Changes in the Activity of Certain Enzymes of Acer pseudoplatanus L. Cells at four Stages of Growth in Suspension Culture

The activities of twelve enzymes were studied in Acer psendoplatanum L. cells grown in suspension cultures for 6, 12, 19 and 22 days. The enzyme activities of cells (6 and 12 days) in the phase of rapid cell division were rather different from that of cells in the stationary phase (19, 22 days). The activity of aldolasc was highest at the first two stages of growth, when the amount of RNA and protein per cell was highest, and after this decreased steeply. The curves of specific activities of several phosphatases (glucosc-6-phosphatasc, α-glycerophosphatase, acid phosphatase and ATPase) and ribonuclease were almost the reverse of the curve of aldolase, showing a minimum in 12-day-old cells and a maximum in cells 19 and 22 days old. The activity of peroxidase was also high in ageing cells. Glutamate: oxaloacetate transaminase and aminopeptidases (leucine and alanine) had synchronous maxima (6 and 19 days) of specific activity but the activity of aminopeptidases decreased gradually during ageing of the suspension, if the enzyme activities are calculated per 10⁷ cells. Glutamate: pyruvate transaminasc activity was very low and no dcoxyribonuclease activity could be detected.     

High Activities of Glutamine Transaminase K (Dichlorovinylcysteine β-Lyase) and ω-Amidase in the Choroid Plexus of Rat Brain

Abstract: Certain halogenated hydrocarbons, e.g., dichlo-roacetylene, are nephrotoxic to experimental animals and neurotoxic to humans; cysteine-S-conjugate β-lyases may play a role in the nephrotoxicity. We now show that with dichlorovinylcysteine as substrate the only detectable cysteine-S-conjugate β-lyase in rat brain homogenates is identical to glutamine transaminase K. The predominant (mitochondrial) form of glutamine transaminase K in rat brain was shown to be immunologically distinct from the predominant (cytosolic) form of the enzyme in rat kidney. Glutamine transaminase K and ω-amidase (constituents of the glutaminase II pathway) activities were shown to be widespread throughout the rat brain. However, the highest specific activities of these enzymes were found in the choroid plexus. The high activity of glutamine transaminase K in choroid plexus was also demonstrated by means of an immunohistochemical staining procedure. Glutamine transaminase K has a broad specificity toward amino acid and α-keto acid substrates. The ω-amidase also has a broad specificity; presumably, however, the natural substrates are α-ketoglutaramate and α-ketosuccinamate, the α-keto acid analogues of glutamine and aspara-gine, respectively. The high activities of both glutamine transaminase K and ω-amidase in the choroid plexus suggest that the two enzymes are linked metabolically and perhaps are coordinately expressed in that organ. The data suggest that the natural substrate of glutamine transaminase K in rat brain is indeed glutamine and that the metabolism of glutamine through the glutaminase II pathway (i.e., l -glutamine and α-keto acid α-ketoglutarate and l -amino acid + ammonia) is an important function of the choroid plexus. Moreover, the present findings also suggest that any explanation of the neurotoxicity of halogenated xenobiotics must take into account the role of glutamine transaminase K and its presence in the choroid plexus.     

Mode of Action of α, β Unsaturated Carbonyl Compounds

The toxicity of the α, β unsaturated carbonyl compounds (α, β UCC_s) (patulin, penicillic acid, parasorbic acid, tulipalin and plumbagin) towards Pythium sp. group F (Van Der Plaat -Niterinks 1981) was neutralized by the addition of an excess of cysteine. This suggests that the mode of action of these compounds could be due to a binding of the α, βi UCC_s to sulphydryl groups in enzymes or other macromolecules. Alcohol dehydrogenase (ADH), an enzyme with a sulphydryl group at the active site, was assayed spectrophotometrically and all the α, β UCC_s inhibited ADH.     

