Existence of a Novel Hemagglutinin Having No Protease Activity in Vibrio mimicus

The protease elaborated by Vibrio mimicus is known to possess hemagglutinating ability to chicken erythrocytes, the well-known HA/protease. A non-protease hemagglutinin (HA) with strong agglutinating ability towards rabbit erythrocytes was obtained from 32 hr culture supernatant of a pathogenic environmental strain of V. mimicus. This HA (V. mimicus HA: VMHA) appeared stable at relatively higher temperature and agglutinated the erythrocytes from rabbit, guinea pig and mouse but not the erythrocytes from chicken, bovine, horse and sheep. Simple sugars, metal ions and chelating agents failed to inhibit the activity of VMHA. The activity of VMHA was found to be sensitive to digestion by proteolytic enzymes including HA/protease. These results provide evidence for the existence of novel HA other than HA/protease in V. mimicus.     

Vascular permeability enhancement by Vibrio mimicus protease and the mechanisms of action.

Vibrio mimicus, a causative agent of gastroenteritis, has also been reported to attribute to extraintestinal infections. Recently we have purified a metalloprotease produced by the pathogen: however, the role of the protease in V. mimicus infection has not been documented. The V. mimicus protease (VMP) was found to enhance vascular permeability and form edema when injected into the dorsal skin of guinea pig and rat. The permeability enhancement by VMP was observed in a dose-dependent manner in both guinea pig and rat skin. In guinea pig, an inhibitor of the angiotensin-converting enzyme was found to augment the permeability enhancement reaction. The permeability enhancement was significantly blocked by soybean trypsin inhibitor (SBTI), an inhibitor of plasma kallikrein reaction. In vitro conversion of plasma prekallikrein to kallikrein by VMP was also noted. In rat skin, the permeability enhancement reaction was not blocked by antihistamine or SBTI. However, the reaction was partially blocked when a mixture of antihistamine and SBTI was administered with VMP. It is apparent from the study that in guinea pig skin, VMP enhances vascular permeability through activation of plasma kallikrein-kinin system which generates bradykinin, whereas in addition to the activation of plasma kallikrein-kinin cascade in the case of rat, stimulation of histamine release from mast cells and other unknown mechanism seem to be also a cause of the permeability enhancement reaction. These results suggest that VMP may play a role in extraintestinal infections with edema caused by the pathogen.     

A gene for the enterotoxin zonula occludens toxin is present in Vibrio mimicus and Vibrio cholerae O139

Abstract The presence of the zonula occludens toxin (ZOT) gene, which encodes an enterotoxin produced by serotype O1 strains of the pathogenic bacterium, Vibrio cholerae , in addition to cholera toxin, was investigated in selected strains of V. mimicus and the new pandemic V. cholerae non-O1 serotype O139. The zot gene was detected by polymerase chain reaction (PCR) amplification, using sets of primers based on the sequence of the V. cholerae O1 zot sequence. PCR amplification of genomic DNAs of both cholera toxin gene ( ctx ) positive and ctx⁻ strains of V. mimicus detected the presence of zot gene. An Acc -I- Eco RV V. cholerae zot gene fragment designed to overlap PCR products was used as a probe. Southern hybridization studies confirmed that the PCR fragments from V. mimicus and V. cholerae O139 were strongly homologous to the V. cholerae O1 zot gene. The zot gene was found with 3 to 5 strains of V. mimicus of which only one strain harbored the ctx gene. The presence of a zot gene in ctx⁻ toxigenic V. mimicus indicates a possible role of ZOT in the toxigenicity of this species. We conclude that, in addition to ctx, V. mimicus and V. cholerae O139 have the potential to produce ZOT.     

Maintenance of broad host range vector pKT230 in marine unicellular cyanobacteria

Clinical isolate of Vibrio mimicus were examined for production of cell-associated hemagglutinin (HA) and pili and for adherence to formalin-fixed human intestinal mucosa. V. mimicus grown on CFA agar for 3 h at 37 degrees C possessed HA and adhered better to the mucus layer than to the epithelial cell surface. A significant correlation was found between the HA titers and adherence ability to the epithelial cell surface of villi (P less than 0.05); adherence to the ileal lymphoid follicle-associated epithelium occurred at higher levels. In contrast, V. mimicus grown on CFA agar for 20 h at 37 degrees C exhibited lower levels of HA and reduced adherence ability. The production of pili was more pronounced after 20 h of incubation than after 3 h of incubation. In comparison with V. cholerae 01 and V. cholerae non-01 cultured under similar conditions, V. mimicus showed inferior adherence, but with similar HA production or piliation.     

