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Philosophers of science diverge on the question what drives the growth of scientific knowledge. Most of the twentieth century was dominated by the notion that theories propel that growth whereas experiments play secondary roles of operating within the theoretical framework or testing theoretical predictions. New experimentalism, a school of thought pioneered by Ian Hacking in the early 1980s, challenged this view by arguing that theory-free exploratory experimentation may in many cases effectively probe nature and potentially spawn higher evidence-based theories. Because theories are often powerless to envisage workings of complex biological systems, theory-independent experimentation is common in the life sciences. Some such experiments are triggered by compelling observation, others are prompted by innovative techniques or instruments, whereas different investigations query big data to identify regularities and underlying organizing principles. A distinct fourth type of experiments is motivated by a major question. Here I describe two question-guided experimental discoveries in biochemistry: the cyclic adenosine monophosphate mediator of hormone action and the ubiquitin-mediated system of protein degradation. Lacking underlying theories, antecedent data bases, or new techniques, the sole guides of the two discoveries were respective substantial questions. Both research projects were similarly instigated by theory-free exploratory experimentation and continued in alternating phases of results-based interim working hypotheses, their examination by experiment, provisional hypotheses again, and so on. These two cases designate theory-free, question-guided, stepwise biochemical investigations as a distinct subtype of the new experimentalism mode of scientific enquiry., (© 2022. Springer Nature Switzerland AG.)

1. Endocytosis of formaldehyde-treated bovine serum albumin by rat liver sinusoidal cells has been followed by injecting rats with the protein labelled with 125I-tyramine cellobiose (125I-TCfBSA). 125I-TCfBSA is quickly taken up by the liver; the radioactivity present in the organ reaches a plateau 5-10 mm after injection and is maintained for up to at least 180 mm. During the first 5 mm most of radioactivity remains acid-precipitable. After which, labelled acid-soluble components are produced at a constant rate for up to 30-40 mm. 2. Differential centrifugation shows that radioactivity is first recovered mainly in the microsomal fraction. Within a few minutes it exhibits a distribution pattern similar to that of lysosomal enzymes, being chiefly located in the mitochondrial fractions. 3. Isopycnic centrifugation in a sucrose gradient of the microsomal fraction isolated 1 min after injection indicates a similar distribution for radioactivity and alkaline phosphodiesterase. Later, the microsomal radioactivity distribution curve is shifted towards higher densities and becomes distinct from that of the plasma-membrane enzyme. After isopycnic centrifugation in a sucrose gradient of the total mitochondrial fraction a considerable overlapping of acid-precipitable and acid-soluble radioactivity distributions is observed without significant changes with time. The same is observed in a Percoll gradient except that after a relatively long lime (> 30 mm) of injection a marked shift of radioactivity distribution towards higher densities occurs. 4. A pretreatment of rats with Triton WR 1339, a density perturbant of liver lysosomes, causes a striking shift of acid-soluble radioactivity distribution in a sucrose gradient towards lower densities while having markedly less influence on the acid-precipitable distribution. As a result, a distinction between the distribution of both kinds of radioactivity becomes clearly apparent. A preinjection of yeast invertase, modifies the acid-soluble distribution without having a significant effect on the acid-precipitable distribution up to 30 mm after 125I-TCfBSA injection. 5. Glycyl-1-phenylalanine-2-naphthylamide largely releases acid-soluble radioactivity associated with the mitochondrial fraction, whatever the time after 125I-TCfBSA injection. On the other hand the proportion of acid- precipitable radioactivity present in the fraction that can be released is almost zero at 10 mm after injection, and it later increases. 6. The results presented here are best explained by supposing that, after being trapped in small pinocytic vesicles, 125I-TCfBSA is quickly delivered to the endosomes. It is then transferred at a constant rate from these organelles to a first population of secondary lysosomes, where it is rapidly hydrolysed. In a later step the hydrolysis products and a small proportion of labelled compounds that remain acid-precipitable accumulate in a second population of lysosomes. [ABSTRACT FROM AUTHOR]

We have investigated the effect of the temperature of centrifugation on the behavior of rat-liver mitochondria during isopycnic centrifugation in a sucrose gradient. From the distribution curves of the reference enzymes, it may be deduced that the damaging effect of high-speed centrifugation on mitochondria can be prevented by increasing the temperature of centrifugation. The results of the morphological examinations are in good agreement with the biochemical determinations. As previously described Wattiaux et al. (1971) the hydrostatic pressure generated by centrifugation is the main causal agent of the deterioration of mitochondria. The effect of temperature suggests that the pressure acts either on the physical state of the mitochondrial membrane lipids or by disrupting hydrophobic bonds essential for the mitochondrial membrane stability. From a practical point of view, our results show that it is possible to prevent the damaging effects of centrifugation on rat-liver mitochondria by adjusting the temperature. [ABSTRACT FROM AUTHOR]

It would not be practical to attempt to deal with all the advances that have informed our understanding of the behavior and functions of this enzyme over the past 90 years. This account concentrates key advances that explain why the monoamine oxidases remain of pharmacological and biochemical interest and on some areas of continuing uncertainty. Some issues that remain to be understood or are in need of further clarification are highlighted. [ABSTRACT FROM AUTHOR]

Background and aims: Hepatic cholesterol deposition drives inflammation and fibrosis in non-alcoholic steatohepatitis (NASH). The Niemann-Pick type C2 (NPC2) protein plays an important role in regulating intracellular cholesterol trafficking and homeostasis. We hypothesized that intravenous NPC2 supplementation reduces cholesterol accumulation, hepatic inflammation and fibrogenesis in a nutritional NASH rat model. Methods: Rats were fed a high-fat, high-cholesterol (HFHC) diet for four weeks resulting in moderately severe NASH. Animals were treated with intravenous NPC2 or placebo twice weekly for either the last two weeks or the entire four weeks. End-points were liver/body- and spleen/body weight ratios, histopathological NASH scores, fibrosis, serum liver enzymes, cholesterol, lipoproteins, cytokines, and quantitative polymerase chain reaction derived hepatic gene expression related to cholesterol metabolism, inflammation, and fibrosis. Results: HFHC rats developed hepatomegaly, non-fibrotic NASH histopathology, elevated liver enzymes, serum cholesterol, and pro-inflammatory cytokines. Their sterol regulatory element binding factor 2 (SREBF2) and low-density lipoprotein receptor (LDL-R) mRNAs were down-regulated compared with rats on standard chow. NPC2 did not improve liver weight, histopathology, levels of serum liver enzymes or pro-inflammatory tumor necrosis factor-α (TNFα), Interleukin (IL)-6, or IL-1β in HFHC rats. Two weeks of NPC2 treatment lowered hepatic TNFα and COL1A1 mRNA expression. However, this effect was ultimately reversed following additional two weeks of treatment. Four weeks NPC2 treatment of rats raised ATP-binding cassette A1 (ABCA1) and low-density lipoprotein receptor (LDLR) mRNAs in the liver, concurrent with a strong tendency towards higher serum high-density lipoprotein (HDL). Furthermore, the peroxisome proliferator activated receptor-ɣ (PPARG) gene expression was reduced. Conclusions: NPC2 proved inefficient at modifying robust hepatic NASH end-points in a HFHC NASH model. Nonetheless, our data suggest that hepatic ABCA1 expression and reverse cholesterol transport were upregulated by NPC2 treatment, thus presenting putative therapeutic effects in diseases associated with deregulated lipid metabolism. [ABSTRACT FROM AUTHOR]