α-Glucosidase Activity Located at the Cell Surface in Callus of Convolvulus arvensis

Cell wall preparations of Convolvulus callus were found to contain α-glucosidase activity, the bulk of which could be solubilized by solutions of high ionic strength. Callus tissue incubated in 0.5 M KC1 released α-glucosidase activity into the washing medium as distinct from tissue incubated in 1.0 M sorbitol. The wall-bound activity of KCl-treated tissue was found to be less than that of sorbitol-treated tissue, while the difference between both activities proved to be equal to the enzyme activity found in the washing medium of KCl-treated tissue. Since no trace of cell leakage was observed, it is concluded that α-glucosidase activity is located at the cell surface. The level of this surface-located enzyme was not affected by the presence of maltose in the nutrient medium.     

ADH, α-GPDH and SOD enzyme activities of second and third chromosomal genotypes from two geographically different populations of Drosophila melanogaster

ADH, α-GPDH and SOD enzyme activities have been measured in lines of Drosophila melanogaster homozygous and/or heterozygous for chromosomes extracted from two different populatioi Globally the results demonstrate that factors other than structural genes are determining the observed pattern of enzyme activities. ADH and α-GPDH activities are, however, more affected than SOD by these factors. Geographic origin, sex, chromosome, genetic background of the lines, containing regulatory genes in a broad sense, can be mentioned as the more relevant factors that influencing enzyme activities. A high and significant correlation is detected between ADH and α-GPDH enzyme activities and it can be interpreted as due to linkage disequilibrium among these two loci. SOD activity shows a lesser correlation with ADH and α-GPDH because it is less variable within population, i.e. it is a more canalized character. Finally, a principal component analysis, using the three enzyme systems shows that both populations are clearly separated, with a first principal component explaining 71.1 percent of the observed variance.     

Characterization of spinach leaf α-l-arabinofuranosidases and β-galactosidases and their synergistic action on an endogenous arabinogalactan-protein

Two β-galaclosidases (β-Galase-I and -II, EC 3.2.1.23) and two α-l -arabinofuranosidases (α-l -Arafase-I and -II. EC 3.2.1.55). were purified from mesophyll tissues of spinach (Spinacia oleracea L.), using chromatography on DEAE-cellulose, lactose-conjugated Sepharose CL-4B, and Sephadex G-100, or on hydroxylapatite and Sephadex G-150. The apparent molecular mass (M_r) of β-Galase-I and -II, respectively, were estimated to be 38 000 and 58 000 on SDS-PAGE and 64 000 and 60 000 on gel-permeation chromatography, indicating that the former was a dimeric protein. The isoelectric points of β-Galase-I and -II were 6.9 and 5.2, respectively. Both enzymes hydrolyzed maximally p-nitrophenyl (PNP) β-galactoside at pH 4.3, and were activated about 2-fold in the presence of BSA (100 μg ml^?1). The activity of both enzymes was inhibited strongly by heavy metal ions and p-chloromercuriberszoate (p-CMB). d -Galactono-(1→4)-lactone and d -galactal served as potent competitive inhibitors for the enzymes. β-Galase-I and -II could be distinguished from each other in their relative rates and kinetic properties in the hydrolysis of aryl β-galactosides as well as of lactose and galacto-oligosaccharides. In particular. β-Galase-I exhibited a preferential exowise cleavage of β-1,6-galactotriose and β-1.3-galactan. α-l -Arafase-l (M_r 118000) and -II (M, 68 000) were optimally active on PNP α-l -arabinofuranoside at pH 4.8 and gave K_m values of 1.2 and 2.2 mM. respectively. l -Arabino-(1 → 4)-lactone. Ag⁺, and SDS acted as inhibitors for the isozymes. α-l Arafase-I was characterized by its activity to hydrolyze PNP β-d -xylopyranoside besides PNP α-l -arabinofuranoside. inhibition by d -xylose and d -glucono-(1 → 5)-lactone. and less sensitivity to Hg²⁺. Cu²⁺, and p-CMB. Sugar beet arabinan was hydrolyzed rapidly by α-l Arafase-II at one-half the rate for PNP α-l arabinofuranoside, while the polysaccharide was less susceptible to α-l Arafase-I. A spinach leaf arabinogalactan-protein was practically resistant to the action of β-Galases, but its susceptibility to the enzymes increased remarkably after prior hydrolysis with α-l Arafase-Il.     

