Cellular location,activity states,and macromolecular organization of glucoamylase in Clostridium thermosaccharolyticum

By application of immunocytochemical techniques at the electron microscope level, glucoamylase was localized to the cell periphery in Clostridium thermosaccharolyticum during and following growth on starch, sucrose or glucose. Levels of immunolabelling were found to be relatively independent of growth substrate and of phase of growth, whereas previous studies had demonstrated strong dependence of glucoamylase activity on growth conditions; previously high levels of glucoamylase activity had been detected after growth on starch (i.e. during the stationary phase after growth) and only very low activities detected during exponential growth and following growth on glucose. The results presented demonstrate that levels of the glucoamylase protein are independent of measurable enzyme activity, and imply that the protein is constitutive. This indicates that the protein can exist in active and inactive states in the cell. By analogy with similar systems, we consider it likely that maturation or activation of newly synthesized glucoamylase occurs during (or following) transport through the cytoplasmic membrane. Electron microscopy of individual protein molecules which had been subjected to negative staining revealed that the enzyme consists of two domains of approximately equal size which are linked by a hinge region.     

Genetic characterization of a new splice variant of the β2 subunit of the voltage-dependent calcium channel

This study reports a novel splice variant form of the voltage-dependent calcium channel ₂ subunit (_2g). This variant is composed of the conserved amino-terminal sequences of the _2a subunit, but lacks the -subunit interaction domain (BID), which is thought essential for interactions with the ₁ subunit. Gene structure analysis revealed that this gene was composed of 13 translated exons spread over 107 kb of the genome. The gene structure of the ₂ subunit was similar in exon-intron organization to the murine ₃ and human ₄ subunits. Electrophysiological evaluation revealed that _2a and _2g affected channel properties in different ways. The _2a subunit increased the peak amplitude, but failed to increase channel inactivation, while _2g had no significant effects on either the peak current amplitude or channel inactivation. Other subunits, such as ₃ and ₄, significantly increased the peak current and accelerated current inactivation.     

Tissue-specific developmental regulation of superoxide dismutase (SOD-1 and SOD-2) activities in genetic strains of mice

The activity levels of CuZn superoxide dismutase (SOD) (SOD-1) and Mn SOD (SOD-2) in liver, kidney, and lung were assessed in newborn and 3-, 10-, 25-, and 70-week-old females from seven genetic strains (BALB/c, Cs^b, C3H/HeSnJ, C3H/S, C57BL/6J, Swiss-Webster, and 129/ReJ) of mice. Total SOD enzyme activity was high at birth and declined somewhat with old age (70 weeks) in the liver and increased in both kidney and lung from newborn to 25 weeks. The activity level of SOD-1 was found to be highly variable among strains at different ages in liver, with little change associated with aging in the kidney, and showed a strain-specific increase during aging in the lung. In general, SOD-2 activity was lower than SOD-1 activity in liver and lung but levels of the two forms of this enzyme were similar in the kidney. The SOD-2 activity increased with age with little variation among strains in kidney. The increase in this form of the enzyme with age was relatively small and strain specific in lung and highly variable among strains in the liver. The Cs^b genotype (acatalasemic) at age 70 weeks showed an exceptionally high SOD-1 activity associated with an exceptionally low SOD-2 activity in the liver. Changes in enzyme activity with age in different tissues associated with differences in activity level among genotypes (of the type reported here for SOD-1 and SOD-2) may be indicative of a complex system of enzyme regulation. Further studies are needed to explain fully the genetic/molecular mechanism(s) for SOD regulation.This study was financially supported by an NSERC operating grant to S.M.S. N.J.S. was the recipient of a postgraduate scholarship from the NSERC during this period.     

ATP synthases—Structure of the F1-moiety and its relationship to function and mechanism