Impairment of the autophagy pathway has been observed during the pathogenesis of Alzheimer’s disease (AD), a neurodegenerative disorder characterized by abnormal deposition of extracellular and intracellular amyloid β (Aβ) peptides. Yet the role of autophagy in Aβ production and AD progression is complex. To study whether increased basal autophagy plays a beneficial role in Aβ clearance and cognitive improvement, we developed a novel genetic model to hyperactivate autophagy in vivo. We found that knock-in of a point mutation F121A in the essential autophagy gene Beclin 1/Becn1 in mice significantly reduces the interaction of BECN1 with its inhibitor BCL2, and thus leads to constitutively active autophagy even under non-autophagy-inducing conditions in multiple tissues, including brain. Becn1F121A-mediated autophagy hyperactivation significantly decreases amyloid accumulation, prevents cognitive decline, and restores survival in AD mouse models. Using an immunoisolation method, we found biochemically that Aβ oligomers are autophagic substrates and sequestered inside autophagosomes in the brain of autophagy-hyperactive AD mice. In addition to genetic activation of autophagy by Becn1 gain-of-function, we also found that ML246, a small-molecule autophagy inducer, as well as voluntary exercise, a physiological autophagy inducer, exert similar Becn1-dependent protective effects on Aβ removal and memory in AD mice. Taken together, these results demonstrate that genetically disrupting BECN1-BCL2 binding hyperactivates autophagy in vivo, which sequestrates amyloid oligomers and prevents AD progression. The study establishes new approaches to activate autophagy in the brain, and reveals the important function of Becn1-mediated autophagy hyperactivation in the prevention of AD. [ABSTRACT FROM AUTHOR]

The human papillomavirus type 16 (HPV16) L2 protein acts as a chaperone to ensure that the viral genome (vDNA) traffics from endosomes to the trans-Golgi network (TGN) and eventually the nucleus, where HPV replication occurs. En route to the nucleus, the L2/vDNA complex must translocate across limiting intracellular membranes. The details of this critical process remain poorly characterized. We have developed a system based on subcellular compartmentalization of the enzyme BirA and its cognate substrate to detect membrane translocation of L2-BirA from incoming virions. We find that L2 translocation requires transport to the TGN and is strictly dependent on entry into mitosis, coinciding with mitotic entry in synchronized cells. Cell cycle arrest causes retention of L2/vDNA at the TGN; only release and progression past G2/M enables translocation across the limiting membrane and subsequent infection. Microscopy of EdU-labeled vDNA reveals a rapid and dramatic shift in vDNA localization during early mitosis. At late G2/early prophase vDNA egresses from the TGN to a pericentriolar location, accumulating there through prometaphase where it begins to associate with condensed chromosomes. By metaphase and throughout anaphase the vDNA is seen bound to the mitotic chromosomes, ensuring distribution into both daughter nuclei. Mutations in a newly defined chromatin binding region of L2 potently blocked translocation, suggesting that translocation is dependent on chromatin binding during prometaphase. This represents the first time a virus has been shown to functionally couple the penetration of limiting membranes to cellular mitosis, explaining in part the tropism of HPV for mitotic basal keratinocytes. [ABSTRACT FROM AUTHOR]

Transglutaminase 2 (TG2) is a multifunctional protein that is primarily engaged in cell adhesion/signaling or shows Ca2+-dependent transglutaminase activity in the extracellular space of tissues. This latter action leads to the cross-linking of the extracellular matrix (ECM) proteins. The enhanced extracellular expression of TG2 is associated with processes such as wound healing, fibrosis or vascular remodeling that are also characterized by a high deposition of dermatan sulfate (DS) proteoglycans in the ECM. However, it is unknown whether DS may bind to TG2 or affect its function. Using the plasmon surface resonance method, we showed that DS chains, especially those of biglycan, are good binding partners for TG2. The interaction has some requirements as to the DS structure. The competitive effect of heparin on DS binding to TG2 suggests that both glycosaminoglycans occupy the same binding site(s) on the protein molecule. An occurrence of the DS-TG2 interaction was confirmed by the co-immunoprecipitation of this protein with native decorin that is a DS-bearing proteoglycan rather than with the decorin core protein. Moreover, in vivo DS is responsible for both TG2 binding and the regulation of the location of this protein in the ECM as can be suggested from an increased extraction of TG2 from the human fascia only when an enzymatic degradation of the tissue DS was conducted in the presence of the anti-collagen type I antiserum. In addition, DS with a low affinity for TG2 exerted an inhibitory effect on the protein transamidating activity most probably via the control of the accessibility of a substrate. Our data show that DS can affect several aspects of TG2 biology in both physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

Osteoclasts are giant bone-resorbing cells originating from monocytes/macrophages. During their differentiation, they overexpress two lysosomal enzymes, cathepsin K and TRAP, which are secreted into the resorption lacuna, an acidified sealed area in contact with bone matrix where bone degradation takes place. Here we report that the acid hydrolase HYAL1, a hyaluronidase able to degrade the glycosaminoglycans hyaluronic acid (HA) and chondroitin sulfate, is also upregulated upon osteoclastogenesis. The mRNA expression and protein level of HYAL1 are markedly increased in osteoclasts differentiated from RAW264.7 mouse macrophages or primary mouse bone marrow monocytes compared to these precursor cells. As a result, the HYAL1-mediated HA hydrolysis ability of osteoclasts is strongly enhanced. Using subcellular fractionation, we demonstrate that HYAL1 proteins are sorted to the osteoclast lysosomes even though, in contrast to cathepsin K and TRAP, HYAL1 is poorly mannose 6-phosphorylated. We reported previously that macrophages secrete HYAL1 proforms by constitutive secretion, and that these are recaptured by the cell surface mannose receptor, processed in endosomes and sorted to lysosomes. Present work highlights that osteoclasts secrete HYAL1 in two ways, through lysosomal exocytosis and constitutive secretion, and that these cells promote the extracellular residency of HYAL1 through downregulation of the mannose receptor. Interestingly, the expression of the other main hyaluronidase, HYAL2, and of lysosomal exoglycosidases involved in HA degradation, does not increase similarly to HYAL1 upon osteoclastogenesis. Taken together, these findings point out the predominant involvement of HYAL1 in bone HA metabolism and perhaps bone remodeling via the resorption lacuna. [ABSTRACT FROM AUTHOR]

