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R Kleine

Publications and source records attributed to R Kleine.

At least 19 recordsLinked to original sources
PubMed1993

Production and immobilization of a proteinase-reduced cyclodextrin glycosyltransferase preparation.

Cyclodextrin glycosyltransferase (CGTase) was produced by a 3-day cultivation of Bacillus macerans growing in a natural medium containing grated-potatoes. Besides CGTase the culture supernatant contained a mixture of serine proteinases with a predominant subtilisin-like activity. By fractionated precipitation with ammonium sulphate the CGTase (molecular mass approximately 70 kDa) was concentrated and largely separated from the proteinases (molecular mass approximately 28 kDa). Among the various immobilization methods and carrier materials tested the enriched CGTase was covalently bound preferably onto porous glass beads using glutardialdehyde as cross-linker. The discontinuous conversion of soluble starch into beta-cyclodextrin was carried out with native as well as immobilized CGTase over 24 h. The batch re-usability of the fixed enzyme proved to be at least 20 times with a residual CGTase activity of 65%.

Bacillus↗
PubMed1990

[Pharmacokinetic studies of a new 20% fat emulsion containing 70% medium-chain triglycerides].

The aim of the study was to acquire basic knowledge on pharmacokinetic, metabolism and tolerance of a new 20% fatty emulsion with a 70% proportion of medium-chain triglycerides (MCT) and a 30% proportion of long-chain triglycerides (LCT) in the postoperative phase after a trauma of medium severity. - 12 female patients who had an elective rectal amputation and who needed parenteral nutrition postoperatively, were studied. The nutritional regime consisted of 4.8 g/kg/day of glucose and 1 g/kg/day of amino acids. On the second postoperative day the patients were given 0.06 g/kg body weight/h and on the third day 0.12 g/kg body weight/h of new 20% fatty emulsion during a time period of eight hours. Blood samples for the evaluation of triglycerides, free fatty acids, phospholipids, beta-hydroxybutyrate (beta-OHB), acetoacetate, cholesterol, glucose, pyruvate and lactate were taken before and after the fat application. Ketone were measured semiquantitatively. Side effects and complications were not observed. Simultaneously to the administrated triglycerides an increase in serum triglycerides was observed. After four hours fat emulsion was infused under steady state conditions. Under the graphically measured half-life of 17 minutes for the MCT/LCT emulsion, rapid and complete elimination could be seen after the infusion had been stopped. Simultaneously with the high clearance of the infused triglycerides, free fatty acids increased significantly in the plasma without reaching a plateau; 30 minutes after the fat application the laboratory results returned to the initial levels.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxybutyric Acid↗
PubMed1989

Caloric requirements in burned patients.

Adequate nutritional support is an important goal for the management of burn patients. Caloric requirements are higher than in any other major trauma or disease. The development of hypermetabolism and hypercatabolism leads to an increase in resting energy expenditure which depends on the severity of the injury. Therefore, with the intent to counteract these developments and their complications, a balanced carbohydrate, fat and protein intake should be adjusted to the actual needs. This paper gives a survey of the metabolic consequences of burns, reviews the underlying pathophysiology and tries to give practical guidelines for the nutritional support of the burn patient.

Burns↗
PubMed1989

[Metabolic problems and therapeutic approaches in multiple organ failure].

Multiple-system organ failure is associated with progressive defects of cellular metabolism involving several organ systems. The metabolic failure is caused by a neural-hormonal reaction to trauma and sepsis and by humoral mediators damaging cell metabolism. Involved are catabolic hormones like catecholamines, cortisol and glucagon as well as the humoral mediators interleukin and arachidonic acid metabolites. There is an increase in resting energy expenditure and a derangement of utilization and production of adenine nucleotides. Severe injury, sepsis and multiple-system organ failure are associated with a 30-40% decreased content of energy rich phosphates in different tissues. The low energy charge potential is caused by the inability to use nutritional substrates adequately. Relative clearance and oxidation of glucose will advance fatty infiltration of the liver. Clearance and oxidation of fat is normal or often increased. If illness is progredient fat utilization will be disturbed. Although protein synthesis is increased in critical ill patients, due to excessive proteolyses net protein loss occur. In preterminal patients the ability of the liver to synthetize protein decreases, concentrations of several free amino acids in plasma increase, while the clearance for amino acids decreases.

Critical Care↗
PubMed1985

[The kinetics of hydrolysis of alanine peptide esters and -p-nitroanilides by thermitase, a thermostable serine protease from Thermoactinomyces vulgaris: secondary specificity, influence of temperature and solute].

