Regulation of phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae by CDP-diacylglycerol

Regulation of the 45- and 55-kDa forms of Saccharomyces cerevisiae membrane-associated phosphatidylinositol (PI) 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase) by phospholipids was examined using Triton X-100/phospholipid-mixed micelles. CDP-diacylglycerol and phosphatidylglycerol inhibited 45-kDa PI 4-kinase activity in a dose-dependent manner. Kinetic analyses of the 45-kDa PI 4-kinase showed that phosphatidylglycerol was a competitive inhibitor with respect to PI (Ki = 2 mol %), and CDP-diacylglycerol was a mixed type of inhibitor with respect to PI (Ki = 4 mol %) and MgATP (Ki = 5 mol %). 55-kDa PI 4-kinase activity was not significantly affected by phospholipids. The physiological relevance of CDP-diacylglycerol inhibition of 45-kDa PI 4-kinase activity was examined using plasma membranes from inositol auxotrophic (ino1) cells. Immunoblot analysis showed that 45-kDa PI 4-kinase expression in plasma membranes was not affected by inositol starvation of ino1 cells. However, both 45-kDa PI 4-kinase activity and its product PI 4-phosphate were reduced in plasma membranes from inositol-starved ino1 cells. The CDP-diacylglycerol concentration (9.6 mol %) in plasma membranes of inositol-starved ino1 cells was 12-fold higher than its concentration (0.8 mol %) in plasma membranes of inositol-supplemented cells. Plasma membranes of inositol-starved ino1 cells also had increased levels of phosphatidate, phosphatidylserine, phosphatidylethanolamine, and cardiolipin. However, these phospholipids did not affect pure 45-kDa PI 4-kinase activity. The concentration of CDP-diacylglycerol in plasma membranes of inositol-starved ino1 cells was in the range of the inhibitor constants determined for CDP-diacylglycerol by kinetic analyses using pure 45-kDa PI 4-kinase. These results raised the suggestion that 45-kDa PI 4-kinase activity may be regulated in vivo by CDP-diacylglycerol.     

Recombinant protein production via fed-batch culture of the yeast Saccharomyces cerevisiae

Protein production with the recombinant yeast Saccharomyces cerevisiae in fed-batch culture is investigated in this work using beta-galactosidase as a model protein. Segregational instability was negligible during the observed culture periods. The final volumetric productivity, as determined by both cell concentration and gene expression, was strongly affected by the time course of the glucose levels in the bioreactor. It was found that an average glucose feed rate of 1.31 g glucose h-1 resulted in both the maximum beta-galactosidase production rate of 831-950 units ml-1 h-1 and the maximum cell production rate of 0.520-0.524 mg ml-1 h-1.     

Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by sphingoid bases

The regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity by sphingoid bases was examined using Triton X-100/lipid-mixed micelles. Sphingosine, phytosphingosine, and sphinganine inhibited purified preparations of the 104- and 45-kDa forms of phosphatidate phosphatase in a dose-dependent manner. The structural requirements for the sphingoid base inhibition of phosphatidate phosphatase activity were a free amino group and a long chain hydrocarbon. A detailed kinetic analysis was performed to determine the mechanism of phosphatidate phosphatase inhibition by sphingoid bases. The phosphatidate phosphatase dependence on phosphatidate was cooperative (Hill numbers of approximately 2) in the absence and presence of sphingoid bases. Sphingosine, phytosphingosine, and sphinganine were parabolic competitive inhibitors of phosphatidate phosphatase activity. This indicated that more than one inhibitor molecule contributed to the exclusion of phosphatidate from the enzyme. The aKi values (inhibitor constants) for sphingosine, phytosphingosine, and sphinganine were 1.5, 0.4, and 0.2 mol %, respectively, and the Km value for phosphatidate was 2.2 mol %. The cellular concentrations of free phytosphingosine and sphinganine were 0.16 and 0.53 mol %, respectively, relative to the total phospholipids in S. cerevisiae. The cellular concentrations of phytosphingosine and sphinganine were in the range of the aKi values for these sphingoid bases. These results raised the suggestion that phosphatidate phosphatase activity may be regulated in vivo by sphingoid bases.     

