Developmental changes in the modification of lysosomal enzymes in Dictyostelium discoideum

Evidence has been found for a generalized change in the post-translational modification of lysosomal enzymes during development of Dictyostelium discoideum. The physical and antigenic properties of four developmentally regulated lysosomal enzymes, N-acetylglucosaminidase, beta-glucosidase, alpha-mannosidase, and acid phosphatase, have been examined throughout the life cycle. In vegetative cells, a single major isoelectric species is detected for each enzymatic activity on native nonequilibrium isoelectric focusing gels. Between 6 and 10 hr of development, all activities, including the preformed enzyme, become less negatively charged, resulting in a modest but reproducible shift in the isoelectric focusing pattern. This alteration is not detected by native gel electrophoresis at constant pH. As development continues, the specific activity of beta-glucosidase, alpha-mannosidase, and acid phosphatase continues to increase and coincidentally, new, less acidic isozymic bands of activity can be observed on both gel systems. Some of these new isozymes accumulate preferentially in anterior cells, while others accumulate preferentially in posterior cells of migrating slugs. N-Acetylglucosaminidase does not increase in specific activity late in development and no new isozymic species appear. Using a monoclonal antibody that reacts with sulfated N-linked oligosaccharides shared by vegetative lysosomal enzymes in D. discoideum, the antigenicity of the developmental isozymes has been characterized. All of the enzymatic activity present during vegetative growth and early development is immunoprecipitable. However, the less negatively charged isozymes that accumulate after aggregation are not recognized by the antibody. Nonantigenic acid phosphatase and alpha-mannosidase are found in both anterior and posterior cells from migrating pseudoplasmodia. Since each enzyme is coded by a single structural gene, these results suggest that the isozymes present late in development arise from the synthesis of the same polypeptides with altered post-translational modifications. The appearance of anterior and posterior specific isozymes is likely to be the result of cell type specific changes in the glycoprotein modification pathway for newly synthesized proteins.     

Characterization and developmental regulation of beta-galactosidase isozymes in Dictyostelium discoideum.

Two isozymes of β-galactosidase (EC 3.2.1.23) have been identified during growth and development of the cellular slime mold, Dictyostelium discoideum. The isozymes have been partially purified and differ in a variety of physical and enzymatic properties. β-Galactosidase-1 is present in vegetative cells. The specific activity is reduced during early development and then increases again during culmination. The specific activity of β-galactosidase-2 increases in early development and then again during culmination and spore maturation. The specific activity of β-galactosidase-2 is extremely dependent upon growth conditions and is regulated over a 160-fold range. The accumulation of both isozymes is dependent on concomitant RNA and protein synthesis.     

Cell-specific differences in membrane beta-glucosidase from normal and Gaucher cells.

Two isozymes of membrane-bound beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) with activity towards 4-methylumbelliferyl-beta-D-glucopyranoside have been identified in human cells. One of these isozymes was found to have a pH optimum of 5.0, a Km of 0.4 mM and to be rapidly inactivated at pH 4.0 ("acid-labile"). The second isozyme had a pH optimum of 4.5, a Km of 0.8 mM and was stable at pH 4.0 ("acid-stable"). Cultured long-term lymphoid lines and peripheral blood leukocytes contained both isozymes while cultured skin fibroblasts contained only the "acid-stable" form in detectable amounts. The specific activity of the "acid-stable" isozyme was severely reduced in cultured skin fibroblasts, cultured long-term lines and peripheral leukocytes from patients with Gaucher's disease. The specific activity of the "acid-labile" enzyme in the latter two cell types was apparently unaffected. The beta-glucosidase activity in all three cell types examined was predominantly particulate but the enzyme could be solubilized with low concentrations of Triton X-100. The solubilized enzyme required sodium taurocholate (0.2%) for maximum activity. Solubilized beta-glucosidase did not exhibit the cell-specific differences in pH optimum and Km shown by the membrane-bound enzyme.     

Independent expression of cardiac type I and II cyclic AMP-dependent protein kinase during murine embryogenesis and postnatal development.

