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In order to extend the available information on the ontogenic significance of the interactions between aldolase and cellular structure, the nature and extent of these associations have been studied in the tissues of the guinea pig during development, along with analyses of the isozyme status in the bound and soluble compartments. In all tissues investigated, a significant degree of binding was evident, along with a considerable variation in the degree of association of aldolase with structure during development. Binding was particularly extensive in the early foetal stages and, in general, binding preference was directed towards A-type activity over the B- and C-type of enzyme. The significance of these ontogenic phenomena have been discussed in relation to the variations in phenotype of individual tissues during maturation and the metabolic correlations of this biphasic micro-organization.

The effects of heat-induced interactions between milk fat globule membrane components and skim milk proteins in whole milk on the structure of the membrane were examined by isopycnic sucrose density gradient centrifugation and by using Triton X-100 as a membrane probe. Skim milk components were incorporated into all the lipoprotein fractions separated by density gradient centrifugation. High density complexes, higher in density than those found in the natural milk fat globule mcmbranc, were formed during the heat treatment. Losses of natural membrane polypeptides from the medium and low density lipoproteins were observed on heating. Heating whole milk also altered the rate of release of membrane components by detergent, with decreases in protein released and an increase in phospholipid constituents released. Studies on washed cream indicated that some of the changes in the membrane on heating whole milk occurred due to thc heat treatment alone, independent of the interactions with skim milk proteins. Reproduced with permission from Cambridge University Press.

SummaryHeating raw milk at 80 °C for 2·5–20 min was found to result in compositional changes in the milk fat globule membrane (MFGM). The yield of protein material increased with the duration of heating, owing to incorporation of skim milk proteins, predominantly β-lactoglobulin, into the membrane. Lipid components of the MFGM were also affected, with losses of triacylglycerols on heating.

1. In reviewing the microenvironmental factors involved in the binding of the glycolytic enzymes to contractile filaments, consideration has been given to the significance of molecular crowding in maintaining these interactions under cellular conditions, and the influence of hormones, metabolites, pH and enzyme modifications on these phenomena. 2. Overall, these data serve to emphasize the biological reality of these associations, and their micro-organizational adaptations during physiological activities.

The peroxisomal compartment in mouse liver was investigated using rate sedimentation of liver subfractions on sucrose density gradients. Treatment of mice with clofibrate, a hypolipidemic agent and peroxisome proliferator, resulted in the formation of small particles which were devoid of catalase and urate oxidase, but which were identified as peroxisomal on the basis of content of the clofibrate-induced peroxisomal β-oxidation enzymes (fatty acyl-CoA oxidase, hydratase/dehydrogenase bifunctional protein, and thiolase) and the 68 kDa peroxisomal integral membrane protein. Immunoelectron microscopy confirmed the membrane-bound organellar nature and enzyme composition of these particles. These particles were absent in normal mice, and were increased to a maximal level within 2 days of clofibrate treatment. These data have been taken as indicative of a role of these particles in the mechanism of drug-induced peroxisome proliferation.

Apolipoprotein E (apoE) has been implicated in the pathology of AD ever since inheritance of the epsilon4 allele was shown to be an important risk factor for the development of AD. Apolipoprotein D (apoD) is elevated in association with several central nervous system disorders, including Alzheimer's disease (AD), and has been proposed to be an especially robust marker for brain regions specifically affected by particular neuropathologies. Progressive cognitive decline is the core clinical feature of AD and is associated with disturbances in the prefrontal cortex.We measured apoD levels in prefrontal cortex samples obtained postmortem from 20 autopsy-confirmed AD subjects and 40 control subjects.Enzyme-linked immunosorbent assay analysis revealed a significant increase in apoD expression in AD subjects compared with control subjects (.218+/-.029 microg/mg protein vs.117+/-.011 microg/mg protein; p=0003). There was no significant difference in apoD expression between early-onset and late-onset Alzheimer's subjects. Apolipoprotein D expression levels were not correlated with apoE levels, nor were they correlated with inheritance of the APOE epsilon4 allele.These findings suggest that apoD may be related to the cognitive decline observed in AD patients and that apoD and apoE likely play different roles in the pathogenesis of AD.