Continuous sucrose hydrolysis by yeast cells immobilized to wool

A novel immobilized biocatalyst with invertase activity was prepared by adhesion of yeast cells to wool using-glutaraldehyde. Yeast cells could be immobilized onto wool by treating either the yeast cells or wool or both with glutaraldehyde. Immobilized cells were not desorbed by washing with 1 M KCl or 0.1 M buffers. pH 3.5-7.5. The biocatalyst shows a maximum enzyme activity when immobilized at pH 4.2-4.6 and 7.5-8.0. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous full-scale sucrose hydrolysis. The influence of temperature, sugar concentration and flow rate on the productivity of the reactor and on the specific productivity of the biocatalyst was studied. The system demonstrates a very good productivity at a temperature of 70 degrees C and a sugar concentration of 2.0 M. The increase of the volume of the biocatalyst layer exponentially increases the productivity. The productivity of the immobilized biocatalyst decreases no more than 50% during 60 days of continuous work at 70 degrees C and 2.0 M sucrose, but during the first 30 days it remains constant. The cumulative biocatalyst productivity for 60 days was 4.8 x 10(3) kg inverted sucrose/kg biocatalyst. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors.     

Molecular cloning of the actin gene from yeast Saccharomyces cerevisiae

Two overlapping DNA fragments from yeast Saccharomyces cerevisiae containing the actin gene have been inserted into pBR322 and cloned in E.coli. Clones were identified by hybridization to complementary RNA from a plasmid containing a copy of Dictyostelium actin mRNA. One recombinant plasmid obtained (pYA102) contains a 3.93-kb Hindlll fragment, the other (pYA208) a 5.1-kb Pstl fragment, both share a common 2.2-kb fragment harboring part of the actin gene. Cloned yeast actin DNA was identified by R-loop formation and translation of the hybridized actin mRNA and by DNA sequence analysis. Cytoplasmic actin mRNA has been estimated to be about 1250 nucleotides long. There is only one type of the actin gene in S.cerevisiae.     

Insulin signaling in the yeast Saccharomyces cerevisiae. 3. Induction of protein phosphorylation by human insulin

A low affinity insulin-binding protein in the plasma membrane of Saccharomyces cerevisiae has been identified recently (Müller, G., Rouveyre, N., Upshon, C., Gross, E., and Bandlow, W., preceding paper in this issue). Since the mammalian insulin receptor functions as a tyrosine kinase with autophosphorylation capacity, kinase studies were performed with the partially purified insulin-binding protein preparation. Incubation with [gamma-32P]ATP in vitro led to phosphorylation of the 53-kDa insulin-binding protein on serine but not on tyrosine residues. In addition, a 70-kDa polypeptide, copurified with the insulin-binding protein preparation, was tyrosine-phosphorylated under the same conditions. Phosphorylation of both proteins was enhanced by human insulin. These results obtained by immunoprecipitation and immunoblotting using specific anti-phosphoserine/threonine/tyrosine antibodies were confirmed by phosphoamino acid analysis of the individual immunoprecipitated and gel-purified 32P-labeled phosphoproteins. During gel filtration, the 53-kDa protein coeluted as a 300-kDa complex together with the 70-kDa phosphotyrosine-containing protein and was coimmunoprecipitated with the latter using an anti-phosphotyrosine antibody, strongly arguing for complex formation between the two proteins. The data presented raise the possibility that stimulation of glycogen synthesis by insulin in yeast is mediated by a 53-kDa insulin-binding protein and a 70-kDa phosphotyrosine-containing protein which are organized in a large plasma membrane-bound signaling complex. Elucidation of the function and molecular mode of interaction of these components in yeast may help to understand metabolic insulin signaling in mammalian cells.     

Adenosine-5'-tetraphosphate and guanosine-5'-tetraphosphate: new substrates of the cytosolic exopolyphosphatase of the yeast Saccharomyces cerevisiae

A cytosolic preparation of Saccharomyces cerevisiae is capable of hydrolyzing adenosine-5'-tetraphosphate and guanosine-5'-tetraphosphate with activities which are 1.5-2 times greater than that with polyP15. The apparent K(m) values for hydrolysis of adenosine-5'-tetraphosphate and guanosine-5'-tetraphosphate are 100 and 80 microM, respectively. A comparative study of inhibitors shows that these activities are inherent characteristics of these exopolyphosphatases.     

Kinetics of the hydrolysis of synthetic substrates by horse urinary kallikrein and trypsin

The kinetic constants for horse urinary kallikrein and trypsin hydrolysis of BAEE, TAME, bradykinin methyl ester and bradykinyl-Ser-Val-Gin-Val-Ser were determined. The values of the ratio kcat/Km show that (1) kallikrein is catalytically less efficient than trypsin for all the substrates (2) the three esters are equally good substrates for trypsin while horse urinary kallikrein is 100-fold more effective on bradykinin methyl ester than on the other substrates (3) for both enzymes the ester of bradykinin is a better substrate than the tetradecapeptide.     