The kinetic parameters Km and kcat and the resulting proteolytic coefficients kcat/Km for the hydrolysis of blocked alanine peptide esters (X(Ala)nOMe) and -p-nitroanilides (X(Ala)n-pNA) of variable length (n = 1 to 5 alanine residues) by the cationic, microbial serine protease thermitase are determined in order to delineate the number of subsites involved in catalysis. Thermitase has at least five secondary subsites (S1 to S5) being hydrophobic in S1 to S4. Arrhenius plots for both, esterase and amidase activity were biphasic with a break at 30 degrees C, followed by a downward bend. The influence of dimethylformamide, solute for many substrates, on the thermitase-catalyzed esterolysis of Z(Ala)2OMe was also investigated. In contrast to the kcat values being unaffected by 5 to 30% dimethylformamide, the Km values increased logarithmically with enhancing its concentration.

Alanine↗
PubMed1984

[The PABA test].

PABA test has proved to be an easy and reliable test for determination of exocrine pancreatic insufficiency. N-benzoyl-L-tyrosyl-p-aminobenzoic acid or 4-(N-acetyl-L-tyrosyl) aminobenzoic acid are split by action of chymotrypsin in the small intestine. N.O-diacetyl-L-tyrosyl-p-aminobenzoic acid is converted easy in vivo in 4(N-acetyl-L-tyrosyl) aminobenzoic acid. The amount of 4-aminobenzoic acid (PABA) in urine collected for 6-10 hours is used as an index of chymotrypsin production. The concentration of PABA (and aromatic amines) is estimated in urine by the Bratton and Marshall method. p-dimethylamino cinnamaldehyde is less useful for the determination of urinary PABA. 60 min are necessary as time for acid hydrolysis of conjugated PABA metabolites. False abnormal test results are found for instance in patients with inflammatory bowel diseases, small bowel resection, impaired liver function, anorexia nervosa, lambliasis or renal insufficiency. The PABA test appears in consideration of these restrictions to be an useful simple method in the assessment of exocrine pancreatic function.

4-Aminobenzoic Acid↗
PubMed1982

Preparation and characterization of proteases from Thermoactinomyces vulgaris. V. Investigations on autolysis and thermostability of the purified protease.

Thermitase, the main component of the proteases of the culture medium from Thermoactinomyces vulgaris, is degraded by autolyses (increase of liberated amino groups) and thereby inactivated especially at elevated temperature, at alkaline pH-values and in the absence of added substrates. As shown by polyacrylamide gel electrophoresis autolysis is an essential part during heat inactivation (complete disappearance of the thermitase band after heating the enzyme at 85 degrees C for 5 min). The quantitative comparison of autolysis and heat inactivation as well as the kinetics of reversible inhibition of the enzyme by HgCl2 at different temperatures showed that above 60 degrees C thermal denaturation of the enzyme protein contributes to thermitase inactivation. Ca2+-ions (20 mM) have a stabilizing effect against both autolysis and thermal denaturation (inactivation) of thermitase.

Calcium↗
PubMed1982

Separation and comparative characterization of the cationic protease and anionic protease from the culture medium of Thermoactinomyces vulgaris.

The anionic protease component which frequently contaminates preparations of routinely isolated cationic protease (thermitase) from Thermoactinomyces vulgaris was purified, virtually to homogeneity, by rechromatography on controlled pore glass (CPG-10). Starting materials were column eluates with anionic protease, contaminated with residual thermitase activity. The purified anionic enzyme shares several properties with thermitase, such as size, sensitivity against phenylmethanesulfonyl fluoride and Hg2+, UV-spectral, immunological and pH behavior. On the other hand, the isoelectric point (at pH 6.5), temperature dependence (more heat stable) and enzymatic activity (less active) of anionic protease differ significantly from thermitase. At pH 8 or 6 and 25 degrees or 4 degrees C anionic protease is hydrolysed completely by thermitase. Like other protein substrates, anionic protease simultaneously acts as a stabilizer for thermitase. In contrast to thermitase, the anionic enzyme partially changes spontaneously during long-term storage at 4 degrees C and pH 6 to a cationic protein species endowed with proteolytic activity.

Drug Stability↗
PubMed1982

Investigations on the substrate specificity of thermitase, a thermostable serine-protease from Thermoactinomyces vulgaris.