Abscisic Acid as Inhibitor of α-Amylase from Aspergillus and Bacillus subtilis

Abscisic acid (ABA) inhibited the activity of α-amylase from both Aspergillus and Bacillus subtilis in vitro if ABA and enzyme solutions were allowed to react with each other before adding to the starch solution. If the ABA solution was put to starch before adding the enzyme, no inhibition occurred. The inhibition increased with increasing time between mixing ABA and enzyme solutions and adding the mixture to starch. It was not the absolute amounts of enzyme and ABA which were of importance for the inhibition, but the concentrations of ABA and enzyme in the ABA + enzyme mixture. Within certain limits the inhibition was proportional to the concentration of ABA, so that it should be possible to use the inhibition in quantitative tests for inhibitors. Dialysis of a mixture of ABA and enzyme showed that ABA is bound to the enzyme. The enzyme was still inhibited after dialysis for 25 h. On the other hand, partitioning with diethylether from acid water solution could free the enzyme from all ABA. Supposedly ABA acts as an allosteric inhibitor. The results may offer the foundation for one possible way to explain why inhibitors in plants sometimes inhibit growth and sometimes do not. If inhibitor, enzyme and substrate are compartmentalized, the degree of reaction should depend upon the sequence in which the three components meet each other.     

Brain α-Ketoglutarate Dehydrotenase Complex Activity in Alzheimer's Disease

We measured the activity of the a-ketoglutarate dehydrogenase complex (α-KGDHC), a rate-limiting Krebs cycle enzyme, in postmortem brain samples from 38 controls and 30 neuropathologically confirmed Alzheimer's disease (AD) cases, in both the presence and absence of thiamine pyrophosphate (TPP), the enzyme's cofactor. Statistically significant correlations between brain pH and lactate levels and α-KGDHC activity in the controls were observed, suggesting an influence of agonal status on the activity of α-KGDHC. As compared with the controls, mean α-KGDHC activity, with added TPP, was significantly (p < 0.005) reduced in AD brain in frontal (-56%), temporal (-60%), and parietal (-68%) cortices, with the reductions (-25 to -53%) in the occipital cortex, hippocampus, amygdala, and caudate failing to reach statistical significance. In the absence of exogenously administered TPP, mean a-KGDHC activity was reduced to a slightly greater extent in all seven AD brain areas (-39 to -83%), with the reductions now reaching statistical significance in the four cerebral cortical areas and hippocampus. A statistically significant negative correlation was observed between α-KGDHC activity and neurofibrillary tangle count in AD parietal cortex, the brain area exhibiting the most marked reduction in enzyme activity; this suggests that the enzyme activity reduction in AD brain may be related to the disease process and severity. In each brain area examined, TPP produced a greater stimulatory effect on α-KGDHC activity in the AD group (23–280% mean stimulation) as compared with the controls (-4 to ±50%); this TPP effect could be explained by reduced endogenous TPP levels in AD brain. Reduced brain α-KGDHC activity could be consequent to loss of neurons preferentially enriched in α-KGDHC, a premortem reduction in TPP levels (which may have affected enzyme stability), elevated brain levels of the α-KGDHC inhibitor ammonia, or an actual failure in the expression of the gene encoding the enzyme. We suggest that a defect in this key Krebs cycle enzyme could contribute to an impairment of cerebral energy metabolism and the brain dysfunction in AD.     