Purification and characterization of DNA helicase III from the yeast Saccharomyces cerevisiae

Two forms of DNA helicase activity, Rad3 and ATPase III, were previously purified from the yeast Saccharomyces cerevisiae and characterized. Here, we have identified and purified an additional DNA helicase activity from S. cerevisiae to near homogeneity. This helicase differs from those described previously in its chromatographic behavior, molecular weight, enzymatic properties, and genetic properties. Thus, we named it DNA helicase III. Its apparent molecular mass is about 120 kDa as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. DNA helicase III requires a divalent cation Mg2+ or Mn2+, either ATP or dATP, and a single-stranded portion on the duplex substrate. Helicase III moves in the 5'-->3' direction on single-stranded portions of the substrate and unwinds the strand of DNA in the 3'-->5' direction. It also has an intrinsic DNA-dependent ATPase (dATPase) activity that hydrolyzes either ATP or dATP to ADP or dADP and orthophosphate in the presence of DNA. DNA helicase III activity was not affected by either rad3 or radH mutations, suggesting that it is encoded by a gene different from RAD3 and RADH.     

Complex formation by all five homologues of mammalian translation initiation factor 3 subunits from yeast Saccharomyces cerevisiae

The PRT1, TIF34, GCD10, and SUI1 proteins of Saccharomyces cerevisiae were found previously to copurify with eukaryotic translation initiation factor 3 (eIF3) activity. Although TIF32, NIP1, and TIF35 are homologous to subunits of human eIF3, they were not known to be components of the yeast factor. We detected interactions between PRT1, TIF34, and TIF35 by the yeast two-hybrid assay and in vitro binding assays. Discrete segments (70-150 amino acids) of PRT1 and TIF35 were found to be responsible for their binding to TIF34. Temperature-sensitive mutations mapping in WD-repeat domains of TIF34 were isolated that decreased binding between TIF34 and TIF35 in vitro. The lethal effect of these mutations was suppressed by increasing TIF35 gene dosage, suggesting that the TIF34-TIF35 interaction is important for TIF34 function in translation. Pairwise in vitro interactions were also detected between PRT1 and TIF32, TIF32 and NIP1, and NIP1 and SUI1. Furthermore, PRT1, NIP1, TIF34, TIF35, and a polypeptide with the size of TIF32 were specifically coimmunoprecipitated from the ribosomal salt wash fraction. We propose that all five yeast proteins homologous to human eIF3 subunits are components of a stable heteromeric complex in vivo and may comprise the conserved core of yeast eIF3.     

Stabilization of microsatellite sequences by variant repeats in the yeast Saccharomyces cerevisiae

We examined the effect of a single variant repeat on the stability of a 51-base pair (bp) microsatellite (poly GT). We found that the insertion stabilizes the microsatellite about fivefold in wild-type strains. The stabilizing effect of the variant base was also observed in strains with mutations in the DNA mismatch repair genes pms1, msh2 and msh3, indicating that this effect does not require a functional DNA mismatch repair system. Most of the microsatellite alterations in the pms1, msh2 and msh3 strains were additions or deletions of single GT repeats, but about half of the alterations in the wild-type and msh6 strains were large (> 8 bp) deletions or additions.     

Pre-steady-state analysis of ATP hydrolysis by Saccharomyces cerevisiae DNA topoisomerase II. 1. A DNA-dependent burst in ATP hydrolysis

When bound to DNA, topoisomerase II from Saccharomyces cerevisiae exhibits burst kinetics with respect to ATP hydrolysis. Pre-steady-state analysis shows that the enzyme binds and hydrolyzes two ATP per reaction cycle. Our data indicate that at least one of the two ATP is rapidly hydrolyzed prior to the rate-determining step in the reaction mechanism. When DNA is not bound to topoisomerase II, the rate-determining step shifts to become either ATP binding or hydrolysis. Two possible mechanisms are proposed that agree with our observations.     

