[Combined sinus lift procedure and lateral augmentation. A treatment concept for the surgical and prosthodontic rehabilitation of the extremely atrophic maxilla]. / Kombination der Sinusliftoperation mit der lateralen Kieferkammaugmentation. Ein Behandlungskonzept zur chirurgisch-prothetischen Rehabilitation der extremen Oberkieferalveolarkammatrophie.

PURPOSE: This paper describes a surgical and prosthetic procedure for treating the extremely atrophic maxilla. It explains a two-staged surgical technique, donor and recipient site morbidity, implant survival, and the implant-retained prosthetic rehabilitation of the patients. PATIENTS AND METHODS: A total of 57 consecutive patients were treated with a sinus lifting procedure and a simultaneous lateral augmentation using autogenous corticocancellous block and particulate bone grafts from the iliac crest. After a 6-month bone healing period, a total of 284 endosteal Titanium screw implants were inserted. Following a 3-month osseointegration period, the implants were exposed and loaded with either fixed or removable prostheses. RESULTS: In three cases a partial bone graft loss was observed; however, enosseous implantation was possible as planned. During the observation period none of the 284 implants was lost; 3 implants exhibited treatable peri-implant infection. Complications at the donor and recipient sites were minimal and did not negatively influence the overall clinical result of the treatment. CONCLUSION: The combination of sinus lift procedure and lateral augmentation for the treatment of the extremely atrophied maxilla proved to be a safe method that produces good and reliable clinical results.

Structural study on the carbohydrate moiety of calf intestinal alkaline phosphatase.

Surprisingly alkaline phosphatase (AP) (EC 3.1.3.1) of calf intestine is found in large amounts, e.g. 80%, within chyme. Most of the enzyme is present as a mixture of four differently hydrophobic anchor-bearing forms and only the minor part is present as an anchorless enzyme. To investigate whether changes in the N-glycosylation pattern are signals responsible for large-scale liberation from mucosa into chyme, the glycans of the two potential glycosylation sites predicted from cDNA were investigated by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry in combination with exoglycosidase treatment after tryptic digestion and reversed-phase chromatography. The glycans linked to Asn249 are at least eight different, mainly non-fucosylated, biantennary or triantennary structures with a bisecting N-acetylglucosamine. For the most abundant glycopeptide (40%) the following glycan structure is proposed: [carbostructure: see text]. The glycans linked to Asn410 are a mixture of at least nine, mainly tetraantennary, fucosylated structures with a bisecting N-acetylglucosamine. For the most abundant glycopeptide (35%) the following glycan structure is proposed: [carbostructure: see text]. For the structures the linkage data were deduced from the reported specificities of the exoglycosidases used and the specificities of the transglycosidases active in biosynthesis. The majority of glycans are capped by alpha-galactose residues at their non-reducing termini. In contrast to the glycans linked to other AP isoenzymes, no sialylation was observed. Glycopeptide 'mass fingerprints' of both glycosylation sites and glycan contents do not differ between AP from mucosa and chyme. These results suggest that the observed large-scale liberation of vesicle-bound glycosylphosphatidylinositol (GPI)-anchored AP from mucosa into chyme is unlikely to be mediated by alteration of glycan structures of the AP investigated. Rather, the exocytotic vesicle formation seems to be mediated by the controlled organization of the raft structures embedding GPI-AP. (c) 2001 John Wiley & Sons, Ltd.

[Resorbable osteosynthesis material in craniosynostosis]. / Resorbierbares Osteosynthesematerial bei Kraniosynostosen.