A great deal of progress has been made in understanding both the structure and the mechanism of F₁-ATPase. The primary structure is now fully known for at least five species. Sequence comparison between chloroplast, photobacteria, aerobic bacteria, and mitochondrial representatives allow us to infer more general functional relationships and evolutionary trends. Although the F₁ moiety is the most studied segment of the H⁺-ATPase complex, there is not a full understanding of the mechanism and regulation of its hydrolytic activity. The subunit is now known to contain one and probably two nucleotide binding domains, one of which is believed to be a catalytic site. Recently, two similar models have been proposed to attempt to describe the active part of the subunits. These models are mainly an attempt to use the structure of adenylate kinase to represent a more general working model for nucleotide binding phosphotransferases. Labelling experiments seem to indicate that several critical residues outside the region described by the adenylate kinase part of this model are also actively involved in the ATPase activity. New models will have to be introduced to include these regions. Finally, it seems that a consensus has been reached with regard to a broad acceptance of the asymmetric structure of the F₁-moiety. In addition, recent experimental evidence points toward the presence of nonequivalent subunits to describe the functional activity of the F₁-ATPase. A summary diagram of the conformational and binding states of the enzyme including the nonequivalent subunit is presented. Additional research is essential to establish the role of the minor subunits—and of the asymmetry they introduce in F₁—on the physiological function of the enzyme.     

Regional Activities of Phospholipase C after Experimental Brain Injury in the Rat

Regional activities of phosphoinositide-specific phospholipase C (PLC) were measured after lateral fluid percussion (FP) brain injury in rats. The activity of PLC on phosphatidylinositol 4,5-bisphosphate (PIP₂) in the rat cortex required calcium, and at 45 M concentration it increased PLC activity by about ten-fold. The activity of PLC was significantly increased in the cytosol fraction in the injured (left) cortex (IC) at 5 min, 30 min and 120 min after brain injury. However, in the same site, increases were observed in the membrane fraction only at 5 min after brain injury. In both the contralateral (right) cortex (CC) and ipsilateral hippocampus (IH), the activity of PLC was increased in the cytosol only at 5 min after brain injury. These results suggest that increased activity of PLC may contribute to increases in levels of cellular diacylglycerol and inositol trisphosphate in the IC (the greatest site of injury), and to a smaller extent in the IH and CC, after lateral FP brain injury. It is likely that this increased PLC activity is caused by alteration in either the levels or activities of one or more of its isozymes (PLC, PLC, and PLC) after FP brain injury.     

Effects of Aging and Amyloid-β Peptides on Choline Acetyltransferase Activity in Rat Brain

Choline acetyltransferase (ChAT, acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6), involved in the learning and memory processes is responsible for the synthesis of acetylcholine. There are many discrepancies in literature concerning ChAT activity during brain aging and the role of amyloid beta peptides in modulation of this enzyme. The aim of the study was to investigate the mechanism of ChAT regulation and age-related alteration of ChAT activity in different parts of the brain. Moreover the effect of A peptides on ChAT activity in adult and aged brain was investigated. The enzyme activity was determined in the brain cortex, hippocampus and striatum in adult (4-months-old), adult-aged (14-months-old) and aged (24-months-old) animals. The highest ChAT activity was observed in the striatum. We found that inhibitors of protein kinase C, A, G and phosphatase A2 have no effect on ChAT activity and that this enzyme is not dependent on calcium ions. About 70% of the total ChAT activity is present in the cytosol. Arachidonic acid significantly inhibited cytosolic form of this enzyme. In the brain cortex and striatum from aged brain ChAT activity is inhibited by 50% and 37%, respectively. The aggregated form of A 25-35 decreased significantly ChAT activity only in the aged striatum and exerted inhibitory effect on this enzyme in adult, however, statistically insignificant. ChAT activity in the striatum was diminished after exposure to 1 mM H₂O₂. The results from our study indicate that aging processes play a major role in inhibition of ChAT activity and that this enzyme in striatum is selectively sensitive for amyloid beta peptides.     

Replication in Drosophila chromosomes

It is widely known that the bulk of the pericentromeric heterochromatin (-heterochromatin) does not replicate during polytenization in Drosophila. However, a recent DNA-Feulgen cytophotometric study (Dennhöfer 1982a) has claimed equal polytenization of all heterochromatin regions. To re-examine this issue, the amount of Hoechst 33258-bright heterochromatin in non-polytene and polytene nuclei in salivary glands and Malpighian tubules of late third instar larvae of D. nasuta has been compared by cytofluorometry. Since the amount of Hoechst 33258-bright heterochromatin is similar in non-polytene and polytene nuclei in spite of the latter having an enormously high euchromatin DNA content, it is concluded that the -heterochromatin does not replicate during polytenization. The present results further indicate that in the polytene nuclei of Malpighian tubules the -heterochromatin remains at the 2C level whereas in salivary gland polytene nuclei it varies between the 2C and 4C levels.I would like to dedicate this paper to the memory of E. Heitz to commemorate 50 years of - and -heterochromatin     