The interactions between the omega-3 unsaturated fatty acids and peroxisomal function have been reviewed, in order to update and integrate knowledge in this area. Following a brief retrospective of the major clinical involvements of these fatty acids, the participation of the peroxisome in their metabolism has been appraised - the peroxisome being shown to exert a major influence on both the synthesis and degradation of the omega-3 fatty acids, with these effects flowing on to the widespread physiological implications of the derivative eicosanoids. Interactions between the omega-3 and omega-6 families of fatty acids have been discussed, as have the interdependent phenomena of peroxisome proliferation, membrane remodelling and cellular signalling. Amongst the signalling involvements covered were those of steroid hormone receptor superfamily, the phosphatidy1choline cycle, and the regulatory influences of oxygen free radicals. Comment has also been included on the separate biological roles of the individual omega-3 fatty acids, their influence on differential gene function, and on the molecular mechanisms of their pharmacological effects. It is concluded that the peroxisome is intimately involved in directing the metabolism and physiological influence of the omega-3 unsaturated fatty acids, and that this organelle merits much greater emphasis in future research aimed at unravelling the profound biological effects of these unique and multipotent compounds.

This article reviews the role of the peroxisome in cellular signalling, with particular emphasis on the unique contributions of this organelle to the complex regulatory inter-relationships of cellular processes within the mammalian organism. Among the topics covered are the close alignments between the signalling systems governing peroxisome proliferation and those of the steroid hormone/thyroid hormone/vitamin D nuclear-receptor superfamily; the regulation of the permeability of the peroxisomal membrane; the involvements of lysophosphatidic acid as an intra- and inter-cellular messenger; the special role of the phosphatidylcholine cycle and its derivative messengers in relation to peroxisomal metabolism; peroxisomal contributions to the regulation of oxygen free radical levels in tissues and the significance of these radicals as second messengers; the evidence of peroxisomal influences on inter-cellular signalling from metabolic turnover studies; modifications of the regulatory significance of fatty acids by the peroxisome; the commonalities in metabolic relationships between the peroxisome and other cellular organelles; and regulatory shuttles associated with peroxisomal function. It is concluded that the peroxisome displays several significant interconnections with the cellular-signalling apparatus, that it is capable of imprinting a characteristic influence on the regulatory network in the cell, and that the contributions of this organelle deserve greater consideration in future investigations of cell-signalling phenomena.

Peroxisomes are subcellular organelles that are present in all eukaryotic organisms. These organelles are the focus of much contemporary interest among cellular and medical biologists - an interest which coincides with the realization of their vital role in higher organisms, their unique metabolic and biogenetic characteristics, and their widespread involvement in genetic and degenerative disease. This article reviews some of the major recent developments in peroxisome biology.

This article reviews the available data on the role of the peroxisome in the growth, differentiation and degeneration of mammalian tissues. Developmental progressions of peroxisomes are described, along with the influence of inhibitors of peroxisomal enzymes, peroxisome proliferators and morphogenetic agents on the ontogeny of experimental animals. The role of the peroxisome in protecting tissues from damage by oxygen free radicals is also described, as is the changing role of the peroxisome in the ageing animal. Amongst the degenerative diseases which have been associated with free radical damage are cancer, atherosclerosis, muscular dystrophy, rheumatoid arthritis and the senile degeneration of brain function. In all these conditions, the major characteristics of molecular damage have been considered, along with the particular role of the peroxisome in alleviating these effects. Proposals for further research into peroxisomal function during ontogeny and the degenerative changes associated with ageing are developed, and the possibility of palliative treatments discussed.

Publisher Summary There is increasing evidence that many of the cytoplasmic enzymes exist in vivo as part of an organized structural system that provides a framework for the coordination of metabolic activities. This chapter focuses on the need for modification of many of the classical views of cytoskeletal function and the regulation of intermediary metabolism. The chapter reviews the available data on the microcompartmentation of carbohydrate metabolism, and comments on localized enzyme associations, the heterogeneity of cytoskeletal structure, the covalent modification of enzymes and structure, energy requirements during signal transduction, the perturbations of micro-organization during cellular dysfunction, and the role of the cytoskeleton in modulating intermediary metabolism. There is evidence that in the cell the sequence of glycolytic enzymes may often operate as a composite of shorter metabolic sequences, each with a degree of independent function and associated with the cytoskeleton. The influence of a number of metabolic effectors (specific metabolites, hormones, second messengers) on the binding of glycolytic enzymes to the cytoskeleton has been tested by the digitonin-treated cell technique. Key protein components of the signaling pathways associated with glycolytic events also bind to the cytoskeleton and exist in multiple enzyme forms, and it may be commented that enzyme heterogeneity is a general phenomenon, which is common to most areas of metabolism in the cell.