Characterization of enterotoxin and soluble hemagglutinin from Vibrio mimicus: identity with V. cholerae O1 toxin and hemagglutinin

Abstract Eight strains of Vibrio mimicus isolated from patients with diarrhoea in Bangladesh were all found to produce an extracellular toxin identical to cholera toxin produced by Vibrio cholerae O1 bacteria, with regard to subunit structure and immunological properties. Like cholera toxin, but in contrast to heat-labile enterotoxin from Escherichia coli most of the toxin from V. mimicus was found extracellularly and was proteolytically 'nicked' in its A subunit. This may relate to the finding that V. mimicus also produced an extracellular hemagglutinin which was immunologically indistinguishable from the soluble hemagglutinin/nicking protease of V. cholerae O1.     

Vibrio mimicus diarrhea following ingestion of raw turtle eggs.

Clinical and epidemiological characteristics of diarrhea associated with Vibrio mimicus were identified in 33 hospitalized patients referred to the Costa Rican National Diagnostic Laboratory Network between 1991 and 1994. The relevant symptoms presented by patients included abundant watery diarrhea, vomiting, and severe dehydration that required intravenous Dhaka solution in 83% of patients but not fever. Seroconversion against V. mimicus was demonstrated in four patients, from whom acute- and convalescent-phase sera were obtained. Those sera did not show cross-reaction when tested against Vibrio cholerae O1 strain VC-12. All the V. mimicus isolates from these cases produced cholera toxin (CT) and were susceptible to commonly used antibiotics. Attempts to isolate this bacterium from stool samples of 127 healthy persons were not successful. Consumption of raw turtle eggs was recalled by 11 of the 19 (58%) individuals interviewed. All but two V. mimicus diarrheal cases were sporadic. These two had a history of a common source of turtle (Lepidochelys olivacea) eggs for consumption, and V. mimicus was isolated from eggs from the same source (a local market). Among the strains, variations in the antimicrobial susceptibility pattern were observed. None of the strains recovered from market turtle eggs nor the four isolates from river water showed CT production. Further efforts to demonstrate the presence of CT-producing V. mimicus strains in turtle eggs were made. Successful results were obtained when nest eggs were tested. In this case, it was possible to isolate CT- and non-CT-producing strains, even from the same egg. For CT detection we used PCR, enzyme-linked immunosorbent assay (ELISA), and Y-1 cell assay, obtaining a 100% correlation between ELISA and PCR results. Primers Col-1 and Col-2, originally described as specific for the V. cholerae O1 ctxA gene, also amplified a 302-bp segment with an identical restriction map from V. mimicus. These results have important implications for epidemiological surveillance in tropical countries where turtle eggs are used for human consumption, serving as potential sources of cholera-like diarrhea.     

Evidence that the interaction between insulin-like growth factor (IGF)-II and IGF binding protein (IGFBP)-4 is essential for the action of the IGF-II-dependent IGFBP-4 protease

A variety of human cell types, including human osteoblasts (hOBs), produce an IGFBP-4 protease, which cleaves IGFBP-4 in the presence of IGF-II. Recently, the pregnancy-associated plasma protein (PAPP)-A has been determined to be the IGF-II-dependent IGFBP-4 protease produced by human fibroblasts. This study sought to define the mechanism by which IGF-II enhances IGFBP-4 proteolysis. Addition of PAPP-A antibody blocked the IGFBP-4 proteolytic activity in hOB conditioned medium (CM), suggesting that PAPP-A is the major IGFBP-4 protease in hOB CM. Pre-incubation of IGFBP-4 with IGF-II, followed by removal of unbound IGF-II, led to IGFBP-4 proteolysis without further requirement of the presence of IGF-II in the reaction. In contrast, prior incubation of the partially purified IGFBP-4 protease from either hOB CM or human pregnancy serum with IGF-II did not lead to IGFBP-4 proteolysis unless IGF-II was re-added to the assays. To further confirm that the interaction between IGF-II and IGFBP-4 is required for IGFBP-4 protease activity, we prepared IGFBP-4 mutants, which contained the intact cleavage site (Met135-Lys136) but lacked the IGF binding activity, by deleting the residues Leu72-His74 in the IGF binding domain or Cys183-Glu237 that contained an IGF binding enhancing motif. The IGFBP-4 protease was unable to cleave these IGFBP-4 mutants, regardless of whether or not IGF-II was present in the assay. Conversely, an IGFBP-4 mutant with His74 replaced by an Ala, which exhibited normal IGF binding activity, was effectively cleaved in the presence of IGF-II. Taken together, these findings provided strong evidence that the interaction between IGF-II and IGFBP-4, rather than the direct interaction between IGF-II and IGFBP-4 protease, is required for optimal IGFBP-4 proteolysis.     