Genetics and ontogeny of α-glycerophosphate dehydrogenase isozymes in Drosophila melanogaster

-Glycerophosphate dehydrogenase (GPDH) occurs in Drosophila melanogaster in three isozymic forms. These are separable by starch gel electrophoresis and have been tentatively numbered 1, 2, and 3. GPDH-1 is most concentrated in the adult thorax and GPDH-3 in the abdomen; 1 and 3 are in approximately equal amounts in the head. GPDH-2 is relatively weak in all preparations. In larvae, only GPDH-3 is present. Purified GPDH-1 has optimal activity at pH 6.7–7.0. GPDH-3 at pH 7.5, and GPDH-2 is intermediate. Changes in total GPDH activity parallel larval growth, pupal histolysis, and differentiation of adult tissues. In the latter period the ratio of activity at pH 6.7 to pH 7.6 increases, reflecting the shift from GPDH-3 to GPDH-1. Two types of homozygous GPDH patterns which differ in the electrophoretic mobilities of all three isozymes have been found in inbred strains. In heterozygous adults six bands, the parental forms of GPDH-1 and GPDH-3 and hybrid forms of each, can be resolved. Analysis of F₂ and backcross progeny suggests that a single genetic locus affects all three isozymes. Heterozygous embryos have only the maternal form of GPDH-3 until just before they hatch as first instar larvae. At this stage they have maternal and paternal GPDH-3 plus an intermediate band.This project was supported in part by National Institutes of Health research grant GM-15597.     

Production,purification, and properties of β-glucosidase from Candida wickerhamii

Summary Candida wickerhamii growing on cellobiose produced -glucosidase with high activity against -nitrophenyl glucoside (PNPG) but low activity against cellobiose. -glucosidase production was constitutive, and was repressed by -glucosides and glucose. -glucosides containing an aromatic moiety in the aglycon were the best substrates for -glucosidase indicating that the enzyme is an aryl--glucosidase. A -glucosidase from C. wickerhamii cells was purified by (NH₄)₂SO₄ precipitation, dialysis, ion-exchange chromatography and gel filtration. The purified enzyme was homogeneous as shown by sodium-dodecyl-sulphate polyacrylamide gel electrophoresis and discontinuous gel electrophoresis. The purified enzyme hydrolysed PNPG but not cellobiose. The K_m of the enzyme was 0.185 mM. Glucose inhibited the enzyme competitively and the K_i was 7.5 mM. The apparent molecular mass was 97,000. The optimum pH and temperature for enzyme activity were between pH 7 and 7.4 and 40°C respectively. At temperatures of 45°C and greater the enzyme was inactivated. The activation energy of the enzyme was 29.4 kJ · mol^-1.     

Enzymes in pancreatic islets that use NADP(H) as a cofactor including evidence for plasma membrane aldehyde reductase

Recent evidence of a pyruvate malate shuttle capable of transporting a large amount of NADPH equivalents out of mitochondria in pancreatic islets suggests that cytosolic NADP(H) plays a role in beta cell metabolism. To obtain clues about these processes the activities of several NADPHutilizing enzymes were estimated in pancreatic islets. Low levels of pyrroquinolone quinone (PQQ) and low levels of enzyme activity that reduce PQQ were found in islets. Low activities of palmitoylCoA and stearoylCoA desaturases were also detected. Significant activities of glutathione reductase, aldose reductase (EC.1.1.1.21) and aldehyde reductase (EC.1.1.1.2) were present in islets. Potent inhibitors of aldehyde and aldose reductases inhibited neither glucoseinduced insulin release nor glucose metabolism in islets indicating that these reductases are not directly involved in glucoseinduced insulin reaction. Over 90% of aldose reductase plus aldehyde reductase enzyme activity was present in the cytosol. Kinetic and chromatographic studies indicated that 60–70% of this activity in cytosol was due to aldehyde reductase and the remainder due to aldose reductase. Aldehyde reductaselike enzyme activity, as well as aldose reductase immunoreactivity, was detected in rat islet plasma membrane fractions purified by a polyethylene glycolDextran gradient or by a sucrose gradient. This is interesting in view of the fact that voltagegated potassium channel beta subunits that contain aldehyde and aldose reductaselike NADPH-binding motifs have been detected in plasma membrane fractions of islets [Receptors and Channels 7: 237–243, 2000] and suggests that NADPH might have a yet unknown function in regulating activity of these potassium channels. Reductases may be present in cytosol to protect the insulin cell from molecules that cause oxidative injury.     