As a prerequisite to investigating the specification and differentiation of cardiac tissue in vitro, the ontogeny of a number of putative cardiac-specific, and striated muscle-specific gene transcripts has been studied. The probes used include cDNAs of α-actins, myosin heavy chains, myosin light chains, α-tropomyosin, troponin-T and atrial natriuretic factor. The expression of these genes was monitored by Northern analysis of heart and various other tissues at three developmental ages, viz, adult, neonatal and mid-foetal. The aim of this exercise was to confirm the efficacy of a number of markers to represent a cardiac-specific subset of gene expression in our mammalian model, the guinea pig. Our results indicate predominantly cardiac expression for the mRNA transcripts of cardiac α-actin (cα-actin), cardiac myosin heavy chain-α (MHCα), cardiac myosin heavy chain-β (MHCβ), myosin light chain-1A (MLC 1A ), myosin light chain-1V (MLC 1v ), α-tropomyosin (αTM), cardiac troponin-T (cTnT) and atrial natriuretic factor (ANF). Furthermore, cardiac-specific expression at the midfoetal time point was observed for five gene transcripts, MLC 1v , MHCα, MHCβ, striated αTM and ANF. No genes were expressed exclusively in cardiac tissue; for example, expression of the genes for cα-actin, both cardiac MHCs, both MLCs, αTM and cTnT was evident in skeletal and vascular smooth muscles at some stages of development. An interesting difference between this species and those of previous studies was the minor contribution of skeletal α-actin to cardiac phenotype. As well, our observation of transcripts of cardiac MHCs and ANF in vascular smooth muscle has not been previously reported. By providing a comprehensive survey of the ontogenic expression of this group of genes, our study has been able to identify a number of useful cardiac differentiation markers which, as a group, may provide a more definitive indication of authentic cardiac cellular differentiation, appropriate to some in vitro experimental situations.

In this paper, the main features of the cellular activities of the glycolytic enzymes during growth and tissue differentiation are summarized, and correlated with the occurrence of multiple forms of these enzymes, and with their degree of interaction with subcellular structure. The substantial evidence for micro-organization of the glycolytic sequence is described, as well as its significant contribution to the diverse physiological situations encountered during development. Based on this evidence, a modular, biphasic model of glycolytic activity has been developed, with associated features of microcompartmentation and segmentation. Evidence has been provided that these phenomena play important roles in meeting the special needs of emerging cell types during early ontogeny, as well as offering the potential for increased flexibility and control of glycolysis in specialized physiological situations in the adult organism.

Catalase leakage from its particulate compartment within the light mitochondrial fraction of liver was used as an index of the integrity of peroxisomes in untreated mice and in mice treated with the peroxisome proliferators clofibrate(ethyl-p-chlorophenoxyisobutyrate), Wy-14,643(4-chloro-6[2,3-xylidino)-2-pyrimidinylthio]acetic acid) and DEHP(di-(2-ethylhexyl)phthalate). Catalase leakage represented about 2% of the total catalase activity when fractions from untreated mice were incubated at 4 degrees C, increasing to about 5% during 60 min incubation at 37 degrees C. In fractions from livers of mice treated with peroxisome proliferators, catalase leakage was significantly higher, being 7-11% at 4 degrees C and increasing to approximately 20% after 60 min incubation at 37 degrees C. The pattern of release was similar for all proliferators. Parallel data were obtained for catalase latency in these fractions, i.e. following 60 min incubation at 37 degrees C, free (non-latent) catalase activity was 18% in control mice and 65, 67, and 83% in fractions from clofibrate-, Wy-14,643- and DEHP-treated mice, respectively. Differences in catalase leakage from peroxisomes in fractions from untreated mice and clofibrate-treated mice were also apparent following treatments designed to effect membrane permeabilization, as in freeze-thawing, osmotic rupture, and extraction with Triton X-100 and lysophosphatidylcholine. These data are consistent with a significant alteration in the integrity of the membranes of peroxisomes in livers of mice which have been treated with peroxisome proliferators, and furthermore indicate a commonality of effect of these agents.