Sequences of the vesicular stomatitis virus matrix protein involved in binding to nucleocapsids.

The purpose of these experiments was to study the physical structure of the nucleocapsid-M protein complex of vesicular stomatitis virus by analysis of nucleocapsid binding by wild-type and mutant M proteins and by limited proteolysis. We used the temperature-sensitive M protein mutant tsO23 and six temperature-stable revertants of tsO23 to test the effect of sequence changes on M protein binding to the nucleocapsid as a function of NaCl concentration. The results showed that M proteins from wild-type, mutant, and three of the revertant viruses had similar NaCl titration curves, while the curve for M proteins from the other three revertants differed significantly. The altered NaCl dependence of M protein was correlated with a single amino acid substitution from Phe to Leu at position 111 compared with the original temperature-sensitive mutant and was not correlated with a substitution of Gly to Glu at position 21 in tsO23 and the revertants. To determine whether protease cleavage sites in the M protein were protected by interaction with the nucleocapsid, nucleocapsid-M protein complexes were subjected to limited proteolysis with trypsin, chymotrypsin, or Staphylococcus aureus V8 protease. The initial trypsin and chymotrypsin cleavage sites, located after amino acids 19 and 20, respectively, were as accessible to proteases when M protein was bound to the nucleocapsid as when it was purified, indicating that this region of the protein does not interact directly with the nucleocapsid. Furthermore, trypsin or chymotrypsin treatment released the M protein fragments from the nucleocapsid, presumably due to conformational changes following proteolysis. V8 protease cleaved the M protein at position 34 or 50, producing two distinct fragments. The M protein fragment produced by V8 protease cleavage at position 34 remained associated with the nucleocapsid, while the fragment produced by cleavage at position 50 was released from the nucleocapsid. These results suggest that the amino-terminal region of the M protein around amino acid 20 does not interact directly with the nucleocapsid and that conformational changes resulting from single-amino-acid substitutions at other sites in the M protein are important for this interaction.     

三种免疫制剂对真鲷弧菌病的免疫保护性

菌体疫苗按不同的方式对真鲷进行免疫2周后,对实验鱼均具有免疫保护性,免疫保护性最好的免疫组,免疫保护率在初次免疫后高达60%,强化免疫后免疫保护率可提高到80%;粗制LPS经去毒处理后初次免疫真鲷,不同浓度的LPS对实验鱼具有不同程度的免疫保护性,强化免疫后,免疫保护率均有明显的提高,浓度越高,免疫保护性越强,对真鲷的免疫保护率最高可达90%,最小弧菌产生的外毒素经福尔马林灭活后制成毒素苗,这种毒素苗能产生较好的免疫保护性,其免疫保护率可达80%,这表明外毒素不仅是最小弧菌产生的毒力因子,同时也是菌体产生的有效保护性抗原。     

Presence of LuxS/AI-2 based quorum-sensing system in Vibrio mimicus : luxO controls protease activity

Presence of the quorum-sensing regulation system in Vibrio mimicus was investigated. The culture supernatants of V. mimicus strains were found to possess AI-2 autoinducer like activity, and the strains were found to harbor the genes which are homologous to luxS, luxO, and luxR of V. harveyi. These genes of V. harveyi have been shown to be important components of V. harveyi-like quorum-sensing system. The luxO gene homologue known to encode LuxO, the central component of the regulation system, was disrupted, and effects on protease and hemolysin activity were studied. Disruption of luxO gene resulted in the increased protease activity, but the hemolysin activity did not vary considerably.     