Selective neuronal vulnerability is characteristic of most degenerative disorders of the CNS, yet mechanisms underlying this phenomenon remain poorly characterized. Many forms of cerebellar degeneration exhibit an anterior-to-posterior gradient of Purkinje cell loss including Niemann-Pick type C1 (NPC) disease, a lysosomal storage disorder characterized by progressive neurological deficits that often begin in childhood. Here, we sought to identify candidate genes underlying vulnerability of Purkinje cells in anterior cerebellar lobules using data freely available in the Allen Brain Atlas. This approach led to the identification of 16 candidate neuroprotective or susceptibility genes. We demonstrate that one candidate gene, heat shock protein beta-1 (HSPB1), promoted neuronal survival in cellular models of NPC disease through a mechanism that involved inhibition of apoptosis. Additionally, we show that over-expression of wild type HSPB1 or a phosphomimetic mutant in NPC mice slowed the progression of motor impairment and diminished cerebellar Purkinje cell loss. We confirmed the modulatory effect of Hspb1 on Purkinje cell degeneration in vivo, as knockdown by Hspb1 shRNA significantly enhanced neuron loss. These results suggest that strategies to promote HSPB1 activity may slow the rate of cerebellar degeneration in NPC disease and highlight the use of bioinformatics tools to uncover pathways leading to neuronal protection in neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Protein metabolism, including the interrelated processes of synthesis and degradation, mediates the growth of an animal. In ectothermic animals, protein metabolism is responsive to changes in both biotic and abiotic conditions. This study aimed to characterise responses of protein metabolism to food deprivation that occur in the coldwater salmonid, Arctic charr, Salvelinus alpinus. We compared two groups of Arctic charr: one fed continuously and the other deprived of food for 36 days. We measured the fractional rate of protein synthesis (KS) in individuals from the fed and fasted groups using a flooding dose technique modified for the use of deuterium-labelled phenylalanine. The enzyme activities of the three major protein degradation pathways (ubiquitin proteasome, lysosomal cathepsins and the calpain systems) were measured in the same fish. This study is the first to measure both KS and the enzymatic activity of protein degradation in the same fish, allowing us to examine the apparent contribution of different protein degradation pathways to protein turnover in various tissues (red and white muscle, liver, heart and gills). KS was lower in the white muscle and in liver of the fasted fish compared to the fed fish. There were no observable effects of food deprivation on the protease activities in any of the tissues with the exception of liver, where the ubiquitin proteasome pathway seemed to be activated during fasting conditions. Lysosomal proteolysis appears to be the primary degradation pathway for muscle protein, while the ubiquitin proteasome pathway seems to predominate in the liver. We speculate that Arctic charr regulate protein metabolism during food deprivation to conserve proteins. [ABSTRACT FROM AUTHOR]

We examined the change in the subcellular distribution of a lysosomal enzyme, β-glucuronidase (β-G), caused by decreased cholesterol levels in mouse melanoma cells using an HMG-CoA reductase inhibitor, lovastatin and lipoprotein-deficient serum (LDS). There was a decrease in the cholesterol content of the cells and increased secretion of the mature form of β-G located in lysosomes, as documented by Percoll density gradient fractionation, digitonin permeabilization and immunoprecipitation. Furthermore, another lysosomal enzyme, cathepsin H, was found to be released in the medium from cells treated with lovastatin. Both the precursor and mature forms of cathepsin H were detected in the medium of treated cells. Next, when cells were treated with LDS without lovastatin, concomitantly with the decrease in the levels of cholesterol and β-G activity in the cells, β-G activity in the medium increased. Also, the ratio of β-G (3.2-fold) released in the medium from cells treated with Dulbecco's modified Eagle medium (D-MEM) containing lovastatin and LDS was higher than that (2.3-fold) on treatment with D-MEM containing LDS without lovastatin. From these results, it was suggested that the exocytosis of mature enzymes from lysosomes into the medium or mis-sorting of the lysosomal precursor forms to the medium was caused by the lovastatin- and/or LDS-induced decrease in the cholesterol content of the cells, although the mechanism of secretion by lysosomal enzymes differed somewhat. [ABSTRACT FROM PUBLISHER]

While chromium was proposed to be an essential trace element over 40 years ago and if essential should possess a specific transport and distribution mechanism, the details of its transport from the bloodstream to the urine have not been elucidated. However, chromium is known to be maintained in the bloodstream bound to transferrin and to be excreted in the urine bound to the oligopeptide chromodulin or a similar chromodulin-like species. Injection of51Cr-labeled transferrin into the bloodstream resulted in a rapid and insulin-sensitive movement of chromium into the tissues as Cr transferrin; greater than 50% of the Cr is transported to the tissues within 30 min. Tissue levels of Cr are maximal 30 min after injection; decreases in tissue Cr with time are mirrored by increases in urine Cr. Approximately 50% of the51Cr appears in the urine within 360 min of injection in the absence of added insulin; insulin treatment concurrent with injection of51Cr-labeled transferrin results in approximately 80% of the label appearing in the urine within 180 min. The removal of51Cr from the blood is faster than the appearance of51Cr in the urine; the lag in time indicates that the Cr transferrin in the blood and Cr in the urine are not in direct equilibrium and that intermediates in the transport of Cr must be involved. This establishes a clear pathway of transport of Cr starting from transport by transferrin from the bloodstream into the tissues, followed by release and processing in the tissues to form chromodulin, excretion into the bloodstream, rapid clearance of chromodulin or a similar species into the urine, and ultimately excretion as this species. Insulin stimulates the processing of Cr in the tissues. [ABSTRACT FROM AUTHOR]