1. 1. The influence of protein depletion and starvation on the turnover parameters of total soluble protein and lactate dehydrogenase in mouse tissues has been studied. 2. 2. Animals on a 20% protein diet reached a new steady state after 2 weeks, and this was characterized by a generalized decrease in the rates of both degradation and synthesis. 3. 3. Some tissues (e.g. kidney) were less affected than others (e.g. muscle, brain), and lactate dehydrogenase was more highly conserved than total protein, in general. 4. 4. Markedly different turnover characteristics were observed in starvation, with increased degradation of total protein being common in the terminal stages. 5. 5. These results have been discussed in relation to the individuality of the tissue responses and possible intracellular mechanisms.

1. 1. As an extension of previous studies on the adsorption of aldolase (EC 4.1.2.13) in rat brain, and the correlation between such adsorption and the expression of aldolase activity, the interaction between purified aldolase A4 and a microsomal membrane fraction which was the main locus of aldolase binding has been studied. 2. 2. The observed effects of pH, salts and metabolites in this binding support the proposition that electrostatic forces are of primary importance in the adsorption process, although more subtle interactions involving the active site of the enzyme are also indicated. 3. 3. The enzyme remained firmly bound to these membranes even after phospholipase and ribonuclease treatment, suggesting that the compounds removed by these enzymes were not primarily involved in the aldolase binding. It also became evident, however, that RNA possessed the ability to associate with either free or bound aldolase under the assay conditions employed, and that a correlation existed in whole brain homogenate between the degree of nucleic acid association with both the soluble and bound forms of the enzyme and expression of activity, rather than between enzyme adsorption and expression of activity as previously suggested. 4. 4. These phenomena are discussed in relation to the structural and metabolic features of this tissue.

The interactions of aldolase with regulatory proteins of rabbit skeletal muscle were investigated by moving-boundary electrophoresis. A salt-dependent interaction of troponin, tropomyosin and the tropomyosin-troponin complex with aldolase was detected, the tropomyosin-troponin complex displaying a greater affinity for the enzyme than did either regulatory protein alone. The results indicate that aldolase possesses multiple binding sites (three or more) for these muscle proteins. Quantitative studies of the binding of aldolase to actin-containing filaments showed the interaction to be influenced markedly by the presence of these muscle regulatory proteins on the filaments. In imidazole/HCl buffer, I 0.088, pH 6.8, aldolase binds to F-actin with an affinity constant of 2 × 10(5) M-1 and a stoicheiometry of one tetrameric aldolase molecule per 14 monomeric actin units. Use of F-actin-tropomyosin as adsorbent results in a doubling of the stoicheiometry without significant change in the intrinsic association constant. With F-actin-tropomyosin-troponin a lower binding constant (6 × 10(4) M-1) but even greater stoicheiometry (4:14 actin units) are observed. The presence of Ca2+ (0.1 mM) decreases this stoicheiometry to 3:14 without affecting significantly the magnitude of the intrinsic binding constant.

The processes associated with the biogenesis of peroxisomes in mouse liver have been studied by following the incorporation of radiolabelled leucine into major enzymic components of this organelle. Maximal incorporation of label into peroxisomal catalase and urate oxidase occurred within 2 hr, with the urate oxidase being labelled before catalase, but subsequent to the incorporation of phospholipid into this organelle. Subsequently, immunoprecipitation of catalase from the large granular fraction of mouse liver was shown to result in the isolation of a catalase molecule which had lost a peptide of approx. 2000 dalton from each subunit by comparison with the newly-synthesized enzyme. It was observed that the modification of catalase was obviated by the presence of leupeptin and iodoacetamide and this information has enabled the purification of both modified and unmodified forms of the enzyme. The possible significance of these data has been discussed and the major features incorporated into a working model of peroxisomal biogenesis.

1. 1. An affinity chromatography technique for the determination of turnover parameters has been applied to the study of carbonic anhydrase in a variety of mouse tissues. 2. 2. The methodology has allowed the establishment of the relative turnover characteristics in these tissues, and the evaluation of the half lives and degradation rate constants. 3. 3. Considerable variation was evident between the individual tissues, with synthesis being indicated as most rapid in intestine while degradation was highest in liver. 4. 4. These data have been discussed in relation to available comparisons in the literature, and the known physiological correlations of carbonic anhydrase in these tissues.