The ability of two different Vibrio spp. bacteriophages to infect Vibrio harveyi, Vibrio cholerae and Vibrio mimicus

AIMS: To determine the host range of the Vibrio harveyi myovirus-like bacteriophage (VHML) and the cholera toxin conversion bacteriophage (CTX Phi) within a range of Vibrio cholerae and V. mimicus and V. harveyi, V. cholerae and V. mimicus isolates respectively. METHODS AND RESULTS: Three V. harveyi, eight V. cholerae and five V. mimicus isolates were incubated with VHML and CTX Phi. Polymerase chain reaction (PCR) was used to determine the presence of VHML and CTX Phi in infected isolates. We demonstrated that it was possible to infect one isolate of V. cholerae (isolate ACM #2773/ATCC #14035) with VHML. This isolate successfully incorporated VHML into its genome as evident by positive PCR amplification of the sequence coding part of the tail sheath of VHML. Attempts to infect all other V. cholerae and V. mimicus isolates with VHML were unsuccessful. Attempts to infect V. cholerae non-01, V. harveyi and V. mimicus isolates with CTX Phi were unsuccessful. CONCLUSIONS: Bacteriophage infection is limited by bacteriophage-exclusion systems operating within bacterial strains and these systems appear to be highly selective. One system may allow the co-existence of one bacteriophage while excluding another. VHML appears to have a narrow host range which may be related to a common receptor protein in such strains. The lack of the vibrio pathogenicity island bacteriophage (VPI Phi) in the isolates used in this study may explain why infections with CTX Phi were unsuccessful. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study has demonstrated that Vibrio spp. bacteriophages may infect other Vibrio spp.     

The latent form of macropain (high molecular weight multicatalytic protease) restores ATP-dependent proteolysis to soluble extracts of BHK fibroblasts pretreated with anti-macropain antibodies

Specific immunoadsorption of the high molecular weight multicatalytic protease, macropain, from postmicrosomal extracts of BHK fibroblasts inhibited ATP-dependent proteolysis of exogenous protein substrates. The immunoprecipitated macropain represented the latent (L) form of the protease because it had low protease activity but was activated by methods that activate purified macropain L. Reconstitution of the antibody-treated extracts with purified macropain L, but not macropain A, from bovine heart or human erythrocytes, completely restored ATP-dependent proteolysis, even though ATP did not directly activate either purified macropain L or the immunoprecipitated protease. Reconstituted ATP-dependent proteolysis was saturable with respect to added macropain and never exceeded the level of proteolysis present in the original extract. These results indicate that macropain L plays a key role in ATP-dependent proteolysis but suggest that the protease may require interaction with or modification by another cellular component to demonstrate this effect.     

黄颡鱼拟态弧菌的鉴定、毒力相关因子及药敏特性

2018年浙江省黄颡鱼主养区暴发了严重的溃疡病, 为查明该病的病原特点, 了解病原的致病因子及药物敏感性, 对溃疡黄颡鱼(Pelteobagrus fulvidraco)上分离的多株优势菌, 分别采用生理生化特性和管家基因16S rRNA、pyrH的系统进化树进行分类鉴定, 通过人工回归感染试验分析菌株的病原性, 利用平板法测定相关的毒力因子并采用PCR法对毒力相关基因进行检测, 应用微量二倍稀释法测定14种抗菌药物的最小抑菌浓度。结果表明, 11株优势菌皆为拟态弧菌, 2株代表菌腹腔注射感染黄颡鱼后均可引发溃疡症状; 11株拟态弧菌均具有溶血活性、蛋白酶、明胶酶和DNA酶活性, 不具有卵磷脂酶活性, 可分泌产生铁载体; 所有菌株均携带vmh、ompU、toxR及matCDB-iucABCD-iutA等毒力相关基因, tcpA、ctxA、st、zot、ace、tdh等毒力基因均未检出。各菌株对氨苄青霉素、磺胺嘧啶、磺胺甲噁唑、磺胺间甲氧嘧啶高度耐药, 对环丙沙星、恩诺沙星、氟苯尼考、多西环素、土霉素、甲砜霉素、红霉素、交沙霉素、磺胺二甲氧嘧啶较敏感, 个别菌株对新霉素和甲砜霉素耐药。以上研究结果表明, 拟态弧菌感染可引起黄颡鱼溃疡病, 不同发病养殖场的分离株具有相同的表型特征及毒力基因型, 并具有较一致的生理生化特性和耐药谱型, 说明它们具有相同的遗传背景或传染源。     

Scrapie prion rod formation in vitro requires both detergent extraction and limited proteolysis.

