Mammalian protein methylesterase. Physical and enzymic properties.

Protein methylesterase (PME) amino acid composition and substrate specificity towards methylated normal and deamidated protein substrates were investigated. The enzyme contained 23% acidic and 5% basic residues. These values are consistent with a pI of 4.45. The product formed from methylated protein by PME was confirmed as methanol by h.p.l.c. The kcat. and Km values for several methylated protein substrates ranged from 20 x 10(-6) to 560 x 10(-6) s-1 and from 0.5 to 64 microM respectively. However, the kcat./Km ratios ranged within one order of magnitude from 11 to 52 M-1.s-1. Results with the irreversible cysteine-proteinase inhibitor E-64 suggested that these low values were in part due to the fact that only one out of 25 molecules in the PME preparations was enzymically active. When PME was incubated with methylated normal and deamidated calmodulin, the enzyme hydrolysed the latter substrate at a higher rate. The Km and kcat. for methylated normal calmodulin were 0.9 microM and 31 x 10(-6) s-1, whereas for methylated deamidated calmodulin values of 1.6 microM and 188 x 10(-6) s-1 were obtained. The kcat./Km ratios for methylated normal and deamidated calmodulin were 34 and 118 M-1.s-1 respectively. By contrast, results with methylated adrenocorticotropic hormone (ACTH) substrates indicated that the main difference between native and deamidated substrates resides in the Km rather than the kcat. The Km for methylated deamidated ACTH was 5-fold lower than that for methylated native ACTH. The kcat./Km ratios for methylated normal and deamidated ACTH were 43 and 185 M-1.s-1 respectively. These results indicate that PME recognizes native and deamidated methylated substrates as two different entities. This suggests that the methyl groups on native calmodulin and ACTH substrates may not be on the same amino acid residues as those on deamidated calmodulin and ACTH substrates.     

Detection and partial purification of two lipases fromCandida rugosa

Summary Two distinct lipases produced byCanadida rugosa were identified and separated by a high resolution anion-exchange column (Mono Q) after an ethanol extraction of the crude lipase. From this Mono Q column, lipase I eluted at 0.05 M NaCl whereas lipase II eluted at 0.15 M NaCl. The less anionic nature of lipase I was also confirmed by native polyacrylamide gel electrophoresis as well as isoelectrophoresis. Both proteins have an apparent molecular weight of 58,000 by SDS-PAGE. The isoelectric points of lipase I and II are 5.6 and 5.8 respectively.     

Separation of colour compounds from lipase in fermentation supernatant by diafiltration

Summary Extracellular lipase was produced by growing Geotrichum candidum. A simple optical method was developed to quantify the dark-brown compounds formed during medium preparation and fermentation. The size of these molecules was in the fractionation range of Sephadex G-50. Diafiltration trials were done to screen ultrafiltration membranes for the most efficient decolonization of the cell-free lipase solution. Membranes were identified which reduced the colour by 80% with less than 5% loss of lipase activity after 4 volume exchanges in continuous diafiltration.     

Characterization of a trypsin inhibitor from equine urine.

A trypsin inhibitor was isolated from pregnant mares' urine by adsorption on bentonite and elution with aqueous pyridine followed by batch DEAE-cellulose treatment and column chromatography. Final purification to an electrophoretically homogenous glycoprotein was achieved by gel permeation chromatography. This equine urinary trypsin inhibitor (E-UTI) is acid- and heat-stable, has a molecular weight of 22 to 23 kDa, an isoelectric point of 4.55, forms a 1:1 molar complex with trypsin and has serine as its N-terminal amino acid. The N-terminal amino acid sequence of this protein is almost identical with that of EI-14, the inhibitor obtained from horse serum by tryptic treatment, except for two extra amino acid residues, Ser-Lys- on the N-terminal end of E-UTI. In its isoelectric point E-UTI differs from EI-14 and the inhibitor from human urine.     