Two forms of N- acetyl -β- d - glucosaminidase were purified from bovine mammary gland by DEAE-cellulose chromatography, Sephadex G-200 gel filtration, affinity chromatography on Con A-Sepharose and preparative isoelectric focusing. The two forms, designated A and B on the basis of their binding to DEAE-cellulose at pH 7, were glycoproteins with different molecular weights as determined by gel filtration and sedimentation equilibrium analysis. The A form had a molecular weight of 118 000, while the B form had a molecular weight of 234 000. Both A and B forms of the purified enzyme showed the presence of two distinct subunits, having apparent molecular weights of 55 000 and 25 000 as determined by sodium dodecyl sulphate-electrophoresis. Amino acid composition of the purified forms showed that a high degree of similarity existed between the two forms. However, the B form had slightly higher levels of serine and threonin than the A form. The structure and possible interrelationship of these two forms in the bovine mammary gland are discussed in relation to the structure of N- acetyl -β- d - glucosaminidase from other sources.

The interaction of GAPDH with cellular structure has been studied in the major tissues of the mouse during development. Overall the data provides a clear indication that interactions between GAPDH and cellular structure are appreciable in all major tissues, at least during early stages of development, and an analysis of the isozyme status of the enzyme in both soluble and bound compartments for all tissues at all developmental stages indicates the presence of only a single GAPDH isozyme in the mouse. Possible reasons for the lack of an extensive multiplicity of this enzyme in mammalian tissues (the only tetrameric glycolytic enzyme to display this restriction) and for the large amounts of GAPDH in many cell types are discussed in relation to the large number of proteins that GAPDH interacts with in the cell.

Ultracentrifugal studies of mixtures of aldolase and the troponin–tropomyosin complex from bovine muscle showed the existence of a labile interaction between these two myofibrillar constituents in imidazole buffers, pH6.8, I 0.02–0.10 (mol/l), and the suppression of the reaction by fructose 1,6-diphosphate. Analysis of the sedimentation-velocity patterns suggests the binding of more than 2 molecules of troponin–tropomyosin/molecule of aldolase. The results illustrate the necessity of considering additional or alternative sites to F-actin to account for the observed binding of aldolase to the thin filaments of skeletal muscle.

1. The influence of fibre-type and an exercise regimen on the turnover characteristics of proteins in mouse skeletal muscle has been studied. 2. Exercise induced a weight increase in most individual muscles and a decrease in lactate dehydrogenase activity which was more marked in white muscle than red. The coincident decrease in M-subunit activity was more noticeable in fast-twitch muscles than in slow-twitch types. 3. Exercise caused a considerable and consistent increase in the rate of degradation of total soluble proteins in individual muscles, with turnover being higher in muscles with a high red fibre content. 4. The response of lactate dehydrogenase to exercise varied considerably between individual muscles as indicated by the comparative rate constants for synthesis and degradation. In general, degradation was greater in muscle with a high slow-twitch or red fibre content. 5. Overall, a considerable redistribution of protein resources was observed during this physiological perturbation (exercise) with the main emphasis away from the pool of total soluble protein towards the myofibrillar constituents. Highly individualistic responses in relation to rates of synthesis and degradation were observed for the specific protein, lactate dehydrogenase, in the separate muscle of this animal.

An in depth study of the turnover characteristics of lactate dehydrogenase and its multiple enzyme forms in several animal tissues has provided a number of insights into the variations of synthesis and degradation of this enzyme in vivo , and the manner in which changes in turnover parameters throughout the body are coordinated during physiological perturbations such as pregnancy and tumour growth.

The interactions of the isozymes of hexokinase with cellular structure have been studied in the major tissues of the mouse during development. Overall, these data provide a clear indication that interactions between hexokinase and cellular structure are appreciable in all major tissues and at all stages of development, and an analysis of the isozyme status of the enzyme in both soluble and bound compartments has been effected. Further evidence in support of the already well documented interaction of hexokinase I to subcellular material in adult brain and kidney tissues is provided and extended to show that such interactions are extensive in both these tissues throughout development. In addition, evidence is provided that considerable hexokinase II activity is present in mouse foetal tissues in both the soluble and bound fractions and this isozyme is also shown to be the predominant "bound" form of the enzyme in adult skeletal muscle. By contrast, hexokinase III and IV are shown to be largely located in the cytosolic fraction of liver. The metabolic implications of these enzyme-structural interactions during development are discussed, as is the possibility of a functional linkage between hexokinase, which is bound to the mitochondria, and other enzymic components of the glycolytic sequence.