Scrapie prion infectivity can be enriched from hamster brain homogenates by using limited proteolysis and detergent extraction. Purified fractions contain both scrapie infectivity and the protein PrP 27-30, which is aggregated in the form of prion rods. During purification, PrP 27-30 is produced from a larger membrane protein, PrPSc, by limited proteolysis with proteinase K. Brain homogenates from scrapie-infected hamsters do not contain prion rods prior to exposure to detergents and proteases. To determine whether both detergent extraction and limited proteolysis are required for the formation of prion rods, microsomal membranes were prepared from infected brains in the presence of protease inhibitors. The isolated membranes were then detergent extracted as well as protease digested to evaluate the effects of these treatments on the formation of prion rods. Neither detergent (2% Sarkosyl) extraction nor limited proteinase K digestion of scrapie microsomes produced recognizable prion amyloid rods. Only after combining detergent extraction with limited proteolysis were numerous prion rods observed. Rod formation was influenced by the protease concentration, the specificity of the protease, and the duration of digestion. Rod formation also depended upon the detergent; some combinations of protease and detergent did not produce prion amyloid rods. Similar results were obtained with purified PrPSc fractions prepared by repeated detergent extractions in the presence of protease inhibitors. These fractions contained amorphous structures but not rods; however, prion rods were produced upon conversion of PrPSc to PrP 27-30 by limited proteolysis. We conclude that the formation of prion amyloid rods in vitro requires both detergent extraction and limited proteolysis. In vivo, amyloid filaments found in the brains of animals with scrapie resemble prion rods in their width and their labeling with prion protein (PrP) antisera; however, filaments are typically longer than rods. Whether limited proteolysis and some process equivalent to detergent extraction are required for amyloid filament formation in vivo remains to be established.     

烷烃溶剂对耐有机溶剂极端微生物地衣芽孢杆菌YP1产胞外蛋白酶的影响

考察了添加5%(V/V)浓度的正庚烷、正辛烷、正癸烷、十二烷、十四烷、十六烷等烷烃溶剂对耐有机溶剂极端微生物地衣芽孢杆菌(Bacillus licheniformis)YP1的生长及产胞外蛋白酶的影响.结果表明5%(V/V)浓度的各种烷烃溶剂对YP1蛋白酶的稳定性及菌体生物量均无显著影响,正庚烷、正辛烷、正癸烷等溶剂显著抑制YP1产蛋白酶,而十二烷、十四烷,十六烷能提高YP1产蛋白酶1倍以上.发酵液中十四烷的浓度(1%-8%,VIV)与蛋白酶的活力呈正相关性,添加十四烷后发酵过程中蛋白酶活力的显著增加出现在菌体生长的对数后期.培养过程中添加十四烷能导致YP1菌体形态显著变小.首次报道了烷烃溶剂对极端微生物产蛋白酶的影响.     

Elucidation of cathepsin B-like activity associated with extracts of human myelin basic protein

Myelin basic protein (MBP) extracted from human delipidated white matter was found to be degraded at pH 3.0 by endogenous proteolytic activities of extracts. Electrophoretic peptide patterns were consistent with limited proteolysis of MBP. Based on pH, activation by EDTA and DTE, and inhibition by p-CMPS, E-64 and, in particular, by leupeptin, the protease involved was tentatively identified as cathepsin B or a cathepsin B-like enzyme. As pepstatin failed to inhibit acid proteolysis of MBP cathepsin D was ruled out.     

Genome sequencing reveals unique mutations in characteristic metabolic pathways and the transfer of virulence genes between V. mimicus and V. cholerae