The amount of total cyclic AMP-dependent protein kinase and of the protein kinase isozymes present in mouse heart changes during development. During embryogenesis, the total cardiac protein kinase activity increases most markedly during the 6 days prior to birth. A maximum kinase level is achieved in the 7 day-old neonate, and then activity progressively declines to an adult level approximating that of the mid-embryo. The type II kinase exhibits a moderate increase during late embryogenesis which declines by the time of birth. The type I isozyme increases throughout embryogenesis and the first neonatal week to a maximum specific activity five-fold higher than the mid-embryogenesis level. The isozyme level then falls to an adult activity similar to the mid-embryonic. These changes in isozyme profile are reflected in a changing type I to type II kinase ratio of 1.1 at 13--14 days embryogenesis, 2.4 at birth, 3.0 in the 7 day-old neonates, and 1 in the adult heart. Thus, the two protein kinase isozymes change in association with the developmental process in an independent fashion.     

Structure and function of beta-blucosidases in Dictyostelium discoideum.

There are two isozymes of beta-glucosidase in developing cells of Dictyostelium discoideum. A procedure for screening large numbers of clones for beta-glucosidase activity was utilized to obtain mutations which directly affect the activity. We recovered seven strains which lack both isozymes and four strains with residual activity in which enzymatic and physical properties of both isozymes are altered. Beta-Glucosidase appears to act as a block to selfing in macrocyst formation as shown by the fact that ssite mating type to form macrocyst-like structures. Immunological evidence utilizing antisera prepared against purified beta-glucosidase-1 demonstrates that most of the glycosidases in Dictyostelium discoideum share a common antigenic determinant which appears to be added post-translationally. The two isozymes of beta-glucosidase share common protein subunits but the antigenic determinant is either lacking or masked in beta-glucosidase-2. This may account for some of the enzymatic and physical differences between the two isozymes.     

Isozymes of β-Glucosidase in Dictyostelium discoideum

The activity of beta-glucosidase (EC 3.2.1.21) in extracts of Dictyostelium discoideum was investigated. The specific activity increased early in development, declined during pseudoplasmodium formation, and increased again during sorocarp formation. The beta-glucosidase which was present in growing amoebae and during the first stages of multicellular development was electrophoretically distinct from the enzyme which accumulated during the final stages of morphogenesis. Ribonucleic acid synthesis and protein synthesis during development were required for the accumulation of the later isozyme. Analysis of beta-glucosidase activity in a number of morphological mutants suggests that the enzyme which accumulates late in morphogenesis is developmentally controlled.     

Biosynthesis of two developmentally distinct acid phosphatase isozymes in Dictyostelium discoideum

The presence of a common antigenic determinant on the Dictyostelium discoideum acid phosphatase isozyme 1 (ap 1), and the absence of this determinant on the isozyme ap2 enables separation of the two isozymes. This separation is accomplished by removal of ap1 from samples with a common antigen monoclonal antibody followed by immunoprecipitation of ap2 with an acid phosphatase monoclonal antibody. Application of this separation scheme on cells pulse-labeled early (2 h) and late (18 h) in the developmental cycle reveal that ap1 protein synthesis occurs only early in development and that the protein remains stable throughout development, whereas ap2 protein synthesis occurs only late in development. Furthermore, pulse-chase experiments during both early and late development reveal that both isozymes of acid phosphatase are initially synthesized as precursor molecules (Mr = 60,000) which are then processed to mature forms (Mr = 58,000). The processing event(s) for acid phosphatase begin in less than 5 min compared to 25-30 min for Dictyostelium alpha-mannosidase and 10-15 min for Dictyostelium beta-glucosidase. Endoglycosidase H and Endoglycosidase F treatment of both isozymes reveals identical cleavage patterns for ap1 and ap2, indicating that the amount of carbohydrate on both molecules is equivalent. Preliminary studies to identify modification differences reveal that fucose is not present on either isozyme; however, sulfate is present on the ap1 isozyme and absent on the ap2 isozyme. These results suggest that differences in the modification of newly synthesized acid phosphatase at different times during the Dictyostelium life cycle result in the appearance of two distinct acid phosphatase isozymes.     

Regulation of lysosomal alpha-mannosidase-1 synthesis during development in Dictyostelium discoideum

The cellular specific activity of lysosomal alpha-mannosidase-1 increases dramatically during development in Dictyostelium discoideum. alpha-Mannosidase-1 is composed of two subunits (Mr = 58,000 and 60,000) which are derived from a common precursor polypeptide (Mr = 140,000). Using enzyme-specific monoclonal antibodies we have determined that throughout development (a) the relative rate of precursor biosynthesis closely parallels the rate of accumulation of cellular enzyme activity and (b) the newly synthesized precursor is efficiently processed to mature enzyme (t1/2 less than 10 min). This indicates that the developmental accumulation of alpha-mannosidase-1 activity is primarily controlled by de novo enzyme synthesis. Furthermore, the change in the relative rate of enzyme precursor synthesis can be accounted for by an increase in the cellular level of functional alpha-mannosidase-1 mRNA during development.     