BACKGROUND: The results of using resorbable plates and screws (82% polylactic acid and 18% polyglycolic acid) in craniofacial surgery for the correction of craniosynostosis after more than 4 years of experience are presented. Special attention is focussed on the degree of stability and the clinical tissue response to the material employed to answer the question of whether the material is an adequate alternative to titanium. METHODS: Thirty patients who had been treated with this method for craniosynostoses were examined at regular intervals regarding the shape and stability of the forehead region, visibility and palpability of the plates, and tissue reactions. RESULTS: The technical handling of the osteosynthesis material proved to be simple and reliable. In one case the bone was not strong enough for the screw pitch. After an observation period of a maximum of 4 years and 1 month, the fixations were stable with no signs of adverse reactions. DISCUSSION: If the long-term results remain favorable, we consider the use of resorbable material a promising method for the stabilization of segments in craniofacial surgery in children.

[Peri-osseous intracranial translocation of titanium osteosynthesis plates and screws after fronto-orbital advancement]. / Perossäre intrakraniale Translokation von Titanosteosyntheseplatten und -schrauben nach frontoorbitalem Advancement.

BACKGROUND: Perossoeus intracranial translocation or passive intracranial transmission of titanium osteosynthesis plates and screws in the growing skull following surgical craniosynostosis corrections, also referred to as the PIT effect, has been described in the literature since 1995. It is a phenomenon which has not received due attention until recently and is explained by appositional and resorptional remodeling processes in the growing skull. CASE REPORT AND DISCUSSION: An impressive case of the PIT effect with a total intracranial dislocation of titanium plates and screws is used to demonstrate the problems associated with this phenomenon and to discuss the few clinical case reports in the English-language literature. The obvious advantages of a resorbable material are pointed out; however, it is still uncertain as to whether the resorption process is fast enough to avoid the PIT effect if used clinically.

[Hemostatic wound management in marcumar patients. Collagen fleece vs. tranexamic acid]. / Hämostyptische Wundversorgung bei Marcumarpatienten. Kollagenvlies vs. Tranexamsäure.

A total of 124 patients on oral anticoagulation therapy with coumarin were treated by orosurgical procedures and entered into a study to determine the hemostatic efficiency of different methods. The therapeutic anticoagulation level was determined in accordance with the recommendations of the American Heart Association (low risk: 2.0 < INR < 3.0; high risk: 2.5 < INR < 3.5) and maintained during treatment. In one group, the alveoli were treated with collagen, in a second group a mouthrinse regime with tranexamic acid was implemented. Twenty-three patients had to be excluded because anticoagulation levels differed from the recommended values. The group treated with collagen included 31 patients, the group with tranexamic acid mouthwashes, 32 patients. A third group was analyzed in which a controlled change in the anticoagulation level had been performed and all treated alveoli had been covered by mucosal flaps (n = 38); they were compared to the other two groups. The surgical proceedings were outlined precisely. Patients treated with collagen had a bleeding rate of 19%, patients with tranexamic acid mouthwash 6%, and those treated with mucosal flaps 40%. The data were not suited for statistical evaluation, they were objected to a descriptive analysis: the confidence intervals were determined by tables for binomial distributions. These did confirm the difference in the frequency of bleeding for the tranexamic acid and mucosal flap groups.

Glycosylphosphatidylinositol-specific phospholipase D of human serum--activity modulation by naturally occurring amphiphiles.

The enzymatic properties of glycosylphosphatidylinositol-specific phospholipase D (EC 3.1.4.50) were characterized using a 6,000-fold purified enzyme. This was obtained in 100 microg amounts from human serum with a recovery of 35%. Pure alkaline phosphatase containing one anchor moiety per molecule was used as substrate. The enzyme is stimulated by n-butanol, but in contrast to other phospholipases this activation is not produced by a transphosphatidylation reaction. The previously reported non-linearity of the specific activity with respect to phospholipase concentration in the test was no longer observed upon purification, indicating inhibitor removal. The serum inhibitor(s) co-chromatograph with serum proteins and lipoproteins. The main part of the inhibitory activity was found in the lipid fraction after protein denaturation and can be subfractionated into acid phospholipids, cholesteryl esters and triacylglycerides. Added phosphatidyl-serine, phosphatidylinositol, phosphatidylglycerol, gangliosides, cholesteryl esters, and sphingomyelins turned out to be strong inhibitors, as well as phosphatidic acid. Phosphatidylethanolamine and various monoacylglycerols were found to be activators. The low glycosylphosphatidylinositol-specific phospholipase activity found in native serum did not increase significantly upon 90% removal of phospholipids by n-butanol. High serum concentrations of strongly inhibiting compounds, complex kinetic interactions among aggregates of these substances, and compartmentalization effects are discussed as possible reasons for the observed inactivity.