Molecular modelling studies of lysozyme catalysed hydrolysis of synthetic substrates

Kinetic data for the lysozyme catalysed hydrolysis of aryl chitooligosides were surveyed. Both electron-donating and electron-withdrawing substituents on the departing aryl aglycones enhance the rate of hydrolyses. The parallel pH-rate profiles implicate that identical catalytic residues are involved in the hydrolytic fission of the glycosyl-aryloxy bond of these two groups of synthetic substrates. Molecular modelling studies of lysozyme complexes with aryl diN-acetyl chitobiosides and their intermediates were performed. The two synthetic substrates bearing aryl aglycones with opposite electronic effects bind to the active site of lysozyme in different conformations. Based on the energetic and geometric considerations, the oxocarbonium ion whose pyranose ring D in a sofa conformation is the most plausible reaction intermediate for the lysozyme catalysed hydrolysis of the synthetic substrates. The modelling study also suggests that considerable conformational changes of both the lysozyme binding site and the chitobiosyl group accompany the formation of the glycosyl enzyme intermediate. In particular, the chitobiosyl group undergoes a dislocation of the pyranose ring C from the subsite C and a constraint of the pyranose ring D to form a boat conformer.     

Functional mapping of the U3 small nucleolar RNA from the yeast Saccharomyces cerevisiae

The 2 f1-f2 distortion product otoacoustic emission (DP) was measured in 20 normal hearing subjects and 15 patients with moderate cochlear hearing loss and compared to the pure-tone hearing threshold, measured with the same probe system at the f2 frequencies. DPs were elicited over a wide primary tone level range between L2 = 20 and 65 dB SPL. With decreasing L2, the L1-L2 primary tone level difference was continuously increased according to L1 = 0.4L2 + 39 dB, to account for differences of the primary tone responses at the f2 place. Above 1.5 kHz, DPs were measurable with that paradigm on average within 10 dB of the average hearing threshold in both subject groups. The growth of the DP was compressive in normal hearing subjects, with strong saturation at moderate primary tone levels. In cases of cochlear impairment, reductions of the DP level were greatest at lowest, but smallest at highest stimulus levels, such that the growth of the DP became linearized. The correlation of the DP level to the hearing threshold was found to depend on the stimulus level. Maximal correlations were found in impaired ears at moderate primary tone levels around L2 = 45 dB SPL, but at lowest stimulus levels in normal hearing (L2 = 25 dB SPL). At these levels, 17/20 impaired ears and 14/15 normally hearing ears showed statistically significant correlations. It is concluded that for a clinical application and prediction of the hearing threshold, DPs should be measured not only at high, but also at lower primary tone levels.     

Involvement of cell surface glycosyl-phosphatidylinositol-linked aspartyl proteases in alpha-secretase-type cleavage and ectodomain solubilization of human Alzheimer beta-amyloid precursor protein in yeast

Human beta-amyloid precursor protein (APP) introduced into yeast undergoes alpha-secretase-type cleavage, suggesting that yeast have alpha-secretase-like protease(s). Here we report that two structurally and functionally related glycosyl-phosphatidylinositol-linked yeast aspartyl proteases, Mkc7p and Yap3p (collectively termed yapsin), are responsible for alpha-secretase-type cleavage of APP expressed in yeast, resulting in release of soluble APP into the extracellular space. Disruption of MKC7 and YAP3 in a vacuolar protease-deficient strain abolished this APP cleavage/release, and APP cleavage/release could be restored by introduction of MKC7 or YAP3 on a single copy plasmid. Purified Mkc7p cleaved an internally quenched fluorogenic APP peptide substrate at the alpha-secretase cleavage site. Measurement of proteolytic activity either in yeast homogenates or on the yeast cell surface revealed that most Mkc7p and Yap3p activities were localized at the cell surface. These results establish a molecular basis for alpha-secretase-type cleavage in yeast and support the generally held concept that alpha-secretase cleavage of APP occurs at the cell surface.     

Isolation and in vitro hydrolysis of lentil protein fractions by trypsin

The albumin and globulin fractions from lentil seeds were isolated and characterised by gel filtration. The latter was shown to be homogeneous and the former heterogeneous on PAGE. The amino acid analysis revealed high values of amidic amino acids for both fractions with great differences in the sulphur-containing amino acids. Native albumin, globulin and salt-soluble proteins were markedly resistant to trypsin hydrolysis compared to casein. The SDS-PAGE of native salt-soluble proteins indicated that the globulin fragments (20 to 30 kD) were slowly digested in the presence of albumin. The heating increased the hydrolysis of the proteins in the order: salt-soluble, albumin and globulin. The facilitated hydrolysis of the heated salt-soluble fraction seemed to be due to protein-protein interactions induced by heat.     

大豆酶解制油蛋白酶的筛选及工艺条件的研究

在以大豆乳液为水解底物进行酶水解来提取大豆油的工艺中,应用蛋白酶水解细胞壁中的蛋白成分使得脂蛋白复合体中的油脂得以释放,从而提高油脂的得油率,通过研究选出破壁能力较强的蛋白酶并优化其水解条件,最后得到最佳水解工艺.