The kinetic parameters Km and kcat and the proteolytic coefficients kcat/Km for the hydrolysis of eighteen Z(benzyloxycarbonyl)-dipeptide methyl esters with variation of the residues in P1 and P2 position catalyzed by thermitase at pH 8 and 55 degrees C are reported. The results indicate that an integral part of both subsites, S1 and S2, are hydrophobic areas and that a mutual interaction between the side chains of P1 and P2 for optimal hydrolyisis does exist. Furthermore, the importance of the P2 for the peptidolytic activity of thermitase was shown using N-acylated oligo-alanine peptides and their p-nitroanilides. In all cases dialanine or alanine p-nitroanilide are the main products.

Endopeptidases↗
PubMed1982

Properties of thermitase, a thermostable serine protease from Thermoactinomyces vulgaris.

Some structural and catalytic properties of the extracellular serine protease thermitase, purified by chromatography on porous glass, are reported. The crystal data and the high elastinolytic action point to possible relationship of thermitase with pancreatic elastase. Subsite-mapping studies clearly indicate, however, that thermitase contains an extended binding region and is closely related to the subtilisin group. The enzyme shows maximal stability between pH 6.0 and 7.5 and maximal activity between pH 7.5 and 9.5. The larger the substrate, the higher is its temperature optimum (60 degrees C for esterolysis, 85 degrees C for proteinolysis). The stability of thermitase is significantly improved by acetates and chlorides at 1 M concentration. Besides its high hydrolytic action on soluble proteins thermitase is capable for efficient degradation of the insoluble proteins elastin and collagen.

Collagen↗
PubMed1981

On the negative straining of the protein crystal structure.

It has been shown that in some cases negative staining reveals structure with details down to 0.4 nm in size, the nature of protein playing a key role. Various stains seem to interact with different parts of a thermitase (a serine protease) molecule, which results in intensity changes in electron diffraction patterns.

Crystallization↗
PubMed1978

[Characterization of a protease from Thermoactinomyces vulgaris (thermitase). 3. Substrate specificity and properties of partially purified thermitase].

During the process of cultivation of Th. vulgaris several proteases are formed. In the present investigation the extensively purified major component was used. The substrate specificity was determined by means of 7 proteins, 7 amino acid esters, 5 fatty acid esters and 15 amino acid 4-nitroanilides. Among the protein substrates tested, urea denaturated hemoglobin was split best, followed by gelatin, casein, field bean protein, serum albumin and gluten. The weakest rate of hydrolysis was observed with elastin. In contrast to this acetyl-(L-ala)3-methylester, that is a substrate for elastase, was split best from all the esters tested. Only 8% of this activity could be found with the chymotrypsin substrates acetyl-L-tyr-ethylester and acetyl-L-phe-ethylester and 1% of the above activity with the trypsin substrates tosyl-L-arg-methylester and benzoyl-L-arg-methylester. The fatty acid esters and the p-nitroanilides were hydrolyzed much more slowly. The pH-optimum of thermitase was found in the weakly alkaline region of pH 7 to 9. There were only small differences between the individual high and low molecular substrates. The temperature optimum was between 60 and 75 degrees C for esters and p-nitroanilides as substrates and at 90 degrees C for casein. It should be mentioned that the enzyme was quickly inactivated at temperatures above 70 degrees C.

Hydrogen-Ion Concentration↗
PubMed1976

[Behavior of leucine aminopeptidase from bovine eye lens in guanidine hydrochloride. Dissociation and reassociation].

The dissociation and reassociation behavior of the hexameric leucine aminopeptidase (LAP) in solutions of 0.5 to 6 M guanidine-HC1 (Gu-HC1) was investigated by means of thin layer chromatography on Sephadex G-200 superfine. Up to 0.5 M Gu - hc1 the hexameric LAP-structure remains intact. In 0.75 to 2 M Gu - HC1 the enzyme dissociates nearly completely into its half-molecules (LAP-trimers, MW 160000 +/- 10,000). In 2.5 and 2.75 M Gu - HC1 a mixture of LAP-monomers (subunits) (MW 60000 +/- 5000) and trimers was to be found. Treatment with beta-mercapto ethanol increases the portion of monomers. Only monomers occurred in 3 to 6 M Gu - HC1. Parallel to the dissociation, a loss of the essential zinc (half life: 10.5 and 3.5 hours in 1 M and 4 M Gu - HC1 resp.) and a decrease of the enzymatic activity (30 min after treatment with 1 M and 4 M Gu - HC1 50% and 1% resp. residual activity) of the observed. On the other hand, the enzyme was activated by dilute solutions of Gu - HC1 (0.1 to 0.3 M: max. increase 50%). Removal of the denaturant causes reassociation of all types of fragments to hexameric LAP which was indistinguishable from the native LAP in Ouchterlony immune diffusion test. The reassociation was accompanied by a small increase in activity only.