Gel chromatographic characterization of the hydrophobic interaction of glycosylphosphatidylinositol-alkaline phosphatase with detergents.

The interaction of a glycosylphosphatidylinositol (GPI) protein with different detergents was studied for the first time with a purified protein. Four differently hydrophobic fractions of GPI-alkaline phosphatase (GPI-AP) from calf intestine were used as model proteins. The mode of interaction was determined by investigating (i) the self-aggregation behaviour of the GPI-AP fractions, (ii) the interference of detergents with GPI-AP binding to octyl-Sepharose, and (iii) the elution of GPI-AP bound to octyl-Sepharose. It was shown that polyoxyethylene-type detergents surprisingly interact much stronger than n-octylglucoside with GPI-AP, which is in contrast to the known behaviour of GPI-proteins in natural membranes. Gel filtration chromatography of Triton X-100 at concentrations above the critical micellar concentration yields three different micelle species with apparent molecular weights of about 166, 54, and 16 kDa. GPI-AP fraction II, which is shown to bear only one anchor per dimer, does not bind to any of these micelles. We demonstrate that a complex is formed containing about 150 Triton X-100 molecules and about 4700 molecules of water per molecule of GPI-AP dimer. The experimental findings are in accordance with a simple geometrical model based on the physical data of fatty acids and the arrangement, mean size, and shape of Triton X-100 molecules.

[Preliminary results of the use of resorbable plates and screws in craniofacial surgery]. / Resorbierbare Platten und Schrauben in der kraniofazialen Chirurgie. Vorläufige Ergebnisse.

In ten patients with craniosynostoses resorbable plates and screws (Lactosorb) consisting of poly-L-lactic acid (82%) and poly-glycolic acid (18%) were used to stabilize the segments after frontoorbital advancement. As our experience increased, an exact adaptation of the plates and simple handling proved to be possible. The plates were stable enough to retain a favorable functional and aesthetic result after redraping the soft tissue envelope. In one patient with Chotzen's syndrome the intended use of the resorbable material was abandoned: the thin osseous structures did not offer enough primary stability to the high pitch of the screws. During an observation period of up to 21 months no infection, exposure, instability or dislocation was observed. The clinical use of the resorbable material in frontoorbital advancement proved to be a stable method of segment fixation if the bone was of sufficient thickness. These promising preliminary results will have to observed in a larger group and over a longer period of time.

Microheterogeneity of the hydrophobic and hydrophilic part of the glycosylphosphatidylinositol anchor of alkaline phosphatase from calf intestine.