Group 2 major mite allergens Der f 2 and Der p 2 are classified into the recently identified group of MD-2-related lipid-recognition (ML) proteins, but the ligands and biological functions of these allergens are unknown. We have obtained a high-quality NMR structure for Der f 2, and found that it is more similar to the crystal structure of NPC2, a distant homologue, than to that of Der p 2, in terms of the separation and angle between the two major β-sheets. This made us propose that ML proteins undergo clamshell-like motions that change the sizes of ligand-binding spaces inside their immunoglobulin-fold β-sandwich to accommodate lipid molecules. This type of motion in lipopolysaccaride recognition of MD-2 is suggested to be likely as well by structural models. We also report the applicability of NMR differential exchange broadening experiments for complexes of intact monoclonal antibodies and antigens; using this technique, we have detected the conformational epitopes for monoclonal antibodies 15E11 and 13A4 as two separate surface patches. [ABSTRACT FROM PUBLISHER]

Today, many scientific discoveries are made using a top-down experimental approach. The ubiquitin system was discovered using a 'classic' bottom-up approach to tackle the question: 'how are cellular proteins selectively degraded?' A simple proteolytic assay, which used a crude cell-extract, was all that was required to address this question; it was followed by fractionation and reconstitution experiments to decipher the role of the components in this multi-step process. This 'biochemistry at its best' approach, which was published in a periodical that today would not be regarded as highly visible, provided magnificent findings. [ABSTRACT FROM AUTHOR]

Abstract: Classical late-infantile neuronal ceroid lipofuscinosis (LINCL), a progressive and fatal neurodegenerative disease of childhood, results from mutations in a gene (CLN2) that encodes a protein with significant sequence similarity to prokaryotic pepstatin-insensitive acid proteases. We have developed a sensitive protease activity assay that allows biochemical characterization of the CLN2 gene product in various human biological samples, including solid tissues (brain and chorionic villi), blood (buffy coat leukocytes, platelets, granulocytes, and mononuclear cells), and cultured cells (lymphoblasts, fibroblasts, and amniocytes). The enzyme has a pH optimum of 3.5 and is rapidly inactivated at neutral pH. A survey of fibroblasts and lymphoblasts demonstrated that lack of activity was associated with LINCL arising from mutations in the CLN2 gene but not other neuronal ceroid lipofuscinoses (NCLs), including the CLN6 variant LINCL, classical infantile NCL, classical juvenile NCL, and adult NCL (Kufs' disease). A study conducted using blood samples collected from classical LINCL families whose affliction was confirmed by genetic analysis indicates that the assay can distinguish homozygotes, heterozygotes, and normal controls and thus is useful for diagnosis and carrier testing. Analysis of archival specimens indicates that several specimens previously classified as LINCL have enzyme activity and thus disease is unlikely to arise from mutations in CLN2. Conversely, a specimen previously classified as juvenile NCL lacks pepinase activity and is associated with mutations in CLN2. In addition, several animals with NCL-like neurodegenerative symptoms [mutant strains of mice (nclf and mnd), English setter, border collie, and Tibetan terrier dogs, sheep, and cattle] were found to contain enzyme activity and are thus unlikely to represent models for classical LINCL. Subcellular fractionation experiments indicate that the CLN2 protein is located in lysosomes, which is consistent with its acidic pH optimum for activity and the presence of mannose 6-phosphate. Taken together, these findings indicate that LINCL represents a lysosomal storage disorder that is characterized by the absence of a specific protease activity. [ABSTRACT FROM AUTHOR]

Presents abstracts of studies presented at the Belgian Association of Biochemistry and Molecular Biology congress in Brussels, Belgium on November 6, 1999. `Fission Yeast Sk1p Interacts With the Mcs6p-Mcs2p Complex in Two-Hybrid Plus 1,' by S. Bamps et al.; `Characterization of a New Class of Plasma Membrane Transport Proteins in Maize,' by F. Chaumont et al.; `Characterization of YKL165c, a new essential gene of the yeast Saccharomyces cerevisiae,' by X. Delbecq et al.

Presents abstracts on biochemistry and molecular biology. `Use of the yeast YOL112w gene as an experimental tool to elucidate the cellular function of the Tre2 oncogene implicated in the Ewing's sarcoma,' by S. Bach, O. Bouchat, et al ; `Ferritin heavy chain overexpression in brain structures of a genetic model of generalized idiopathic absence epilepsy in rat,' by B. De Borman, B. Lakaye, et al.

Discusses the pathways for nucleic acid biosynthesis and degradation in skin. Aspects of nucleic acid metabolism; Location of DNA and RNA synthesis in the epidermis; Importance of pyrimidine nucleotide salvage pathways for DNA synthesis in the skin.

A metallo-endopeptidase, which appears to be an integral membrane protein of rat kidney, was purified to homogeneity by a series of standard chromatographic procedures. This enzyme significantly hydrolyzed human parathyroid hormone [hPTH(1–84)] and a synthetic substrate Suc-Leu-Leu-Val-Tyr-Mec (Suc = succinyl, Mec = 4-methyl-coumarinyl-7-amide). The purified enzyme had apparent molecular masses of 250 kDa on gel filtration, and 88 kDa and 245 kDa on sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and now reducing conditions, respectively. Its pH optimum for activity was 8.0–8.5 and its isoelectric point was pH 4.9. Its activity was inhibited by EDTA, EGTA and o-phenanthroline, but not by phosphoramidon. The metal-depleted enzyme was reactivated by the addition of metal ions. The enzyme was also inhibited by chymostatin and eglin C, and by thiol compounds. Of the synthetic substrates examined, the enzyme hydrolyzed only Suc-Leu-Leu-Val- Tyr-Mec, one of file synthetic substrates for α-chymotrypsin. It did not hydrolyze synthetic substrates with less than four amino acid residues with tyrosine in the Pl position. The enzyme hydrolyzed hPTH and reduced hen egg lysozyme but did not hydrolyze azocasein or [3H]methyl-casein. NH2-terminal amino acid sequence analyses of the degradation products of hPTH(1–84) and reduced hen egg lysozyme by the purified enzyme revealed that the enzyme preferentially cleaved these peptides at peptide bonds flanked by hydrophilic amino acid residues. Amino acid analyses showed that the main degradation products of PTH were hPTH(I7–29), hPTH(30–38) and hPTH(74–84). The ability of the enzyme to hydrolyze peptide bonds flanked by hydrophilic amino acid residues and its inability to degrade azocssein distinguish it from several other kidney endopeptidases reported, such as endopeptidase 24.11 and meprin. [ABSTRACT FROM AUTHOR]