PURIFICATION AND PROPERTIES OF HUMAN BRAIN α-L-FUCOSIDASE

Human brain α-L-fucosidase has been extracted and the soluble portion has been purified 9388-fold with 25% yield by a two-step affinity chromatographic procedure utilizing agarose-epsilon-aminocaproyl-fucosamine. Isoelectric focusing revealed that all seven isoelectric forms of the enzyme were purified. Trace amounts of eight glycosidases, with hexosaminidase being the largest contaminant (1% by activity) were found in the purified α-L-fucosidase preparation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of a single subunit of molecular weight 51,000 ± 2500. The purified enzyme has a pH optimum of 4.7 with a suggested second optimum of 6.6. The apparent Michaelis constant and maximal velocity of the purified enzyme with respect to the p-nitrophenyl substrate are 0.44 mM and 10.7 μmol/min/mg protein, respectively. Ag²⁺ and Hg²⁺ completely inactivated the enzyme at concentrations of 0.1-0.3 mM. Antibodies made previously against purified human liver α-L-fucosidase cross-reacted with the purified brain α-L-fucosidase and gave a single precipitin line coincident with that from purified liver α-L-fucosidase. From all our studies it appears that at least the soluble portion of brain α-L-fucosidase is identical to human liver α-L-fucosidase.     

α-glucosidase in Mycoplasma mycoides subspecies capri

Abstract Mycoplasma mycoides subsp. capri utilisede maltose in medium lacking serum and hence serum saccharolytic enzymes. The presence of α-glucosidase activity was demonstrated by p-nitrophenyl-α- d -glucoside hydrolysis in toluene-treated cells. Specific activities were approx. 4-fold higher in cells grown in the presence of maltose than in cells grown with other sugars or with glucose plus maltose. Extracellular activity was < 2% of cellular activity in growing cultures. α-Glucosidase activity was also demonstrated in cells grown in medium containing serum. It is suggested that the presence of α-glucosidase might be of value in mycoplasma chatacterisation; in a previous study, α-glucosidase activity was not detected in Mycoplasma mycoides subsp. mycoides .     

Cytokinin Reversal of Abscisic Acid Inhibition of Growth and α-Amylase Synthesis in Barley Seed

The effect of cytokinin, kinetin, on abscisic acid (dormin) inhibition of α-amylase synthesis and growth in intact barley seed was investigated. Abscisic acid at 5 × 10^?5M nearly completely inhibited growth response and α-amylase synthesis in barley seed. Kinetin reversed to a large extent abscisic acid inhibition of α-aniylase synthesis and coleoptile growth. The response curves of α-amylase synthesis and coleoptile growth in presence of a fixed amount of abscisic acid (6 × l0^?6M) and increasing concentrations of kinetin (from 5 × l0^?7M to 5 × 10^?5 M) showed remarkable similarity. Kinetin and abscisic acid caused synergistic inhibition of root growth. Gibberellic acid was far less effective than kinetin in reversing abscisic acid inhibition of α-amylase synthesis and coleoptile growth. A combination of kinetin and gibberellic acid caused nearly complete reversal of abscisic acid inhibition of α-amylase synthesis but not the abscisic acid inhibition of growth. The results suggest that factors controlling α-amylase synthesis may not have a dominant role in all growth responses of the seed. Kinetin possibly acts by removing the abscisic acid inhibition of enzyme specific sites thereby allowing gibberellic acid to function to produce α-amylase.     

Endogenous elemental sulfur (S°) in dormant and aging α-spores of Phomopsis viticola