Digestion of calf intestine alkaline phosphatase with pronase and subsequent dephosphorylation of the released peptidyl-(Etn-P)2-glycosyl-PtdIns with HF generated 8 glycosyl-Ins species the largest of which (G1 and G2) have the following proposed structures: [sequence: see text] G3 and G5 are lower homologues of G1 and G2, respectively, being one alpha 1-2 linked mannopyranosyl residue shorter. G4 is analogous to G2 lacking the N-acetylgalactosaminyl residue and G6 is the next lower homologue of G4. Most of G4 and G6 occur substituted with a palmitoyl (G4, G6) or a myristoyl residue (G6) probably attached to the inositol moiety. Thus, the basic ManxGlc-Ins species are either substituted with an N-acetylgalactosaminyl residue or a fatty acid ester. The structures were deduced from compositional analysis, molecular-mass determination by matrix-assisted laser desorption MS, sequential hydrolysis with appropriate exoglycosidases and treatment with CrO3. Purification of the glycosylinositol species was achieved by a novel reverse-phase HPLC technique using fluorescent fluoren-9-yl-methoxy-carbonyl (Fmoc) derivatives. These stable derivatives were susceptible to hydrolysis with exoglycosidases which allowed sequential cleavages to be carried out and kinetics to be followed at the picomole level. We observed recently that native alkaline phosphatase separates on octyl-Sepharose into four distinct fractions of increasing hydrophobicity (F1-F4). Here we show that all four fractions contain G1-G6. The acylated species G4 and G6 were restricted to F2 and F4 which had been shown earlier to contain, on average, 2.5 and 3 fatty acid residues/subunit, respectively. In all four fractions the diradylglycerol moiety was predominantly diacylglycerol, alkylacylglycerol being less than 10% which is in contrast to most glycosyl-PtdIns--anchored proteins of mammalian origin.

Glycosylphosphatidylinositol-alkaline phosphatase from calf intestine as substrate for glycosylphosphatidylinositol-specific phospholipases--microassay using hydrophobic chromatography in pipet tips.

An electrophoretically homogeneous glycosylphosphatidylinositol- alkaline phosphatase fraction from calf intestine, obtained by hydrophobic chromatography, was used as "enzyme-labeled" substrate for testing phospholipase activity. The reaction products were separated by (i) hydrophobic chromatography in pipet tips and (ii) Triton X-114 phase partitioning. The chromatographic method presented permits high test frequencies, does not need temperature-controlled sample handling, and is only slightly disturbed by detergents, organic solvents, and proteins. The method was used to characterize phosphatidylinositol- specific phospholipase C from Bacillus cereus and phospholipase D from calf serum. Measurement of substrate hydrolysis by phospholipases is apparently linear to enzyme concentration and time. Relative activity of both enzymes is maximum at pH 6.5, corresponding to the optimal pH range found with other glycosylphosphatidylinositol substrates and phosphatidylinositol-specific phospholipases of other sources. Maximum activity of phospholipase C was found at 0.03% Triton X-100, 0.01% Brij 35, and 0.2% n-octylglucoside. The activity is not affected by Ca(2+), NaHCO(3), o-phenanthroline, or EDTA, increasingly inhibited by MgCl(2), MnCl(2), and ZnCl(2), and slightly activated by Na+ and K+. Calf serum phospholipase D shows maximum activity at 0.05% Triton X-100, 0.02% Brij 35, and 0.4% n-octylglucoside. The apparent Km values for phospholipase C (12.25 micron) and phospholipase D (4.94 micron) found with glycosylphosphatidylinositol-alkaline phosphatase are compared with values published for other glycosylphosphatidylinositol substrates.

Characterization of the interaction of alkaline phosphatase with an activity inhibiting monoclonal antibody by progress curve analysis.

Using the enzyme activity inhibiting monoclonal antibody IB 10B8 against alkaline phosphatase of calf intestine (AP), the interaction of a macromolecular antigen with the antibody was studied with different reaction conditions and with different conformations of the antigen, i.e. using (i) different pH values, (ii) different temperatures, (iii) different substrate saturation of the enzyme, (iv) different glycosylphosphatidyl-AP (GPI-AP) aggregates, and (v) membrane-bound species. In the case of antibody excess and negligible substrate consumption enzymic product formation proceeds according to [P] = a + b x t - c x exp(-d x t). By direct progress curve fitting and secondary data evaluation using nonlinear regression, omitting numerical derivation and graphic techniques, kinetic constants of the immune reaction have been estimated. The method does not require any artificial labelling nor any separation of bound and free entities. (i) Upon increasing pH from 9.8 to 11.0, the dissociation constant of the enzyme-antibody complex is increased strongly, mainly due to the decreasing association rate constant. (ii) A temperature increase from 25 degrees C to 37 degrees C produces a marked increase of both the association and dissociation rate constant. (iii) To differentiate between the interaction of the antibody with the free (E) and substrate-bound (ES) enzyme, experiments were done at different substrate concentrations. The results were fitted to a model allowing determination of association and dissociation rate constants of the free and substrate-bound enzyme. The inverse variation of association and dissociation rate constants caused by substrate binding produces a marked increase of the dissociation constant of the antibody-enzyme complex. The antibody-bound enzyme shows a nearly three-fold higher Km value and a six-fold lower catalytic constant as compared to the free enzyme. (iv) Investigations of the interaction of the antibody with anchorless AP, different hydrophobic aggregates of purified GPI-AP (fractions II-V). (v) Membrane-bound GPI-AP show that the epitopes of all species are fully accessible to the antibody and not cryptic. Surprisingly the insertion of the GPI-moiety into the membrane and the aggregation of the different GPI-AP fractions II-V seem to improve antibody binding. Such improvement of binding was not found in control experiments with Fab, indicating only for the bivalent antibody a stronger interaction with the multivalent antigen than with the monovalent antigen.