Increase of enzyme activities in Neurospora crassa during incubation at low temperatures.

The effect of lowering the incubation temperature of sucrose-grown cultures of Neurospora crassa on the level of various enzyme activities was investigated. Of twelve inducible/derepressible activities studied, three, in addition to glycerol kinase, were found to increase during 48 h of incubation at 4-6 degrees C: trehalase (increase in specific activity of 3-10-fold), beta-glucosidase (6-12-fold) and beta-N-acetylglucosaminidase (4 to 6-fold). The maximum increases occurred at 6 degrees C and no increases took place in mycelia incubated at 0 degrees C. The kinetics of the changes in activity were markedly different from those observed previously with glycerol kinase. The increases were inhibited by cycloheximide. Trehalase, beta-glucosidase and beta-N-acetylglucosaminidase activities were not rapidly lost when cultures incubated at 6 degrees C were returned to 26 degrees C.     

Enzyme synthesis in the cellular slime mould Dictyostelium discoideum during the differentiation of myxamoebae grown axenically

1. Myxamoebae of the cellular slime mould Dictyostelium discoideum Ax-2 were grown on different media, and were harvested either in the stationary or exponential phases of the growth cycle to yield samples of myxamoebae differing in enzymic composition. 2. Morphogenesis and cell differentiation phenomena in D. discoideum appear to be similar in myxamoebae grown and harvested under different conditions. 3. The specific activity of the enzymes beta-N-acetylglucosaminidase, acid phosphatase, alpha-mannosidase, beta-glucosidase and alkaline phosphatase have been determined during cell differentiation of myxamoebae grown and harvested under different conditions. 4. The pattern of synthesis of these enzymes, all of which have been claimed to be part of the ;developmental programme', either remains unaffected despite the origin of the myxamoebae (alkaline phosphatase) or is qualitatively similar but quantitatively affected (acid phosphatase, beta-glucosidase) or is both qualitatively and quantitatively affected by changes in the myxamoebae (alpha-mannosidase, beta-N-acetylglucosaminidase). 5. The implications of these results for the concept of a ;developmental programme' are discussed.     

Differentiation of creatine phosphokinase during myogenesis: quantitative fractionation of isozymes

A technique is described for the quantitative measurement of creatine phosphokinase (CPK) isozymes in extracts of chick muscle. The isozymes are fractionated by stepwise elution with increasing salt concentrations from DEAE-Sephadex minicolumns. Isozyme separation was confirmed by polyacrylamide gel electrophoresis followed by enzyme staining. We used this method to determine changes in CPK isozymes during the course of myogenesis in culture. The total specific activity of CPK increases about 20-fold during myogenesis. Quantative analysis of isozyme changes shows that the muscle-specific form (MM) accounts for virtually all of this increase. Activity of MM-CPK is undetectable in 1-day cultures, increases rapidly after myoblast fusion, and comprises more than 70% of total CPK in mature cultures. In contrast, the specific activity of the brain-specific isozyme (BB) remains constant throughout myogenesis. This was interpreted as indicating that the B subunit is expressed in both mononucleated cells and myotubes. We confirmed this by analyzing CPK isozymes in fibroblast cultures and in myotube-enriched cultures. Elimination of most of the mononucleated cells in the cultures produced an increase in the specific activity of CPK, but had no effect on the isozyme pattern and did not decrease the relative amount of the BB isozyme. Pure fibroblast cultures contained very low CPK activity, predominantly the BB isozyme.     

Rates of accumulation of glycosidase activities during growth and differentiation of Dictyostelium discoideum.