Endogenous elemental sulfur (S°) was measured in dormant α-spores of Phomopsis viticola Sacc. (ATCC 44940) from young (25-day-old) and aging (105-day-old) cultures grown on malt extract agar medium enriched with [³⁵S]-MgSO₄. Endogenous S° from the mitochondrial fraction, and the lipid and aqueous cytoplasmic fractions of young and aging α-spores were purified by column chromatography followed by thin-layer chromatography. The purity of mitochondrial pellets were checked by the catalase (EC 1.11.1.6) and acid phosphatase (EC 3.1.3.1) activities. Activities of the mitochondrial enzymes NAD⁺-isocitrate dehydrogenase (EC 1.1.1.41) and cytochrome c oxidase (EC 1.9.3.1) were also measured to determine the distribution of the endogenous S° between mitochondria and cytoplasm. In young dormant α-spores, endogenous S° was mostly found in the cytoplasmic lipid reserves, which were mainly phospholipids. The mitochondrial fraction of these young α-spores contained ca 10% of the total endogenous S°, whereas in aging α-spores stored for 105 days the endogenous S° was mainly (ca 90%) localized in the mitochondrial fraction. This accumulation of S° in mitochondria of aging α-spores was correlated with a sharp decrease in phospholipid reserves, endogenous and exogenous respiratory activities, ATP concentration, uptake of sulfate, and NAD⁺-isocitrate dehydrogenase and cytochrome c oxidase activities. These metabolic changes were correlated with an irreversible loss of germination capacity which leads to the natural death of P. viticolaα-spores. During the first min of the breaking of dormancy, the young α-spores possess a 7.3-fold capacity to reduce exogenous S° with production of hydrogen sulfide, as compared to the aging α-spores. In young α-spores the production of hydrogen sulfide was almost totally inhibited by 40 μM antimycin A (92%), and strongly inhibited by 2mM azide (75%) and by 15 μM 2,4-dinitrophenol (63%). Our work suggests that endogenous S° plays a key role in the regulation of the dormancy and aging processes of α-spores of P. viticola.     

MECHANISM OF ACTION OF β-N-OXALYL-L-α, β-DIAMINOPROPIONIC ACID, THE LATHYRUS SATIVUS NEUROTOXIN

The source of ammonia in the brain tissue of young rats treated with β-N-oxalyl-l -α, β-diaminopropionic acid (ODAP) has been studied. ODAP administration to 12-day-old rats causes a significant increase in the levels of adenylic acid deaminase in the brain. Glutaminase activity also shows an increase under these conditions. An increase in the levels of acid protease and transglutaminase is also observed in the brain of ODAP-treated animals. Glutamate dehydrogenase activity is decreased slightly. Glutamine synthetase enzyme is not affected. Aspartate-α-ketoglutarate transaminase and aspartate-pyruvate transaminase activities are enhanced in the brain tissue of ODAP-treated rats. It is held that protein degradation, especially the cleavage of free and protein-bound amide bonds, may be responsible for excess ammonia liberation in the brain of ODAP-treated young rats.     

Role of the conserved carboxy-terminal α-helix of Rad6p in ubiquitination and DNA repair

RAD6 in the yeast Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme essential for DNA repair as well as for a number of other biological processes. It is believed that the functions of Rad6p require the ubiquitination of target proteins, but its substrates as well as other interacting proteins are largely unknown. Rad6p homologues of higher eukaryotes have a number of amino acid residues in the C-terminal α-helix, which are conserved from yeast to man but are absent from most other yeast ubiquitin-conjugating enzymes (Ubcs). This specific conservation suggests that the C-terminal a-helix is important for the unique activities of the Rad6p family of Ubcs. We have investigated the effects of mutating this highly conserved region on the ubiquitination of model substrates in vitro and on error-free DNA repair in vivo. C-terminal point and deletion mutants of Rad6p differentially affected its in vitro activity on various substrates, raising the possibility that Rad6p interacts with its substrates in vivo by similar mechanisms. The distal part of the C-terminal u-helix is also essential for error-free DNA repair in vivo. Overexpression of Rad18p, a single-stranded DNA-binding protein that also interacts with Rad6p, alleviates the DNA repair defects of the C-terminal α-helix mutants to different degrees. This indicates that the C-terminal α-helix of Rad6p mediates its interaction with Rad18p, an essential step in DNA repair. Models of Rad6p action propose that its ubiquitination function is followed by proteolysis of unknown ubiquitinated targets. Mutants affecting several functions of the 26S proteasome retain wild-type capacity for error-free DNA repair. This raises the possibility that ubiquitination by Rad6p in DNA repair does not target proteins for proteasomal degradation.     