Is the brush border membrane of the intestinal mucosa a generator of "chymosomes"?

1. The microvilli of enterocytes in calf intestine demonstrate high levels of vesiculation activity at the top and at the basal region. 2. The morphology of the vesicles associated with microvilli (100-500 nm diameter, unilamellar, few intramembraneous particles, high AP activity) is very similar to the morphology of vesicles found in the chyme. 3. Vesicles can be purified 6-10 fold from chyme of the calf intestine applying a Mg(++)-precipitation method, used for brush border membrane preparation. 4. Specific activities of alkaline phosphatase and disaccharidases were found to be much higher in chyme vesicles than in the mucosa. 5. Phospholipid content and phospholipid composition is in chyme vesicles different from brush border membrane vesicles. 6. The characterized chyme vesicles are referred to as chymosomes. We consider the mucosa as a large-scale generator of chymosomes, i.e. digestive enzymes bearing vesicles.

Heterogeneity of glycosylphosphatidylinositol-anchored alkaline phosphatase of calf intestine.

A method is described for large-scale purification of glycosylphosphatidylinositol-anchored alkaline phosphatase from intestinal mucosa and chyme to homogeneity. Both enzyme preparations contain approximately 2 mol fatty acid/mol subunit and exhibit a very similar fatty acid composition with octadecanoate and hexadecanoate as prevalent components. No significant differences between native glycosylPtdIns-anchored and hydrophilic alkaline phosphatases from both sources were found regarding Km, Vmax, the type of inhibition and inhibition constants of the amino acids L-leucine, L-phenylalanine, and L-tryptophan. The purified enzymes of both sources yield diacylglycerol and phosphatidic acid, after treatment with phosphatidylinositol-specific phospholipase C (PtdIns-PLC) and glycosylphosphatidylinositol phospholipase D (PLD), respectively. Enzyme preparations of both sources appear as heterogeneous mixtures of five fractions separable by octyl-Sepharose chromatography. Fraction I corresponds to the anchorless enzyme, fractions II-V differ in their susceptibility to phospholipases. Fractions II and IV are completely split by PtdIns-PLC or PLD action, almost 50% of fraction III is split by PtdIns-PLC, while fraction V is resistant. The susceptibility of these two fractions toward the action of PLD is considerably higher. Fatty acid analysis yields molar ratios of fatty acids/alkaline phosphatase subunit of 1.78, 2.58, 2.24, and 3.37 for fractions II, III, IV, and V, respectively. Aggregates of glycosylPtdIns-anchored alkaline phosphatase of all fractions are seen in native PAGE in the presence of Triton X-100. By gel chromatography in the presence of Brij 35, fractions II-V form stable multiple aggregates of dimers and may bind different amounts of the detergent. These data, together with fatty acid analysis, can be interpreted by the following model. Fractions II and IV are tetramers and octamers with two molecules fatty acid/subunit. Fraction III is a tetramer, bearing one additional fatty acid molecule, localized on the dimer. Fraction V is an octamer, containing glycosylPtdIns-anchor molecules with three molecules fatty acids/anchor molecule. The additional fatty acid residue is possibly located on inositol and responsible for the reduced susceptibility to PtdIns-PLC. The similarity of all measured parameters of both enzymes suggests that the glycosylPtdIns-anchored alkaline phosphatase of the mucosa is released into the chyme without changing the anchor molecule constituents.