Effect of dexamethasone on the neonatal adrenal medulla

Summary Newborn male rats were injected with a single dose of dexamethasone on the first day of life. Controls received only the diluent. Two to ten days later the experimental and control animals were sacrificed, and the adrenal medullae were processed for electron microscopy and for histochemical demonstration of catecholamines. Rats that received dexamethasone, as compared to controls showed (1) an earlier appearance of the catecholamine reaction, and (2) a pronounced development of the Golgi complex. These results are further indication that glucocorticoid stimulates the maturation of the chromaffin complex. The Golgi apparatus may play some role in this inductive mechanism.Supported by grants from Consejo Nacional de Investigaciones Científicas y Técnicas Argentina, and Comisión Asesora de Promotión a la Investigatión, Universidad National de Cuyo, Argentina. We are grateful to Teresa H. Fogal Sarmiento for her technical collaborationScientist of Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina     

Immunological comparison of enzymes of the β-ketoadipate pathway

-Carboxy-cis,cis-muconate lactonizing enzyme and -carboxymuconolactone decarboxylase catalyze sequential reactions in the -ketoadipate pathway, the subunit sizes of the enzymes from Pseudomonas putida, biotype A, are 40000 and 13000, respectively. The cross reaction of antisera prepared against the enzymes was tested with the isofunctional enzymes formed by representatives of other bacterial species. Despite the differences in the subunit sizes of the enzymes, the antisera revealed the same general pattern: cross reaction was observed with the corresponding enzymes formed by other strains in the fluorescent Pseudomonas RNA homology group I and generally was not observed with enzymes from other Pseudomonas species or from other bacterial genera. Exceptions were provided by representatives of Pseudomonas cepacia. Members of this species are classified outside the fluorescent Pseudomonas RNA homology group. Nevertheless, the -carboxymuconolactone decarboxylases from these organisms formed precipitin bands with antisera prepared against the corresponding enzyme from P. putida, biotype A; the lactonizing enzymes from the two species did not appear to cross react. Immunodiffusion experiments with -carboxymuconolactone decarboxylase indicated that a common set of antigenic determinants for the enzyme is conserved among strains that have been classified together by other criteria; the relative immunological distances of the decarboxylases of each taxon from the reference P. putida, biotype A, enzyme were indicated by spurring patterns on Ouchterlony plates. These results suggested that the interspecific transfer of the structural gene for the enzyme is not a common event in Pseudomonas.Non-Standard Abbreviations CMLE -carboxy-cis,cis-muconate lactonizing enzyme (EC 5.5.1.2) - CMD -carboxymuconolactone decarboxylase (EC 4.1.1.44) - MLE cis,cis-muconate lactonizing enzyme (EC 5.5.1.1) - MI muconolactone isomerase (EC 5.3.3.4) Dedicated with affection and admiration to Professor R. Y. Stanier on his 60th birthday     

Regional and subcellular distribution of taurine-synthesizing enzymes in the rat central nervous system

The distribution of cysteine oxidase (CO) and cysteine sulfinate decarboxylase (CSD) was examined in 12 regions of the rat central nervous system (CNS). The distribution of CO activity, expressed as mol of cysteine sulfinate formed per h per g, was the following: hypothalamus, superior and inferior colliculi, 94–99 mol/h/g; olfactory bulbs, cerebral cortex, striatum, and hippocampus, 44–51 mol/h/g; cerebellum, 71 mol/h/g; pons-medula and spinal cord, 94 and 60 mol/h/g, respectively. The distribution of CSD activity expressed as mol of cysteine sulfinate decarboxylated per h per g was the following: hypothalamus and colliculi, 14–21 mol/h/g; olfactory bulbs, cerebral cortex, striatum, hippocampus, and cerebellum, 8–13 mol/h/g; pons-medulla, 7.3; and spinal cord, 3.6 mol/h/g. No CSD activity was detected in sciatic nerve. The subcellular distribution of CO and CSD activities was studied in hypothalamus, colliculi, and cerebral cortex. CO activity was localized in synaptosomes, mitochondria, and microsomes. CSD was primarily confined to the crude mitochondrial fraction and after subfraction, recovered mainly in the synaptosomal fraction.     