Utilization of starch and synthesis of a combined amylase/αα-glucosidase by the human colonic anaerobe Bacteroides ovatus

Bacteroides ovatus preferentially utilized starch and pectin when grown on a mixture of polysaccharides in batch culture, indicating that these carbohydrates are important substrates for the bacterium in the human large intestine. Further studies on starch breakdown showed that continuous cultures grew on the polysaccharide when it provided the sole carbohydrate source, to yield a single hydrolytic product at low dilution rates ( D = 0·04 h⁻¹), with an estimated molecular mass of 13 kDa. In contrast, two major types of oligomeric products were formed at higher dilution rates ( D = 0·44 h⁻¹), with approximate molecular weights of 11 and 140 kDa. Analysis of cell-associated starch-degrading enzymes produced by Bact. ovatus using ion exchange chromatography and HPLC gel-filtration showed that amylase and α-glucosidase activities eluted in the same fractions. The single peak containing amylase and α-glucosidase activities obtained by HPLC gel-filtration chromatography corresponded to a molecular mass of approximately 140 kDa, and activity staining of gels for α-glucosidase activity after polyacrylamide gel electrophoresis, in the presence of sodium dodecyl sulphate, gave an estimated molecular mass of 70 kDa, indicating this enzyme to be a dimer. After renaturation, the 70 kDa band was cut from the gels and solubilized. The extract hydrolysed gelatinized starch and p -nitrophenyl-α- D -glucopyranoside.     

Virulence studies on chromosomal α-toxin and Θ-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of α-toxin in Clostridium perfringens-mediated gas gangrene

The pathogenesis of clostridial myonecrosis, or gas gangrene, involves the growth of the anaerobic bacterium Clostridium perfringens in the infected tissues and the elaboration of numerous extracellular toxins and enzymes. The precise role of each of these toxins in tissue invasion and necrosis has not been determined. To enable genetic approaches to be used to study C. perfringens pathogenesis we developed an allelic exchange method which involved the transformation of C. perfringens cells with a suicide plasmid carrying a gene insertionally inactivated with an erythromycin-resistance determinant. The frequency with which double reciprocal crossover events were observed was increased to a workable level by increasing the amount of homologous DNA located on either side of the inactivated gene. Allelic exchange was used to isolate mutations in the‘chromosomal pfoA gene, which encodes an oxygen-labile haemolysin known as Θ-toxin or perfringolysin O. and in the chromosomal pic gene, which encodes the α-toxin or phospholipase C. The resultant mutants failed to produce detectable Θ-toxin or α-toxin activity, respectively, and could be complemented by recombinant plasmids that carried the respective wild-type genes. The resultant strains were virulence tested in a mouse myonecrosis model. The results showed that the pic mutants had demonstrably reduced virulence and therefore provided definitive genetic evidence for the essential role of α-toxin in gas gangrene or clostridial myonecrosis.     

The Influence of the Synthetic Growth Regulator α-Chloro-(β-(3-chloro-o-tolyl) propionitrile on Some Phytochrome-mediated Processes

In intact Sinapis alba seedlings the synthetic growth regulator α-chloro-β-(3-chloro-o-tolyl)propionitrile (PRB-8) inhibits the phytochrome-mediated anthocyanin synthesis, carotenoid synthesis and L-phenylalanine ammonia-lyase activity. This action was compared with that of other auxins and with that of α-chloro-β-(3-chloro-o-tolyl)propionitrile-homologues. From the results it appeared that PRB-8 and some other o-tolyl derivatives are more inhibitive than other known auxins.     