β-Migrating very-low-density lipoproteins (β-VLDL) are cholesteryl-ester-enriched lipoproteins which accumulate in the serum of cholesterol-fed animals or patients with type III hyperlipoproteinemia. In the rat, β-VLDL are rapidly cleared by the liver and parenchymal liver cells form the major site for uptake. In this investigation, β-VLDL were labeled with [3H]cholesteryl esters and the hepatic intracellular transport of these esters was followed. 2 min after injection, the major part of the [3H]cholesteryl esters is already associated with the liver and a significant proportion is recovered in endosomes. Up to 25 min after injection, an increase in radioactivity in the lysosomal compartment is noticed. This radioactivity initially represents cholesteryl esters, while from 25 min onward, radioactivity is mainly present in unesterified cholesterol. Between 45 min and 90 min after β-VLDL injection, specific transfer of unesterified [3H]cholesterol to the endoplasmic reticulum is observed, while by 3 h the majority is located in this fraction. The appearance of radioactivity in the bile was rather slow as compared to the rapid initial uptake and processing, and up to 5 h after injection only 10% of the injected dose had reached the bile (mainly as bile acids). 72 h after injection, the amount of the injected radioactivity recovered in the bile had increased to 50%. Chloroquine treatment of the rats inhibited the hydrolysis of the cholesteryl esters and the appearance of radioactivity in the bile was retarded. It is concluded that β-VLDL are rapidly processed by parenchymal liver cells and that the cholesteryl esters from β-VLDL are hydrolyzed in the lysosomal compartment. Unesterified cholesterol remains associated with the endoplasmic reticulum for a prolonged time, although ultimately the majority will be secreted into the bile as bile acids. The effective operation of this pathway will prevent extrahepatic accumulation of cholesteryl esters from β-VLDL, while the prolonged residence time of unesterified cholesterol in the endoplasmic reticulum might be important for regulation of low-density lipoprotein (LDL) receptors in liver and thus for LDL levels in the blood. [ABSTRACT FROM AUTHOR]

In vitro incubation of the oligomannosyl oligosaccharides Man9GlcNAc and Man5GlcNAc with isolated disrupted lysosomes yields different oligosaccharide isomers resulting from mannosidase hydrolysis. These isomers were isolated by HPLC and characterized by 1H-NMR spectroscopy. The first steps of the degradation involve an (α1–2)mannosidase activity and lead to the formation of one Man8GlcNAc, one Man7GlcNAc, two Man6GlcNAc and two Man5GlcNAc isomers. These reactions do not require Zn2+ as activator. On the other hand, the following steps, which lead to the formation of Man3GlcNAc and Man2GlcNAc, are Zn2+-dependent. This process is characterized by the preferential action of an (α1–3)mannosidase activity, and the formation of Man(α1–6)Man(αl–6)Man(βl–4)(GlcNAc and Man(α1–6)Man(β1–4)GlcNAc. Therefore, the digestion of Man9GlcNAc inside the lysosome appears to follow a very specific pathway, since only nine intermediate compounds can be identified instead of the 38 possible isomers. Our results are consistent both with the existence of several specific enzymes for α1–2, α1–3 and α1–6 linkages, and with the presence of a unique enzyme whose specificity would be dependent either on Zn2+ or on the spatial conformation of the glycan. Nevertheless, previous work on the structural analysis of oligosaccharides excreted in the urine of patients suffering from mannosidosis, demonstrates the absence of the core α1-6-1inked mannosyl residue in the major storage product derived from oligomannosyl oligosaccharides. This observation indicates the presence of a specific (α1–6)mannosidase form, unaffected in mannosidosis. [ABSTRACT FROM AUTHOR]

Presents an author index for the Volumes 161-170, 1986-1988 issues of the "European Journal of Biochemistry."

[14C]Sucrose, introduced into the cytosol of isolated rat hepatocytes by means of electropermeabilization, was sequestered by sedimentable subcellular particles during incubation of the cells at 37°C. The sedimentation characteristics of particle-associated [14C]Sucrose were different from the lysosomal marker enzyme acid phosphatase, suggesting an involvement of organelles of greater size than the average lysosome. Isopycnic binding in isotonic metrizamide/sucrose density gradients resolved two major peaks of radioactivity: a light peak (1.08-1.10 g/ml) coinciding with lysosomal marker enzymes, and a dense peak (1.15 g/ml), coinciding with a mitochondrial marker enzyme. The dense peak was preferentially associated with large-size particles having the sedimentation properties of mitochondria, and it was resistant to the detergent digitonin at a concentration which extracted all of the radioactivity in the light peak. Similarly the autophagy inhibitor 3-methyladenine prevented accumulation of [14C]sucrose in the light peak, while the radioactivity in the dense peak was unaffected. We therefore tentatively conclude that the light peak represents autophagic sequestration of [14C]sucrose into lysosomes (and probably autophagosomes) while the dense peak represents a mitochondrial uptake unrelated to autophagy. [ABSTRACT FROM AUTHOR]

Most lysosomal hydrolases are soluble enzymes. Lamp-II (lysosome-associated membrane protein-II) is a major constituent of the lysosomal membrane. We studied the aggregation of a series of lysosomai molecules. The aggregation-sensitive lysosomal marker enzymes were optimally aggregated at intralysosomal pH. A similar pH dependence was recorded for aggregation of Lamp-II. The pH-dependent loss of solubility of isolated Lamp-II required components of the lysosome extract. Conditions of mild acid pH promoting aggregation triggered the formation of complexes with lipids of lysosomal origin. We fractionated a membrane-free lysosome extract by gel-filtration chromatography and could reconstitute assemblies in vitro from separated fractions. We found some selectivity in the lysosomal proteins binding to complex lipids, phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine being most effective. We propose that the formation at pH 5.0 of such supramolecular assemblies between lysosomal proteins and lipids occurs within the intralysosomal environment. Some possible consequences of such an intralysosomal matrix formation on organelle function are discussed. [ABSTRACT FROM AUTHOR]

In order to gain some insight into mitochondrial flavin biochemistry, rat liver mitochondria essentially free of lysosomal and microsomal contamination were prepared and their capability to metabolise externally added and endogenous FAD and FMN tested both spectroscopically and via HPLC. The existence of two novel mitochondrial enzymes, namely FAD pyrophosphatase (EC 3.6.1.18) and FMN phosphohydrolase (EC 3.1.3.2), which catalyse FAD←FMN and FMN←riboflavin conversion, respectively, is shown. They differ from each other and from extramitochondrial enzymes, as judged by their pH profile and inhibitor sensitivity, and can be separated in a partial FAD pyrophosphatase purification. Digitonin titration and subfractionation experiments show that iFAD pyrophosphatase is located in the outer mitochondrial membrane and FMN phosphohydrolase in the intermembrane space. Since these enzymes can metabolise endogenous FAD and FMN, which are made available by using both Triton X-100 and the effector oxaloacetate, a proposal is made that FAD pyrophosphatase and FMN phosphohydrolase play a major role in mitochondrial flavoprotein turnover. [ABSTRACT FROM AUTHOR]

Presents several abstracts on physiology and biochemistry. 'A Modified AFLP method: A Tool for the Detection of Molecular Markers in Ryegrass Mitochondria,' by E. Adams, I. Roldan-Ruiz, et al. ; 'Study of Defence Responses Expressed by Grapevine During Its Interaction With Botrytis Cinerea,' by F. Baillieul, J-P. Cassart, et al.; 'Promoter-Probe System to Identify CtrA-Regulated Genes From Brucella,' by S. Virot, A. Tibor, et al.

