particulate interactions. In this paper, the aldolase isozyme system has been examined, and evidence of a differential binding of the isozymes in rat brain provided. The adsorption process was shown to be reversible, of an electrostatic type, and to involve the active site region of the enzyme. In pursuing the microlocalization of this isozyme binding, a particular membrane type within the microsomal fraction was identified as the main site, and the characteristics of the adsorption were detailed.

1. 1. The double isotope ratios of individual lipid fractions in major tissues of mice were determined before and after a period of total dietary deprivation. 2. 2. Several significant alterations in these ratios were caused by this treatment, with a marked individuality evident in the response of separate tissues. 3. 3. The increased metabolic flux overall was contributed to by increased rates of lipid degradation in liver, and active mobilization from the peripheral depots. 4. 4. Significant utilization of phospholipid fractions for energy purposes was also noticeable, however. along with tissue specific variation in the patterns of both double isotope ratios and specific radioactivities. 5. 5. These data are discussed in relation to the comparative priorities of synthesis and degradation in the different tissue and lipid sources in response to this physiological perturbation.

1. 1. Catalases in the tissues of six different mammalian species were examined, as well as in a mutant strain of mouse. 2. 2. Considerable variation was observed in regard to total activity, heat stability, urea stability and susceptibility to inhibition of catalase between the individual mammalian species. 3. 3. These results stress that considerable variation in the physicochemical properties of an enzyme can take place even amongst animals that are closely related phylogenetically, and suggest that the heterogeneity observed for the rat and the mouse may reflect an evolutionary mechanism by which these animals have compensated for the increased lability of their catalases.

Peroxisomes were purified from livers of control mice and from mice treated with three agents which induce proliferation of hepatic peroxisomes - namely two structurally unrelated hypolipidemic drugs, clofibrate (ethyl-alpha-p-chlorophenoxyisobutyrate) and Wy-14,643 (4-chloro-6[2,3-xylidino)-2-pyrimidinylthio]acetic acid), and a plasticizer, DEHP (di-(2-ethylhexyl)phthalate). Membranes were isolated from these purified peroxisomes and analysed by SDS-polyacrylamide gel electrophoresis. All membranes which were tested, displayed two predominant integral membrane proteins of apparent molecular weights of 68 kDa and 70 kDa respectively, as well as a number of minor components. Treatment of animals with clofibrate, Wy-14,643 and DEHP was observed to result in each case in an increased proportion of the 70 kDa protein in the peroxisomal membranes. These treatments also resulted in increased peroxisomal fatty acid oxidation in livers and an increase in the proportion of catalase activity in the cytosolic fraction of liver cells. These results have been discussed in relation to alterations in the molecular composition of the membranes, the mechanisms of peroxisome proliferation and the inducibility of peroxisomal membrane proteins.

Recent investigations into the role of peroxisomes in mammalian lipid metabolism have employed double isotope methodologies to examine the influence of peroxisomal agents on lipid turnover in the liver and extra hepatic tissues of the living animal. The action of these agents, all of which caused extensive changes in the flux of lipid metabolism in the treated animals, may best be viewed in relation to their effects on the common pathway of fatty acid oxidation in peroxisomes. Clofibrate, for example, acts through induction of peroxisomal oxidases and catalase; glycolate and ethanol through activation of this pathway; and aminotriazole and allylisopropylacetamide through inhibition of the catalase step in the sequence. The data from these studies provide support for the concept of an important contributory and regulatory role of peroxisomes in relation to the overall balance of lipid metabolism, and emphasize that these organelles play a significant role in the oxidation of common fatty acids, as well as a potential for the elimination of fatty acids that are poorly oxidized by mitochondria. Additionally, the data raise intriguing questions on the extension of peroxisomal influence to include phospholipid metabolism and the substantial degree of inter-tissue communication which is involved in the balance of lipid metabolism in the whole animal.