1. 1. The effects of protein concentration and ionic strength on the adsorption of the individual glycolytic enzymes to F-actin and F-actin-trypomyosin-troponin have been studied. 2. 2. Appreciable association was demonstrated under conditions of physiological ionic strength and high protein concentration, and tropomyosin-troponin established as an important and generalized component of these interactions. 3. 3. Phosphofructokinase, aldolase, pyruvate kinase, lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate isomerase were strongly bound under these conditions, while triosephosphate isomerase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and hexokinase displayed less adsorption to the structural proteins. 4. 4. The influence of a number of parameters on the adsorption phenomena was examined. Ca2+ and fructose 1,6-diphosphate increased the adsorption of aldolase, lactate dehydrogenase and pyruvate kinase, while decreasing the adsorption of the enzymes of the constant-proportion group. 5. 5. Of the other major enzymic components of skeletal muscle, creatine kinase, adenylate kinase and malate dehydrogenase showed no adsorption to F-actin-tropomyosin-troponin under the experimental conditions. Some adsorption was evident, however, in the case of aspartate aminotransferase, (NADP) isocitrate dehydrogenase and α-glycerolphosphate dehydrogenase. 6. 6. These results have been discussed in relation to their functional significance and the roles of enzyme compartmentation in the cell.

1. 1. The double isotope ratios and specific radioactivities of individual lipid fractions in major tissues of mice have been determined before and after ethanol ingestion. 2. 2. Several significant alterations in these ratios were caused by this treatment, with marked increases in most liver lipids and diverse responses in other tissues. 3. 3. These data establish that ethanol ingestion causes widespread perturbations in tissue lipid metabolism, with the main emphasis being directed towards an increase in the rate of synthesis of the liver lipid classes. 4. 4. The data also provides indications of an increase in the peroxisomal oxidation of fatty acids following ethanol ingestion, with the different distribution of ethanol metabolizing systems giving rise to individual metabolic responses in the separate tissues. 5. 5. These results have been discussed in relation to the eatablished involvements of lipid metabolism and tissue interactions in the whole animal.

A method is described for the purification of troponin from beef skeletal muscle. The resultant preparation differs from the troponin of rabbit skeletal muscle in that it contains at least two forms of the tropomyosin-binding component, Troponin-T: these are designated as the 37 000 and 40 000 dalton forms of Troponin-T on the basis of sodium dodecyl sulphate gel electrophoresis. Either of these Troponin-T forms may be used to reconstitute troponin by mixing with the appropriate amounts of the calcium-binding (Troponin-C) and and actomyosin ATPase-inhibitory (Troponin-I) components. These reconstituted troponins are shown to interact with tropomyosin and also to confer full calcium sensitivity on actomyosin ATPase. Despite the existence of proteolysis in troponin preparations, the experimental evidence indicates that the smaller form of Troponin-T is not derived from the 40 000 dalton species by limited degradation. Although both species of Troponin-T have been found routinely in troponin from beef skeletal muscle, only the larger form is detected in troponin preparations from beef cardiac muscle. Further studies are required in order to clarify the functional significance and differential distribution of these multiple forms of Troponin-T.

After existing in the shadow of its fellow oxidative organelles for some decades, the peroxi some is emerging as an important site of carbohydrate, lipid and nitrogen metabolism; the organelle may also have significant regulatory functions.

Two major proteins with subunit molecular masses of 68 and 70 kDa were isolated from the integral membrane protein fraction of peroxisomes purified from mouse liver. The two proteins were shown to be distinct proteins by two criteria: first, immunoblot analysis demonstrated that antisera against the 68 kDa protein did not cross-react with the 70 kDa protein, and vice versa; and second, the partial peptide maps resulting from proteinase digestion of the proteins were different. Immunoblot analyses to test the specificities of the antisera demonstrated that only the expected molecular mass species in purified peroxisomes, and in membranes prepared from these organelles, were recognized; there was no identification of proteins from purified mitochondrial or microsomal fractions. The concentrations of both of these proteins were increased in livers of mice treated with clofibrate, a hypolipidemic drug and peroxisome proliferator, with the effect being greater for the 70 kDa component. The localization of the 68 kDa protein was shown to be completely integral to the peroxisome membrane. Although some 70 kDa protein was integral to the membrane, a significant proportion was released from the membrane by some procedures believed to detach peripheral proteins. The 70 kDa protein was also particularly susceptible to degradation during isolation — in particular, addition of EDTA to media used for isolation of peroxisomes resulted in membranes in which this protein was degraded to smaller immunoreactive fragments. These data have been discussed in relation to the significant clarification which they have provided of the status and characteristics of the major protein components of peroxisomal membranes.