Subcellular fractions from rat anterior pituitary homogenates were obtained by differential and gradient centrifugation, identified with the help of marker enzymes and screened for peptidases capable of hydrolyzing gonadoliberin, thyroliberin, enkephalin and substane P. Since each neuropeptide is susceptible to cleavage by more than one enzyme, specific substrates or inhibitors have been used for the selective determination of the individual peptidasic activities. Among the various enzymes tested, the angiotensin-converting enzyme, the thermolysin-like metalloendopeptidase (‘enkephalinase’), a thyroliberin-degrading enzyme and some aminopeptidasic activities were found to be associated with the plasmamembrane. Other aminopeptidases, a gonadoliberin-degrading and a substance-P-degrading enzyme are associated with the mitochondria and thus are most likely not involved in the biological inactivation of neuropeptides. [ABSTRACT FROM AUTHOR]

The effect of pressure on the heat activation in vivo of trehalase in the spores of Phycomyces blakesleeanus has been investigated in order to obtain information about the molecular mechanism of the activation. For a protein conformational change directly induced in the enzyme by the heat treatment an upward shift with about 2–6 K/1000 atm (1.013 ⊗ 105 kPa) is to be expected in the moderate high-pressure region. On the other hand, for a phospholipid phase transition causing the activation, a continuous upward shift with about 20 K/1000 atm is to be expected. For trehalase activation we find a continuous upward shift of the activation temperature with about 5–9 K/1000 atm. The denaturation of trehalase. which occurs at slightly higher temperatures, is influenced by pressure completely as expected for a protein conformational change. The application of high pressure during spore heat activation makes it possible to break the dormancy of the spores without concomitant activation of trehalase. [ABSTRACT FROM AUTHOR]

The subcellular distribution of aryl-hydrocarbon hydroxylase, NADPH-cytochrome c reductase, NADH :cytochrome c reductase, and NADH :cytochrome b5 reductase activities in mouse liver was studied using the biochemical membrane markers microsomal glucose-6-phosphatase, mitochondrial cytochrome c oxidase, and plasma membrane 5′-nucleotidase. The rate of appearance of activity of 3-methylcholanthrene-induced aryl-hydrocarbon hydroxylase in the microsomes of C57BL/6N mice is more than twice as rapid as that in the nuclear envelope. The nuclear fraction contains less than 1% of the total cellular activities of the hydroxylase and all three reductases. All detectable basal activity of aryl-hydrocarbon hydroxylase in the nuclear fraction of control C57BL/6N and DBA′2N and 3-methylcholanthrene-treated DBA/2N mice and all detectable activities of NADPH :cytochrome c, NADH :cytochrome c, and NADH :catochrome b5 reductase in the nuclear fraction of control and 3-methylcholanthrene-treated C57BL/6N and DBA/2N mice can be completely accounted for by the degree of microsomal fragment contamination (as assessed by the microsomal marker glucose-6-phosphatase). These data raise doubts about certain previous reports of ‘nuclear’ enzyme activities in which microsomal contamination was not taken into account. However, there is more induced activity of aryl-hydrocarbon hydroxylase in the nuclear fraction of 3-methylcholanthrene-treated C57BL/6N mice than can be accounted for by the degree of microsomal membrane contribution. The routine Ah locus is known to regulate the induction by certain polycyclic aromatic chemicals of numerous drug-metabolizing enzyme activities such as aryl-hydrocarbon hydroxylase associated with cytochrome P1-450. The expression of 3-methylcholanthrene-inducible hydroxylase in nuclear membranes, like that in microsomal membranes, thus appears to be controlled by the Ah regulatory gene. [ABSTRACT FROM AUTHOR]

A lipoxygenase has been purified from rabbit reticulocyte-rich anaemic blood cells. It possesses a molecular weight of 78 000 and an isoelectric point of 5.5 and contains 5% neutral sugars and two iron atoms per enzyme molecule. The lipoxygenase has proved to be identical with the inhibitors of respiratory proteins described formerly. The actions of the lipoxygenase on linoleic acid, phospholipids, mitochondrial and erythrocyte membranes and electron transfer particles were studied. A special feature of the reticulocyte lipoxygenase is the suicidal character of its action on lipids. With electron transfer particles the reticulocyte lipoxygenase causes a loss of acid-labile sulfur which accompanies respiratory inhibition; the strong respiratory inhibition is not exerted by soybean lipoxygenase. The reticulocyte lipoxygenase acts preferably on mitochondrial membranes as compared with cell membranes of the erythrocyte; erythrocyte cytosol moderates the action on mitochondrial membranes. Furthermore, the lipoxygenase reaction can concomitantly and irreversibly inactivate sulfydryl enzymes as demonstrated with muscle glyceraldehyde-3-phosphate dehydrogenase. The occurrence of the lipoxygenase here described is restricted to reticulocytes; very low amounts were observed in bone marrow and no lipoxygenase was detectable in normal blood. During the course of an experimental anaemia the lipoxygenase is produced owing to superinduction in large amounts, which may persist for a long time since they escape inactivation. Preliminary evidence was obtained for the occurrence of other lipoxygenases in tissues of lung, spleen, kidney and also epithelial tumours. [ABSTRACT FROM AUTHOR]