β-1,3-Glucanase and dimorphism in Paracoccidioides brasiliensis

Mycelial and yeast forms of P. brasiliensis were tested for several glucohydrolases. In addition to high levels of -blucanases, low amounts of -glucanase, chitinase and maltase were found. Tests for invertase, amylase and lactase were negative. The levels of -1,3-glucanase were higher in the mycelial form. The shift to the mycelial phase correlated with an increase in the levels of -1,3-glucanase. The enzyme was present in the cytoplasm, cell wall and culture medium. The extracellular enzyme was purified 42 fold by ammonium sulphate precipitation and gel filtration. Maximal activity was obtained at 60°C and pH of 5.0 acetate buffer or pH 6.0 (phosphate buffer). Its K _m was 0.205 mg/ml. The cell wall-bound enzyme showed a higher temperature optimum. Optimum pH and K _m were also slightly different. Following treatment of the cell walls with chitinase, -1,3-glucanase was released into the medium.     

Ultrastructural localization of acid phosphatase activity in the small intestinal absorptive cells of adult rats

Summary Ultrastructural localization of acid phosphatase activity was investigated in ultrathin (0.05 m) and semithin (0.5 and 0.75 m) sections of the small intestinal epithelial cells of adult rats. The results showed that the enzyme activity was localized on the membrane of microvilli, lateral cell membranes, lysosomes, the Golgi complex, and the GERL of Novikoff (a part of the smooth-surfaced endoplasmic reticulum located in close proximity to the inner Golgi saccules) of duodenal absorptive cells. The lysosomes contained within the duodenal and jejunal absorptive cells appeared to be mainly heterolysosomes rather than autolysosomes. The enzyme activity of absorptive cells was lower in the jejunum than in the duodenum, and was barely detectable except in the GERL and lysosomes of the ileum. The average numbers of lysosomes having a diameter of 0.21.0 m, per cell profile in sections of 214 duodenal, 226 jejunal and 318 ileal epithelial cells were 8.9±0.189, 6.4±0.155 and 3.5±0.027 (mean±SE), respectively. From these results, it was assumed that both the Golgi apparatus and GERL produce some lysosomes in the duodenal and jejunal absorptive cells, but only GERL does so in the ileum. It was considered also that because of an unexpectedly high number of lysosomes contained within the epithelial absorptive cells of the proximal intestine of adult rats, these cells may possess the strong heterophagic, as well as absorptive capacity.     

Enzymatic and morphological response of the thymus to drugs in normal and zinc-deficient pregnant rats and their fetuses

Summary In the thymus of normally fed pregnant rats the plasma membrane enzymes dipeptidyl peptidase IV (DPP IV) and alkaline phosphatase (alP) were found in cortical and medullary lymphocytes (thymocytes). Plasma membrane aminopeptidase A (APA) and adenosine monophosphate hydrolysing phosphatase (AMPP) were present in cortical reticular cells. In medullary reticular cells, aminopeptidase M (APM), -glutamyl transferase (GGT), adenosine triphosphate (ATPP) and thiamine pyrophosphate (TPPP) cleaving phosphatases were detected. Medullary reticular cells did not contain APA. Lysosomal DPP I and II, acid phosphatase, acid -d-galactosidase, -d-N-acetylglucosaminidase, -d-glucuronidase and non-specific esterases occurred especially in macrophages at the corticomedullary junction. The 21-day-old fetal thymus showed a similar reaction pattern as the maternal organ except for APA which was absent before birth.—After treatment of the pregnant rats with valproic acid (VPA), salicylic acid (SA), streptozotocin (ST) and retinoic acid (RA) APA showed an increase in activity in the thymic cortex. In addition, ST and RA induced AMPP, ATPP and TPPP activity in cortical reticular cells up to the same pattern as in medullary reticular cells. After ethanol (ET) administration severe damages occurred. The thymic cortex was free of DPP IV-positive lymphocytes; the medullary reticular cells showed reduced or no GGT and occasionally an increased APM activity. Dexamethasone (DEXA) given to normal or zinc-deficient rats produced the most severe lesions; thymocytes with DPP IV activity were completely absent in the cortex and medulla. In Zn-deficient pregnant rats similar alterations were observed as after ET. When the drugs were applied to Zn-deficient pregnant rats, the alterations resembled those observed after drug treatment alone. In all cases of severe thymus degeneration, i.e. ET and DEXA treatment and Zn-deficiency, the number of macrophages and activities of lysosomal hydrolases in macrophages and reticular cells were increased; the lysosomal hydrolases were often homogeneously distributed over the cortex. Cell contacts between reticular cells and lymphocytes were reduced. Vacuoles occurred within the reticular cells.—The fetal thymus was reduced in size and the number of macrophages and the activities of their lysosomal enzymes were increased after Zn-deficiency, DEXA treatment and Zn-deficiency combined with ET administration.Supported by the Deutsche Forschungsgemeinschaft (Sfb 174)     