Protein-carboxyl methylation in adrenal medullary cells

1. The protein-carboxyl methylating system has been studied in adrenal medullary cells either using disrupted cell components or with intact cells. Whereas the enzyme protein-carboxyl methylase (PCM) is cytosolic, the majority of its substrates is on or within chromaffin granules. With intact granules, methylation of surface proteins results in solubilization of membrane proteins. 2. Membrane PCM substrates have been identified as two proteins with apparent molecular weights of 55,000 and 32,000. Among the substrates located inside the granules, the chromogranins are excellent substrates, while dopamine beta-hydroxylase is poorly methylated. 3. Under physiological conditions, stimulation of the splanchnic nerve results in an increase in adrenal medullary protein-methyl ester formation as well as in an augmented methanol production. With adrenal medullary cells in culture, carboxyl-methylated chromogranin A is detected in mature chromaffin granules between 3 and 6 hr after labeling. Methylated chromogranins are secreted concomitantly with catecholamines following cholinergic stimulation. 4. These data coupled with those of Chelsky et al. (J. Biol. Chem. 262:4303-4309, 1987) on lamin B suggest that PCM methylates residues other than D-aspartyl and L-isoaspartyl in proteins. They further suggest that methylation may occur on nascent peptide chains before they are injected into the rough endoplasmic reticulum.     

Purification and characterization of two distinct lipases from Geotrichum candidum

Lipase, an enzyme that hydrolyzes triacylglycerol, has been purified and characterized. The purification procedure includes ethanol precipitation and chromatographies on Sephacryl-200 HR, high resolution anion-exchange (mono Q) and Polybuffer exchanger 94. With this procedure, two forms of lipases from Geotrichum candidum were obtained. Lipase I (main enzyme) and lipase II (minor enzyme) were purified 35-fold with a 62% recovery in activity and 94-fold with a 18% recovery in activity, respectively. Their molecular weights have been estimated by polyacrylamide gel electrophoresis under denaturing conditions and by molecular sieving under native conditions at 56,000. Lipase I and II had optimum pH values of 6.0 and 6.8 and isoelectric points of 4.56 and 4.46, respectively. The enzymes are stable at a pH range of 6.0 to 8.0. Monovalent ions had little effect on both enzyme activities, while divalent ions at concentrations above 50 mM inhibited the lipase activities in a concentration-dependent manner. Sodium dodecyl sulfate at a concentration lower than 10 mM completely inhibited the lipase activity.     

Transformation and regeneration of loblolly pine: shoot apex inoculation with Agrobacterium

Loblolly pine (Pinus taeda L.) is the mostimportant tree species in US commerce and has much to gain through geneticengineering. This species can be transformed using particle bombardment andAgrobacterium; however, the regeneration of plants fromtransgenic tissues has been difficult and the recovery of transgenic plants hasbeen rare. A shoot-based and genotype-independent transformation methodemploying Agrobacterium tumefaciens was used to facilitaterecovery of plants and permit the transformation of elite germplasm. Shootsfrom4–6 week old seedlings and adventitious shoots from culture wereinoculated with A. tumefaciens EHA101 (pGUS3), or EHA105(pSSLa.3), subjected to selection and regenerated. Shoots that survivedexhibited expression of the uidA gene (GUS) in a patterncharacteristic of the either the CaMV35S promoter (pGUS3), or the larch RbcSpromoter (pSSLa.3) transferred. Recovered plants were screened using PCRamplification. Southern DNA analyses and amplification of the T-DNA borderjunction confirmed genomic integration of both transferreduidA and nptII genes. In this proofofconcept study, the overall recovery of P. taeda shoots wasfair (10–20%), while recovery of intact rooted plants was poor (>1%)due to difficulty in rooting. Recovery of intact rooted plants from inoculatedshoots of P. eldarica and P. radiatawas more efficient (10–30%). The addition of a shoot multiplication stepand effective rooting protocols will improve the efficiency of this genotypeindependent transformation method in P. taeda, and inotherPinus spp.     

Effect of 5-thio-D-glucose on testosterone biosynthesis in vivo in mice testes

Testicular synthesis of (14C)cholesterol and (14C)testosterone from (14C)acetate were investigated in mice treated with 5-thio-D-glucose at a dose of 33 mg/kg body weight/day for 21 days. The testicular synthesis of free cholesterol as well as steroids were significantly decreased. The steroid synthesizing enzymes, cholesterol esterase, cholesterol side-chain cleaving enzyme, total alpha-hydroxysteroid dehydrogenase and total beta-hydroxysteroid dehydrogenase, were also analysed. Cholesterol esterase and total beta-hydroxysteroid dehydrogenase were significantly reduced whereas total alpha-hydroxysteroid dehydrogenase was unaffected. Hence, a decrease in free cholesterol for steroid synthesis and a decreased activity of the steroidogenic enzyme, beta-hydroxysteroid dehydrogenase, were responsible for the diminished synthesis of testosterone.