Evidence for glycosylphosphatidylinositol anchoring of intralumenal alkaline phosphatase of the calf intestine.

1. Considerable amounts of intestinal alkaline phosphatase (AP) were found intralumenally in all animal species investigated, i.e. calf, pig, goat, rat, mouse, guinea pig, hen and carp. The ratios between the total activity of AP found intralumenally and the total intestinal activity vary considerably. Calves and pigs show the highest, i.e. 0.77 and 0.44, respectively, while rodents have much lower ratios. Only 20-34% of the intralumenal alkaline phosphatase (IAP) of the calf and pig is soluble and not within the sediment after centrifugation at 135,000 x g for 60 min. whereas the IAP of rodents is soluble in the range of 60-72% of the total IAP. 2. For the IAP of the mucosa and chyme of calf, all criteria were found which are generally used, indicating a glycosylphosphatidylinositol (GlcPtdIns) anchor as proved by strong hydrophobicity using Triton X-114 phase partitioning, phenyl-Sepharose binding and enzyme aggregation, and the susceptibility to phosphatidylinositol-specific phospholipase C (PtdIns-PLC) and papain digestion. 3. More than 80% of the mucosa alkaline phosphatase (MAP) of the proximal part of the intestine and of the particulate fraction of IAP exhibit these criteria indicating the presence of the GlcPtdIns-anchor structure, whereas the anchor content of the soluble intralumenal enzyme decreases from the pylorus to the ileocecal junction. 4. MAP partially purified to a specific activity of 1747 IU/mg retains the anchor structure. 5. The results presented indicate that the release of large amounts of AP into the chyme is realized without splitting the GlcPtdIns anchor. The possible intralumenal function of this form of AP is discussed.

Determination of rate constants of monoclonal antibodies to enzymes.

A novel graphical method for determining rate constants of the immune reaction of enzyme-inhibiting or -activating antibodies has been evaluated. The experimental determination of kinetic constants does not require purification of antibody and enzyme nor any separation step of bound and free entities. The resultant enzyme activity is used as a measurement of the extent of enzyme-antibody complex formation. The plot ln magnitude of v - v infinity versus t (v = enzyme activity at time t, v infinity = enzyme activity at infinite time) yields straight lines in the case of antibody excess. Slope and vertical intercept of that primary plot can be used for secondary plots to obtain association (k1) and dissociation (k-1) rate constant, the dissociation constant KD of the complex and the residual enzyme activity of the complex (g/f). The suitability of the graphical method has been established experimentally using the mab IB 10B8 (2) which inhibits alkaline phosphatase activity. With the homogeneous assay in the presence of substrate as well as a microassay in the absence of substrate, k1 and g/f were found to be 4.68 x 10(7) M-1 min-1; 3.74 x 10(7) M-1 min-1 and 0.035; 0.107 respectively. For k-1 and KD, only crude estimates could be derived. The method has been tested for model discrimination using computer simulated data.

Large scale purification of intralumenal alkaline phosphatase from calf intestine by immunosorbent affinity chromatography.

Electrophoretically and immunologically homogeneous alkaline phosphatase (AP) can be obtained by a simple and rapid two-step procedure. Step 1: Immunosorbent affinity chromatography using immobilized polyclonal anti-AP-antibodies and elution of the bound enzyme by triethylamine solution, pH 11.4. After affinity chromatography the specific activity of the extracted crude material increases from 3.5 to 793 IU/mg. Step 2: Final purification to a specific activity of 1524 IU/mg by DEAE-cellulose ion exchange chromatography. One investigator is able to purify 100 mg AP within 3 days. The overall recovery is 65%. Methods for characterization and selection of anti-AP-antiserum using immunoinhibition of AP are described.