Glutamate decarboxylase and GABA transaminase activity in the mitochondrial fraction of the dog brain limbic system during postnatal development

Overall glutamate decarboxylase (GDCase) activity in the initial mitochondrial fraction of the limbic structure is found to be regionally different it increases from the moment of birth up to 1 year, the midbrain reticular formation (RF), where the enzyme activity in the mitochondrial decreases in pups aged 3 month and reincreases in 1 year old dogs being the exception. Overall GABA-transaminase (GABA-T-ase) activity reaches the "adult" level and is the highest: in the hypothalamic and hippocampal mitochondria on the 1st postnatal day; in the limbic cortex (l1 and l2 fields), amygdala and midbrain RF--on the 12-16th postnatal days. During the period from 12-16 postnatal days up to the age of 1 year GABA-T-ase activity in the dog limbic system decreases reliably.     

Discrepancy between the histochemically and biochemically determined 3β-hydroxysteroiddehydrogenase activity in the calf adrenal cortex

Summary In sections of the calf adrenal cortex the histochemically determined3-hydroxysteroiddehydrogenaae activity is lower in the zona glomerulosa than in the zona fasciculata. Biochemically the activity of this enzyme was found in mitochondrial as well as in microsomal fractions. The mitochondrial, respectively the microsomal fractions of the two zones showed identical enzyme activities. So there is a discrepancy between the histochemical results on tissue sections and biochemical results obtained with isolated subcellular fractions.However, if the histochemical determination of 3-hydroxysteroiddehydrogenase activity is carried out on sections of mitochondrial and microsomal pellets of the two zones the results are in agreement with the biochemical findings. Therefore the observed discrepancy rather seems to be related with the state of tissue—intact or cell fractions—than with the used histochemical method.     

Sequence analysis of the upstream regions of Xenopus laevis β-globin genes and arrangement of repetitive elements within the globin gene clusters

The globin gene clusters of Xenopus laevis are interspersed by various different repetitive DNA elements. A specific repeat, the JH12 element, has been mapped by Southern analysis and some of its locations have been subsequently confirmed by nucleotide sequencing. JH12 family members seem to represent mobile genetic elements and display a high degree of divergence. The nucleotide sequences upstream to the adult _I-globin gene and to the two coordinately expressed larval _I- and _II genes have been determined and compared to those of the adult -genes. Besides some repetitive DNA elements and a short sequence of rather weak homology we have found no characteristic sequence motifs to be common to the adult - and -genes. The two larval -genes share one short sequence element being absent from the adult genes. This might reflect completely different sequence requirements for protein interactions and for the regulation of adult and larval globin gene expression.     

Enzymatic synthesis of poly-β-hydroxybutyrate inZoogloea ramigera

The enzyme activity synthesizing poly--hydroxybutyrate (PHB) was mainly localized in the PHB-containing particulate fraction ofZoogloea ramigera I-16-M, when it grew flocculatedly in a medium supplemented with glucose. On the other hand, the enzyme activity remained in the soluble fraction, when the bacterium grew dispersedly in a glucose-starved medium.The soluble PHB synthase activity became associated with the particulate fraction as PHB synthesis was initiated on the addition of glucose to the dispersed culture. Conversely, the enzyme activity was released from the PHB-containing granules to the soluble fraction when the flocculated culture was kept incubated without supplementing the medium with glucose.PHB synthase was also incorporated into the newly formed PHB fraction when partially purified soluble PHB synthase was incubated withd(-)--hydroxybutyryl CoA in vitro.Although attempts to solubilize the particulate enzyme were unsuccessful, and the soluble enzyme became extremely unstable in advanced stages of purification, both PHB synthases had the same strict substrate specificity ford(-)--hydroxybutyryl CoA, and showed the same pH optimum at 7.0.Non-Standard Abbreviations PHB poly--hydroxybutyrate