INHIBITION OF ALANINE AND ASPARTATE AMINOTRANSFERASES BY α-OXODERIVATIVES OF THE BRANCHED-CHAIN AMINO ACIDS

Abstract—

• 1 L-Alanine: α-oxoglutarate aminotransferase was partly purified from rat brain and liver. The enzyme from the brain has about 10 times less activity than that from the liver.
• 2 Both enzymes have identical apparent K_m values for L-alanine, L-glutamate, α-oxoglutarate and pyruvate. Moreover they are competitively inhibited by L-leucine. α-oxoisocaproate and α-oxotsovalerate. Obtained K, values are very similar and do not depend on the course of reaction.
• 3 α-Oxoisocaproate inhibits the activity of crystalline L-aspartate: α-oxoglutarate aminotransferase; Kj is about 4–7 mM.
• 4 The pyridoxamine form of L-alanine: α-oxoglutarate aminotransferase seems to be more sensitive to the inhibitory effect of the compounds investigated.
• 5 The effect of branched-chain amino acids and their α-oxoanalogues on the metabolism of amino groups in maple syrup urine disease is discussed.

     

Synthesis and localization of α-amylase and α-glucosidase in Bacillus licheniformis grown in batch and continuous culture

In batch and continuous cultures of Bacillus licheniformis NC1B 6346 α-amylase was invariably extracellular and could not be detected in the cytoplasm or cell surface. α-Glucosidase however, was largely intracellular but at the end of exponential growth and during slow growth under Mg²⁺ limitation it was detected in the culture fluid. Both enzymes were susceptible to catabolite repression and glucose totally inhibited their synthesis in batch culture. In maltose-limited chemostat culture, synthesis of both enzymes was maximal at D = 0.2/h and declined at higher growth rates. α-Amylase synthesis was constitutive but α-glucosidase synthesis was induced by maltose and maltotriose but not by methyl-α-D-glucoside or phenyl-α-D-glucoside. α-Amylase was synthesized at pH 6.5 and above in maltose-limited chemostat culture but not below this pH. Intracellular α-glucosidase synthesis varied little with pH. Increasing temperature decreased the synthesis of both enzymes in chemostat culture to the extent that α-glucosidase was undetectable at 50° C. Polar lipid composition varied with pH and temperature but there was no correlation between this and enzyme secretion. Moreover cerulenin, an antibiotic that inhibits protein secretion in some bacteria by interacting with the membrane had no effect on α-amylase secretion but decreased the release of α-glucosidase upon protoplast formation.     

Correlation between activities of starch debranching enzyme and α-polyglucan structure in endosperms of sugary-1 mutants of rice

The biochemical lesion of the sugary-1 mutation was examined in five different mutants of rice with varying phenotypes but with mutations at the same locus. The cells in the inner part of the endosperm of all mutants tested contained phytoglycogen instead of starch, while the cells located in the outer part of the endosperm tissue from some mutants were filled with numerous starch granules. The molecular size of phytoglycogen was markedly smaller than that of amylopectin as measured by Sephacryl S-1000 chromatography. Analysis of the distribution of α-1,4 chain lengths revealed that in phytoglycogen the number of A-chains dramatically increased, while long B chains with DP ≥ 37 remarkably decreased or were almost absent, which resulted in the disappearance of the cluster structure. The results suggest that changes in the balance of enzymic activities induced by the mutations brought about a drastic alteration in polyglucan structure and the shape of the polyglucan granule. The greater the extent of phytoglycogen regions in su1 endosperm tissues became, the greater was the phytoglycogen content, and the greater the reduction in the activity of starch debranching enzyme, a type of enzyme referred to as R-enzyme (RE), limit dextrinase or pullulanase. Immunoblot analysis showed that the reduction in RE activity was due to a decrease in the amount of RE protein, and that the reduction in RE was specific since proteins of starch-branching enzymes I and IIa and ADP-glucose pyrophosphorylase were not markedly affected by su1 mutations. The proportion of starch region to the whole endosperm tissue of various su1 mutants was correlated with the RE activity in these endosperms. The results strongly suggest that the reduction in RE activity is involved in the su1 phenotype and that the enzyme plays an essential role in determining the fine structure of the amylopectin molecule