The intracellular localization of exogenously supplied human platelet β-glucuronidase in cultured skin fibroblasts derived from a β-glucuronidase-deficient patient was studied. Four cellular fractions were obtained by differential speed centrifugation. Following two days of incubation, the exogenously supplied enzyme exhibited a distribution pattern identical to that of endogenous β-hexosaminidase. Disruption of membranes by freezing and thawing caused a 35% increase of the enzyme activity, thus indicating a latent activity following the internalization. This indicated localization in the lysosomal fractions. Longer incubation periods led to an intracellular shift of the engulfed enzyme from the lighter lysosomal fraction to heavier particles. Once located in the heavier fraction, the enzyme was relatively stable, and participated in the catabolism of 35S-labeled mucopolysaccharides which had accumulated in the lysosomes of these fibroblasts. A marked reduction in the accumulated mucopolysaccharides of the lysosomal fraction was observed following addition of the enzyme, This was accompanied by the formation of smaller sized molecules. [ABSTRACT FROM AUTHOR]

To localize the membrane-bound, histone-degrading proteinase, which was previously isolated from the mitochondrial fraction, nuclei, mitochondria, lysosomes, peroxisomes, smooth and rough endoplasmic reticulum, ribosomes and plasma membranes were prepared from a rat liver homogenate according to established methods and characterized by marker enzyme activities. The isolated sub- cellular fractions were treated with digitonin, and subjected to a discontinuous sucrose gradient centrifugation. The material, which sedimented through 1.74 M sucrose was analyzed in respect to the various marker enzymes and for proteolytic activity. Proteinase activity was found in the material obtained after digitonin treatment and step gradient centrifugation of mitochondria. This finding shows the occurrence of a proteinase in mitochondria. After fractionation of mitochondria into outer and inner membrane, intermembrane fraction and matrix, the proteinase could be localized exclusively in the inner mitochondrial membrane. A possible physiological function of the enzyme during the biosynthesis of inner membrane constituents is discussed. [ABSTRACT FROM AUTHOR]

Previous studies have shown that mannose incorporation into mitochondrial glycoproteins is catalysed by a mannosyl-transferase, which is located in the mitochondrial inner membrane. It seems important to study the presence of mannosyl-transferases which are able to catalyse the transfer of mannose into other acceptors, such as polyprenic intermediates. Mouse liver mitochondria catalyse the incorporation of mannose from GDP-[14C]mannose into three products: most of the radioactivity is in a product (A) soluble in chloroform/methanol (2/1, by vol.); mannose is also incorporated into product (B) soluble in chloroform/methanol/water (10/10/3, by vol.), and into a product which is insoluble in organic solvents and trichloroacetic acid and which is a glycoprotein. We have tried to localize in mitochondria the mannosyl-transferases implicated in these reactions. Mitochondrial outer membranes were prepared by swelling purified mitochondria in phosphate buffer, and were purified on a discontinuous sucrose gradient. The purified outer membranes catalyse the incorporation of mannose from GDP-[14C]mannose in very large quantities into product A and in small quantities into product B. Different procedures (purification on silicic acid and DEAE-cellulose, hydrolytic procedures and chromatographic properties) demonstrated the identity of product A with authentic dolichol phosphate mannose. Product B is lipid-linked oligosaccharides. The biosynthesis of dolichol phosphate mannose depends on Mn2+. When ethyleneglycol bis(2aminoethyl)N,N'-tetracetic-acid (EGTA) is added to the mixture, the reaction is inhibited. When EGTA is added to a mixture containing preformed endogenous dolichol phosphate [14C]mannose, the biosynthesis of the product stops, and the product is hydrolysed, with a concomitant apparition of lipid-linked-oligosaccharides. These results and the kinetics of the reactions suggest that dolichol phosphate mannose might be the mannosyl donor for the lipid-linked-oligosaccharides. The synthesis of dolichol phosphate mannose is reversed by the addition of GDP. The role of these products as intermediates in mitochondrial glycoprotein biosynthesis is discussed. [ABSTRACT FROM AUTHOR]

It is shown that lipids are responsible for the breaks in the Arrhenius plots of Azotobacter nitrogenase. The physical evidence is that temperature at which the break occurs increases with increasing pressure by 20 K/1000 atm. This is in agreement with the pressure dependence of the transition temperature of several synthetic phospholipids. We also find the same pressure dependence for the broad transitions observed in Esclwrichia coli phosphatidylethanolamine and in the membrane lipids from Azotobacter itself. Detergents and phospholipase remove the break. Reconstruction can be performed only with specific phospholipids. [ABSTRACT FROM AUTHOR]

Homogenates of Chinese hamster fibroblasts have been fractionated by analytical centrifugation techniques. Lysosomes have been characterized and identified by the distinctive behaviour of specific marker constituents. The acid hydrolases of hamster fibroblasts were located in two distinct organelles, characterized by differences in their equilibrium densities and rates of sedimentation. The lighter population of lysosomes with a modal density of 1,10 g cm-3, contained glycosidases, nucleases and acid phosphatase. The denser population, with a modal density of 1.15 g cm-3, contained proteases and arylsulphatase B. Acid hydrolases in both populations showed similar degrees of latency and structure-linked sedimentability. The heterophagic activity of hamster fibroblasts was investigated by growing the cells in the presence of [14C]sucrose, [14C]inulin and Triton WR 1339. These compounds are taken up by the cells and appear to be located in the denser population of lysosomes. The low density of the lighter population of lysosomes appears to result from a high content of cholesterol esters and triacylglycerols. It is suggested that the lysosomes may be the site of storage of a considerable proportion of these lipids in cultured hamster cells. [ABSTRACT FROM AUTHOR]

With a view to the study of the subcellular localization of nucleases, methods ensuring the specificity of enzyme determinations are presented for six types of nuclease activities in liver homogenates. The ribonuclease activity of rat liver is due to three enzymes with different pH optimum. For acid ribonuclease (pH optimum 5.3), it is possible to avoid interference from the other ribonucleases by performing the incubation at pH 5. Neutral ribonuclease (pH optimum 7.6) is differentiated by relying on its sensitivity to the natural inhibitor from the supernatant of liver homogenate. Comparison of activities before and after pretreatment at 50°C in acid medium permits the specific measurement of alkaline ribonuclease (pH optimum 8.8). The optimal conditions for the determination in liver homogenates of two deoxyribonucleases and of an enzyme acting on polyriboadenylate are also described. The activity of these various nucleases is compared and some of their properties are investigated. [ABSTRACT FROM AUTHOR]