1. The rates of accumulation (enzyme units/h per 10(8) cells) of a number of glycosidase activities were studied in Dictyostelium discoideum cells during the growth and differentiation phases of this organism's life cycle. 2. The rates of accumulation of the enzymes beta-N-acetylglucosaminidase, alpha-glucosidase and beta-galactosidase remain unchanged during the growth and early differentiation phases. 3. The considerable changes in specific activity of the enzymes which occur in the early differentiation phase are due to the massive loss of total cellular protein which occurs at this time. 4. Significant alterations can occur in the rates of accumulation of alpha-mannosidase during both the growth and differentiation phases, and since, on the onset of differentiation, beta-glucosidase activity is excreted and degraded, the rate of accumulation of this enzyme differs in the growth and differentiation phases. 5. The characteristic rates of accumulation of all these glycosidases change markedly with changes in the growth conditions of the myxamoebae, and thus these rates of synthesis must be regulated independently; however, addition of cyclic AMP to the growth medium has no effect on them.     

A role for glyceraldehyde-3-phosphate dehydrogenase in the development of thermotolerance in Xenopus laevis embryos

During heat shock, Xenopus laevis embryos exhibit an increase in the rate of accumulation of lactate and a loss of ATP relative to non-heat-shocked control embryos. These results suggest that heat shock stimulates a shift in energy metabolism to anaerobic glycolysis while at the same time causing an increase in the demand for ATP. We have evidence indicating that the embryo may meet such demands placed on it by increasing the levels of some glycolytic enzymes. In this report, we show that heat shock stimulates increases in the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase [( EC 1.2.1.12] GAPDH). The specific activity of GAPDH shows a significant increase after heat shock, which correlates with the accumulation of GAPDH in heat-shocked embryos as detected by immunoblotting. Increases in GAPDH-specific activity are variable, however, and are inversely proportional to the levels of specific activity in control embryos; i.e., constitutive enzyme activity. We further analyzed the heat-enhanced accumulation of GAPDH by electrophoretically separating GAPDH isozymes on nondenaturing polyacrylamide gels. Control embryos exhibit a single isozyme of GAPDH, whereas heat-shocked embryos exhibit two isozymes of GAPDH. When these isozymes are labeled with [35S]methionine, separated by nondenaturing gel electrophoresis, and analyzed by fluorography, a heat-shock protein is found to comigrate with the isozyme unique to the heat-shocked sample. Enzyme activity assays at different temperatures suggest that this isozyme has optimum enzymatic activity only at heat-shock temperatures. We have correlated a 35-kD heat-shock protein (hsp35) with GAPDH using the following evidence: this hsp comigrates with GAPDH on one-dimensional SDS polyacrylamide gels; heat-enhanced increases in GAPDH specific activity correlate with hsp35 synthesis; and hsp35 and GAPDH have similar peptide maps. This relationship also provides a compelling explanation for the restriction of hsp35 synthesis to the vegetal hemisphere cells of heat-shocked early gastrulae reported previously (Nickells, R. W., and L. W. Browder. 1985. Dev. Biol. 112:391-395).     

Regulation of various hydrolase activities in the myxomycete Physarum polycephalum

In a standard growing medium, the specific activities of acid-phosphatase, beta-galactosidase and beta-glucosidase of Physarum polycephalum increase during the growth of the culture. The decrease of the pH of the culture medium during the growth has no effect on the variations of these hydrolase activities. In a glucose-starved medium, the specific activity of beta-galactosidase increases up to 350% of its initial value in 24 h, whereas the specific activities of acid phosphatase and beta-glucosidase stay near their minimal level.     

Changes in activity and mRNA levels of poly(ADP-ribose) polymerase during rat liver regeneration

ADP-ribosylation of nuclear proteins, catalysed by the enzyme poly(ADP-ribose) polymerase, is involved in the regulation of different cellular processes of DNA metabolism. To further clarify the role of the enzyme during proliferating activity of mammalian cells, we have studied the control of gene expression in regenerating rat liver. The changes in activity and mRNA levels were analysed during the early and late phases of the compensatory model. When enzyme activity was measured in isolated liver nuclei obtained at different times after hepatectomy, two different phases were observed: an early wave occurring before the onset of DNA synthesis, and a second one, starting several hours after the onset of DNA synthesis and returning to control values at later times. The evaluation of the enzymatic level in nuclear extracts and by activity gel analysis showed a more gradual increase starting 1 day after hepatectomy, in concomitance with the peak of DNA synthesis. By using a specific murine cDNA probe, a significant enhancement of mRNA levels for poly(ADP-ribose) polymerase was observed during liver regeneration, slightly preceding the onset of DNA synthesis. The results obtained show that changes in poly(ADP-ribose) polymerase activity, during liver regeneration, are associated both to early events preceding the increase in DNA synthesis and to later phases of the cell proliferation process.     