Intralumenal alkaline phosphatase of the calf intestine.

About 80% of the total activity of the alkaline phosphatase of the calf intestine was found within the lumen and only 20% in the mucosa. The specific activity of the intralumenal enzyme fraction was about ten times higher than that of the mucosa enzyme. Differential centrifugation experiments demonstrated that the intralumenal enzyme of the proximal gut segment can be found in a fraction which exhibits sedimentation behaviour like a microsomal fraction, whilst the intralumenal enzyme of the distal gut segment was found to be soluble in the supernatant after centrifugation at 135000 X g for 60 min. Ouchterlony double diffusion analysis and antibody inhibition of alkaline phosphatase by antisera against the mucosa enzyme demonstrate that the two forms of intestinal enzyme have apparently identical antigenicity. Purified alkaline phosphatase obtained from the intralumenal fraction and from mucosa exhibits closely related pH-optima, Michaelis constants, amino acid inhibition type and inhibition constants. The results of large scale release of alkaline phosphatase bound to microvesicles are discussed with regard to results of morphological investigations in different tissues and cell cultures demonstrating the formation of intestinal membrane bodies and cell extrusion.

Dynamic properties of a phosphofructokinase/pyruvate kinase system. Experiments in vitro using the substrate-stat technique.

It is known from theoretical work that very simple enzyme systems may exhibit non-linear dynamic properties like those found in vivo. To realize such a system experimentally, an ATP-producing reaction (pyruvate kinase) was coupled with an ATP-consuming reaction (phosphofructokinase). The substrate-stat technique has been adapted to characterize each single enzyme as well as to follow up one enzyme in the coupled system. If the plots of both pyruvate kinase activity versus [ADP] and phosphofructokinase activity versus [ATP] are hyperbolic, the coupled system approaches a unique steady state as predicted from the single characteristics. Under assay conditions where phosphofructokinase is inhibited by ATP, its characteristics as found in a single assay and in the coupled system are different. For a system with ATP-inhibited phosphofructokinase, a paradoxical behaviour is predicted and is demonstrated experimentally. If the total pyruvate kinase activity is increased over a certain limit, the system is switched from a low [ATP], high-activity steady state into a high [ATP] low-activity steady state.

Improvements of the chamber analysis.

Improvements of the chamber analysis directed to increase the sensitivity by introduction of fluorescence assays, to facilitate the handling of the procedure and to increase the accuracy by introduction of automatic data processing and partially automatized volume dispensing for 50 microliter volumes simultaneously are described. The method is used for measuring of enzyme activities and substrate concentrations in 2--30 microliter sample volumes with final assay volumes lower than 100 microliter. Two variants of the procedure are presented. With variant I all steps of the analysis including start of the reaction by mixing, incubation and measuring are carried out in the measuring chamber. With variant II the measuring chamber is used for the measuring process only, the other steps are performed outside in special chambers. Application examples from biochemical and clinical chemical analysis are demonstrated.

Substrate-stat: a method for characterization of enzymes in concentrated solutions at low substrate concentrations.

A method is proposed, which is suitable for kinetic studies with concentrated enzyme preparations, and its principal features are described. The method makes it possible to keep constant the concentration of substrate(s) or product(s) during the observation time. For this purpose, the increase or decrease of an indicator extinction signal, dependent on enzyme velocity, is monitored and automatic titration is performed using a suitable titer solution. This latter contains equimolar amounts of indicator and substrate(s). The whole system operates in a similar manner as in pH-stat devices. The technical set-up, model reaction systems and practical applications are shown. Operation problems and possible sources of error are examined. Finally, the main limitations as well as possible improvements are discussed.