Several protein kinases which catalyze the phosphorylation of glycogen synthetase, histones, protamine and proteins present in isolated plasma membranes, microsomes and nuclei have been extensively purified from swine kidney homogenates. About 60% of the protein kinase activity in kidney was recovered in the soluble 100000 × g supernatant fraction following differential centrifugation. The remaining 40% of the activity was found to be associated with the plasma membrane, nuclear and microsomal subcellular fractions. The activity of the protein kinases which were bound to the particulate structures was not enhanced by adenosine .3′:5′-mono-phosphate (cyclic AMP) when kidney glycogen synthetase or histone was used as the protein substrate, however the rate of phosphorylation of these substrates by the soluble enzymes was increased 50 to 100% by the addition of 0.5 μM cyclic AMP. The bound protein kinase activity was dissociated from the particulate structures by treatment with 0.7 M NaCl. The soluble and bound protein kinase were purified by a procedure involving treatment with alumina Cγ, adsorption to calcium phosphate gel, and chromatography on diethylaminoethylcellulose. Both protein kinase preparations were separated into two fractions by the last procedure and their elution profiles were very similar. The apparent Km for ATP in the presence of 10 mM MgCl2 varied from 5 to 9 μM when either glycogen s3mthetase or histone was the substrate. All four preparations showed a very broad specificity for protein substrates. They phosphorylated histones, protamine, casein and glycogen synthetases. The molecular weight of the four kidney protein kinase preparations calculated from data obtained by sucrose-density centrifugation and chromatography on Sephadex G-200 was 170000 ± 10000. Upon polyacrylamide-gel electrophoresis in 1 % sodium dodecylsulfate all of the samples showed two major components. The kinetic properties of the bound form of protein kinase was examined with a sonicated preparation of kidney plasma membranes. The molecular weight of the active fragment was greater than 1000000. Proteins present in the membrane were labeled with 32P when the preparation was incubated with magnesium ion and [γ-32P] ATP. This preparation also phosphorylated glycogen synthetase and histone. However, the apparent Km of the bound form of the enzyme for ATP was 2.8 mM when glycogen synthetase was used as the protein substrate. [ABSTRACT FROM AUTHOR]

After differential centrifugation of Ehrlich ascites cells by the de Duve method, NAD nucleosidase activities were found in the nuclear and the microsomal fractions, but not in the lysosomal fraction. The nuclear NAB nucleosidase is inhibited 50% by 0.2 mM nicotinamide and is highly specific for β-NAD. Its activity is markedly decreased after DNase treatment; it is associated with chromatin and is very probably identical with an enzyme that polymerizes the adenosine diphosphate ribose moiety of NAB to an acid-insoluble polymer first described in rat liver nuclei by Chambon, Weill, and Mandel. The microsomal NAD nucleosidase is inhibited 50% by 1.2 mM nicotinamide, and splits NAD, NADP and NMN at a ratio of 1:0.5:0.2. NAD and NADP nucleosidase activities are reciprocally inhibited by NADP and NAD. Evidence is presented that the microsomal enzyme is active mainly at the surface of the cell. Nicotinamide concentration may be one factor regulating the nuclear but not the microsomal NAD nucleosidase. Within the cell, NAD is protected by membrane structure from degradation by the microsomal NAD nucleosidase. [ABSTRACT FROM AUTHOR]

The intracellular distribution of thymidine phosphorylating kinases has been studied ill rat liver at different times after partial hepatectomy. After conventional fractionation in 0.25 M sucrose, thymidine kinase is present in all fractions, the highest proportion being found, however, in the supernatant; phosphorylation of thymidine to TDP and TTP is detected only in the supernatant fraction from 24 h onwards after hepatectomy. Nuclei prepared in 2.2 M sucrose or in non-aqueous media have the same level of thymidine kinase activity and can never phosphorylate thymidine to TDP and TTP. In the nuclear fractions, the absolute activity of thymidine kinase increases from 16 to 46 h after hepatectomy but represents a decreasing proportion of the activity of the homogenate. At the same time, the proportion of kinase activity rises markedly in the supernatant fraction with a maximum of 83% in 40 h regenerating livers. The implications of these results are discussed. [ABSTRACT FROM AUTHOR]

A β-oxidative cleavage of oleyl- and stearyl-CoA has been obtained with soluble enzymes of rat-liver homogenate. The transformation requires NAD+ as an oxido-reductive cofactor and is enhanced by ATP and CoA. Under the reported conditions 2,9-octadecadienoic, 3,9-octadecadienoic, 3-hydroxy-9-octadecenoic, 7-hexadecenoic, 3-hydroxy-7-hexadecenoic and 5-tetradecenoic acids are present after incubation of oleyl-CoA, while no detectable amounts of shorter fatty acids are formed. A similar oxidation pattern is obtained with stearyl-CoA when Mg2+ ions are also added to the system. The cytoplasmic origin of these enzymes is established by comparing their activity with that of the mitochondrial β-oxidation enzymes. [ABSTRACT FROM AUTHOR]

An enzyme degrading dephospho-CoA with 5′-AMP as the reaction product is present in the plasma membranes of rat liver. CoA is degraded at less than 10% of the rate of dephospho-CoA. The presence of plasma membranes in nuclear and microsomal fractions, which also contain significant amounts of the plasma-membrane marker enzymes, probably explains the presence of dephospho-CoA pyrophosphatase in these fractions. The enzyme is different from inorganic pyrophosphatase present in nuclei and microsomes with respect to pH optimum and sensitivity to inhibitors. Dephospho-CoA pyrophosphatase is inactivated by dialysis against EDTA, and is partially reactivated by Mn2+ and a number of other divalent cations. The pyrophosphatase degrading dephospho-CoA and CoA is probably identical with nucleotide pyrophosphatase from plasma membranes. The degradation of dephospho-CoA is inhibited competitively by nucleotides with a pyrophosphate bond; most strongly by NADH, which has the highest affinity for nucleotide pyrophosphatase. Competitive inhibitors of dephospho-CoA degradation are less efficient when a ribose 3′-phosphate is present in the nucleotide. Km for dephospho-CoA is 0.02 mM, for CoA about 0.3 mM. These results indicate that the 3′-phosphate group is a sterical hindrance for the access of the nucleotides to the active site. V for dephospho-CoA in plasma membranes was about 200 nmol × mg protein,-1 × min-1, but for CoA one order of magnitude less. Also nucleotide pyrophosphatase from Crotalus adamanteus degraded CoA at a much lower rate than dephospho-CoA (below 1%). [ABSTRACT FROM AUTHOR]

Presents author index for volume 174, issues 1-4 of the "European Journal of Biochemistry".

Presents the author index for the December 15, 1986 issue of the "European Journal of Biochemistry."

Presents the table of contents of the July 1998 issue of the "European Journal of Biochemistry".

Presents the author index for the December 1985 issue of the "European Journal of Biochemistry."

Presents the table of contents for the March 1984 issue of the "European Journal of Biochemistry."

Presents an author index for the July 1988 issue of the "Journal of Biochemistry".