Animals↗
PubMed1976

[Preparation and some properties of immobilized trypsin from the crayfish Cambarus affinis Say (author's transl)].

The anionic tryptic enzyme from the crayfish (crayfish trypsin) was adsorbed to DEAE-Sephadex A-50 and covalently coupled to BrCN-activated Sepharose 4B and porous glass loaded with isothiocyanate propyl groups (ITC-glass). The relative activities against p-tosylarginine methyl ester (TosArgOMe) were found to be 30 to 100% for DEAE-Sephadex crayfish trypsin, 20 to 53% for Sepharose crayfish trypsin, and 17 to 38% for ITC-glass crayfish trypsin. The relative activities rise with declining protein content of the enzyme matrix complexes. The highest relative proteinase activities (substrate: 1% casein) were obtained with Sepharose crayfish trypsin (74%), followed by DEAE-Sephadex crayfish trypsin (68%) and ITC-glass crayfish trypsin (45%). Similar results are obtained with protamine and native lactate dehydrogenase as substrates. In accordance with the Sepharose bovine trypsin complex the apparent Michaelis constant (Km(app)) of the Sepharose crayfish trypsin with TosArgOMe was found to be markedly higher than that of the native enzyme. The pH-activity profiles of the crayfish trypsin derivatives using TosArgOMe as substrate were shown to be displaced towards more alkaline pH values by 0.5 (ITC-glass crayfish trypsin) and 1 (Sepharose crayfish trypsin) pH units, respectively, or towards more acidic pH values (by 1.5 pH units) with the polycationic derivative (DEAE-Sephadex crayfish trypsin) as compared to the native enzyme (optimum pH 8.6). Concerning the temperature stability of the derivatives, Sepharose crayfish trypsin was more stabile, ITC-glass crayfish trypsin behaves like the native crayfish trypsin, and DEAE-Sephadex crayfish trypsin was more sensitive at elevated temperatures as compared to the soluble enzyme. The properties of the crayfish trypsin derivatives are compared with the properties of the bovine analogues.

Animals↗
PubMed1976

[Isolation and characterization of a microsomal arylaminopeptidase from rat kidney].

The isolation and characterization of a microsomal arylaminopeptidase from rat kidney is reported. By treatment of a microsomal arylaminopeptidase-phosphatase-complex with trypsin and subsequent gel filtration of the solubilized proteins on Sepharose 6B a electrophoretic homogeneous arylaminopeptidase was obtained (yield, 3%; enrichment, 900 times). The following properties of the purified enzyme were determined: 1. Molecular weight: 182000 (gel filtration on Sepharose 6B) to 192000 (SDS-polyacrylamide gel electrophoresis). 2. Subunit structure: In the presence of 6 M guanidine - HC1 + 1% BETA-mercaptoethanol the enzyme dissociates into subunits (MW 46700, ESTIMATED BY SDS gel electrophoresis method). 3. Isoelectric point: 4,71 (agarose gel electrophoresis method). 4. UV characteristics: E 280nm/E260NM=1.3. 5. Substrate specifity: optimal substrates L-alanyl derivatives (anilide, beta-naphthyl amide, p-nitroanilide, 4-(phenylazo)-phenylamide and hydrazide). Among these compounds the anilide derivative was hydrolyzed most rapidly. Furthermore, di- and tripeptides, especially L-methionyl-L-leucine, were also split. No hydrolysis was observed with hemoglobin (pH 4.5 and 7.5) and amino acid- or peptide-ester substrates. 6. Optimal pH: 7.5 +/- 0,1; optimal temperature: 45 to 50 degrees C. 7. The enzyme has no transamidation activity with L-alanyl amide both as aminoacyl donator and -acceptor. 8. Influence of effectors: Heavy metal ions (Ni2+, Cd2+, Cu2+, Zn2+), chelating agents (EDTA, o-phenanthroline) and puromycin inhibit the enzyme significantly. SH-group reagents are without any influence. 9. L-alanyl-L-alanyl-4 (phenylazo)-phenylamide, a dipeptide aryl aminopeptidase substrate, is hydrolyzed by the purified enzyme preparation according to a consecutive or step by step mechanism.

Aminopeptidases↗