The localization of lactate dehydrogenase in the ovaries and reproductive tracts of rats and mice has been studied by a methodology which minimizes loss of this soluble enzyme by diffusion, and allows comment on the subunit composition of the cellular enzyme. The results differ significantly from previous data with conventional methodologies. In particular, the major localization of activity in the present study was identified in interstitial cells, and not the corpora lutea or granulosa cells; and it was noticeable that neither species exhibited massively greater expression of lactate dehydrogenase activity in the oocytes than in adjacent cell types of the reproductive tract. The goblet cells of the Fallopian tube stained intensively for activity of this enzyme. These results have been discussed in relation to the discordant data in the literature, the important role of lactate dehydrogenase in mammalian development, and the evidence for a masking of the activity of this enzyme in oocytes and pre-implantation ova.

1. 1. The distribution and interactions of phosphofructokinase isozymes with cellular structure have been studied in the major tissues of the mouse during development. 2. 2. The ontogenic patterns of isozymes which were obtained were consistent with those observed for other species and are interpreted in terms of the presence of three genes and three homotetrameric forms of the enzyme (A 4 , B 4 and C 4 ) in the tissues of the mouse. 3. 3. In addition, the data provides a clear indication that interactions between the enzyme and cellular structure are appreciable in all major tissues and at all stages of development, with all isozyme types exhibiting such interactions. 4. 4. The significance of the study of subcellular interactions of these isozymes in contributing to a comprehensive physiological rationale for this mammalian enzyme and its multiple forms is discussed.

Purified preparations of bovine plasma arylesterase were obtained by isoelectric focusing of enzyme prepared by (NH4)2SO4 fractionation of plasma and chromatography on DEAE-cellulose and Sephadex G-200. Although the high-density-lipoprotein fraction (HDL2) of serum provides an alternative source of enzyme, the enzymic activity of preparations made from it is much less stable. The purified arylesterase preparation has a molecular weight of 440000 and a partial specific volume of 0.91 ml/g, properties indistinguishable from those of the less highly purified enzyme. Extraction with acetone and ether removes neutral lipids from the enzyme, but the resulting lipid-depleted preparation retains most of the phospholipid present initially. A partial specific volume of 0.81 ml/g and a minimum molecular weight of approx. 100000 were determined for the lipid-depleted preparations of arylesterase. The present results support the concept of bovine plasma arylesterase as a lipoprotein in its own right, rather than as an enzymic polypeptide that is loosely associated with the HDL2 fraction of serum.

1. 1. Turnover parameters (half-lives, rate constants for synthesis and degradation) for tissue catalase have been measured in three vertebrate species (a mammal, bird and fish) by means of the kinetics of return of catalase activity after inhibition with 3-amino-1,2,4-triazole. These results serve to extend previously available data both in terms of the range of tissues and the range of species analyzed. 2. 2. Catalase degradation rates were of similar magnitude in the corresponding mammalian and avian tissues, although significant differences existed between individual tissues in each species. 3. 3. In contrast a wide variation in catalase synthesis rate constants, and in steady-state tissue levels was observed between tissues and species. 4. 4. The amino-triazole method was shown to be unsuited to the determination of turnover parameters for catalase in fish, because of the comparatively slow elimination of this inhibitor from piscine tissues. 5. 5. Administration of allylisopropylacetamide to mice resulted in anomalous responses by contrast with the established behaviour in rats. For example, complete depression of catalase activity was not observed in any of the tissues examined, and in fact, a marked increase occurred in the levels of catalase activity in mouse heart. 6. 6. The significance of these data has been discussed in relation to the individual tissue characteristics of these animals and possible mechanisms of proteolysis.