Developmental studies with the 14-3-2 antigen and the neuron specific enolase (NSE) associated activities—I

We have compared the rodent developmental pattern of the 14-3-2 antigen estimated by a microcomplement fixation technique with that of the cerebral enolases. Chromatographic separation of enolase isozymes on microcolumns demonstrates that the embryonic neuron specific enolase is firstly and mostly represented by the αγ isozyme. The most important increase in 14-3-2 antigen and γγ enolase occurs between post-natal days 7th and 15th. By post-natal day 30, adult levels have been reached. An interesting observation is—during embryonic development—the decrease in the specific activity of the cerebral enolase isozyme αα. This could be explained by the replacement—in neuroblasts—of αα enolase by neuron specific enolase. A comparison between 14-3-2 antigen and neuron specific enolase (γγ) purified by completely different methods is presented. The 14-3-2 antigen exhibits an enolase specific activity comparable to that of purified enzyme and has the same electrophoretic mobility. Antibodies raised against either antigen have an identical specificity. Pre and post-natal developmental pattern in rodent brains are similar for both proteins. Thus neuron specific 14-3-2 antigen is identical to neuron specific enolase.Thus we have precisely described the ontogenic transition between the three cerebral enolase isozymes at the tissue level. This study is completed by the analysis of these transitions at the neuronal cell level, using homogenous cell lines (Part II of this paper).     

beta-Amylase from Mustard (Sinapis alba L.) Cotyledons : Immunochemical Evidence for Synthesis de Novo during Photoregulated Seedling Development

In barley (Hordeum vulgare L.), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) are induced by anaerobiosis in both aleurone layers and roots. Under aerobic conditions, developing seeds of cv Himalaya accumulate ADH activity, which survives seed drying and rehydration. This activity consists almost entirely of the ADH1 homodimer. Activity of LDH also increases during seed development, but the level of activity in dry or rehydrated seeds is very low, indicating that this enzyme may not be involved in anaerobic glycolysis during the initial stages of germination. In contrast to ADH, the LDH isozymes present in developing seeds are similar to those found in uninduced and induced roots. Developmental expression of ADH and LDH was monitored from 0 to 24 days postgermination. Neither activity was induced to any extent in the germinating seeds; however, both enzymes were highly induced by anoxia in root tissue during development. Based on gel electrophoresis, this increase in activity results from the differential expression of different Adh and Ldh genes in root tissue. The changes in ADH and LDH activity levels were matched by changes in the amount of these particular proteins, indicating that the increase in activity results from de novo synthesis of these two proteins. The level of inducible LDH activity in an ADH1⁻ mutant was not found to differ from cv Himalaya. We suggest that although the ADH⁻ plants are more susceptible to flooding, they are not capable of responding to the lack of ADH1 activity by increasing the amount of LDH activity in root tissue.     

Enzymes of DNA biosynthesis in developing sea urchins. Changes in ribonucleotide reductase, thymidine, and thymidylate kinase activities.

The pattern of ribonucleotide reductase, thymidine kinase, and thymidylate kinase activities during development of Paracentrotus lividus eggs and the effect of actinomycin on these enzymatic activities have been studied. Ribonucleotide reductase activity is detectable, though at a low level, in the unfertilized egg; the activity increases sharply soon after fertilization and reaches a peak at the morula stage. Thereafter it decreases and remains at a lower level than that of the unfertilized egg. Actinomycin, at a concentration sufficient to inhibit messenger RNA (mRNA) synthesis does not affect the level of enzymatic activity, indicating that preexisting maternal mRNA is used for the synthesis of this enzyme. Thymidine kinase is present at a low level in the egg; it increases sharply after the hatching blastula until the pluteus stage. Actinomycin does not affect the enzyme activity from fertilization until blastula but prevents the increase in enzyme activity that is observed between blastula and pluteus. Thymidylate kinase activity shows an increase after fertilization, followed by fluctuations throughout development with a considerable decrease at the blastula stage and at the end of gastrulation. Actinomycin has no effect on the activity of thymidylate kinase regardless of when the drug is added to the embryo suspension. Possible regulatory mechanisms of DNA synthesis in sea urchin embryos are discussed: The presence in the unfertilized egg of the most important enzymes controlling the cellular flow of DNA precursors and the availability of dTTP suggest that the block in DNA synthesis observed in the unfertilized egg is due to some particular mechanism that is switched on at fertilization.