Vibrio mimicus, the species most similar to V. cholerae, is a microbe present in the natural environmental and sometimes causes diarrhea and internal infections in humans. It shows similar phenotypes to V. cholerae but differs in some biochemical characteristics. The molecular mechanisms underlying the differences in biochemical metabolism between V. mimicus and V. cholerae are currently unclear. Several V. mimicus isolates have been found that carry cholera toxin genes (ctxAB) and cause cholera-like diarrhea in humans. Here, the genome of the V. mimicus isolate SX-4, which carries an intact CTX element, was sequenced and annotated. Analysis of its genome, together with those of other Vibrio species, revealed extensive differences within the Vibrionaceae. Common mutations in gene clusters involved in three biochemical metabolism pathways that are used for discrimination between V. mimicus and V. cholerae were found in V. mimicus strains. We also constructed detailed genomic structures and evolution maps for the general types of genomic drift associated with pathogenic characters in polysaccharides, CTX elements and toxin co-regulated pilus (TCP) gene clusters. Overall, the whole-genome sequencing of the V. mimicus strain carrying the cholera toxin gene provides detailed information for understanding genomic differences among Vibrio spp. V. mimicus has a large number of diverse gene and nucleotide differences from its nearest neighbor, V. cholerae. The observed mutations in the characteristic metabolism pathways may indicate different adaptations to different niches for these species and may be caused by ancient events in evolution before the divergence of V. cholerae and V. mimicus. Horizontal transfers of virulence-related genes from an uncommon clone of V. cholerae, rather than the seventh pandemic strains, have generated the pathogenic V. mimicus strain carrying cholera toxin genes.     

Probing the yeast 5 S RNA-protein complex by fluorescence and controlled proteolytic digestion

The nature of the interaction between the RNA and the protein component in the yeast 5 S rRNA-L1a complex was assessed using fluorescence and controlled proteolytic and RNase digestion. (a) Influence of L1a on the RNA conformation was monitored by ethidium fluorescence and controlled RNase T1 digestion. The complex was digested with alpha-chymotrypsin, Staphylococcus aureus protease V8, subtilisin, or trypsin. Both termini of L1a in the complex were readily accessible to proteases. Proteolytic digestion of the complex resulted in a reduction in fluorescence intensity if ethidium was added after proteolysis. No change was observed when ethidium was allowed to react with the complex prior to proteolysis. Neither the rate of proteolysis nor the resultant peptide pattern was affected by the presence of ethidium. T1 digestion of intact RNP and trypsin-treated RNP produced different oligonucleotide patterns. Both the fluorescence and the T1 digestion data suggest that the conformation of the RNA moiety was influenced by the protein. (b) Influence of the RNA molecule on L1a conformation in the complex was monitored by limited proteolysis. Whereas the protein in the complex was relatively sensitive to proteases, free protein was completely resistant to digestion under identical conditions. The trypsin sensitivity of L1a in complexes containing different truncated 5 S RNA molecules was studied also. Upon removal of residues 31-49 of the 5 S RNA molecule, L1a in the complex became resistant to proteolysis. These results are interpreted in a model in which specific regions of both the RNA and the protein are involved in the interaction.     

Relationship Among Calmodulin-, Forskolin-, and Guanine Nucleotide-Dependent Adenylate Cyclase Activities in Cerebellar Membranes: Studies by Limited Proteolysis

Adenylate cyclase activity in bovine cerebellar membranes is regulated by calmodulin, forskolin, and both stimulatory (Ns) and inhibitory (Ni) guanine nucleotide-binding components. The susceptibility of the enzyme to chymotrypsin proteolysis was used as a probe of structure-function relationships for these different regulatory pathways. Pretreatment of membranes with low concentrations of chymotrypsin (1-2 micrograms/ml) caused a three- to fourfold increase in basal adenylate cyclase activity and abolished the Ca2+-dependent activation of the enzyme by calmodulin. In contrast, the stimulation of the enzyme by GTP plus isoproterenol was strongly potentiated after protease treatment, an effect that mimics the synergistic activation of adenylate cyclase by Ns and calmodulin in unproteolyzed membranes. Limited proteolysis revealed low- and high-affinity components in the activation of adenylate cyclase by forskolin. The low-affinity component was readily lost on proteolysis, together with calmodulin stimulation of the enzyme. The activation via the high-affinity component was resistant to proteolysis and nonadditive with the Ns-mediated activation of the enzyme, suggesting that both effectors utilize a common pathway. The inhibitory effect of low concentrations (10(-7) M) of guanyl-5'-yl imidodiphosphate [Gpp(NH)p] on forskolin-activated adenylate cyclase was retained after limited proteolysis of the membranes, indicating that the proteolytic activation does not result from an impairment of the Ni subunit. Moreover, in the rat cerebellum, proteolysis as well as calmodulin was found to enhance strongly the inhibitory effect of Gpp(NH)p on basal adenylate cyclase activity. Our results suggest that calmodulin and Ns/Ni interact with two structurally distinct but allosterically linked domains of the enzyme. Both domains appear to be involved in the mode of action of forskolin.