Kallikrein-like prorenin-converting enzymes in inbred hypertensive mice.

Kallikreins are a group of serine proteases and are distinguished by having serine residue at their active site. Their general function is to convert inactive pro-peptide into its biologically active form. In recent years, emerging evidence indicates that some kallikrein-kinin enzymes also play a role in the modulation of renin-angiotensin system. These kallikrein-like prorenin converting enzymes act on renin-angiotensin by converting prorenin into biologically active renin. In this investigation, kallikrein-like prorenin converting enzyme (PRCE C) (mK9) is isolated from genetically inbred high blood pressure (BPH) and their normal counterparts (BPN) mice, and its protein levels are quantitated. Levels of mRNA expression are also compared. Additionally, localization of the enzyme is visualized by in situ hybridization histochemistry. Results indicated higher levels of PRCE C (mK9) enzyme in BPH mice in comparison to their normal counterparts. mRNA expression was also higher in BPH mice. In situ hybridization histochemistry results localized PRCE C (mK9) in the striated duct cells of submandibular gland.

A cost-effective plasmid purification protocol suitable for fluorescent automated DNA sequencing.

A cost-effective, reliable, and reproducible method has been developed to produce good-quality, double-stranded plasmid DNA for automated sequence analysis. The method incorporates modifications to a previously described plasmid-purification protocol used in manual sequencing. The quality of the DNA produced from the present protocol is suitable for automated fluorescent sequencing. Using a dye-terminator sequencing protocol, most runs using plasmid DNA prepared using this protocol produced over 700 bases with greater than 99% base-calling accuracy.

Cleavage specificities of individual members of kallikrein family of proteins on synthetic peptide containing the bradykinin sequence.

We have analyzed the effect on bond specificity of various isolated members of the mouse kallikrein family of proteins on a synthetic peptide containing the bradykinin sequence. The cleavage pattern shows the selected specificity of these proteases toward the synthetic peptide. The Phe-His bond (positions 11-12) in the synthetic peptide was favorably cleaved by most of the members in this family, including gamma nerve growth factor. On the other hand, the Lys-Arg bond (position 3-4) was found to be susceptible only to gamma-NGF. The combination of these cleavages could result in the degradation of bradykinin in vivo.

Analysis of heparin-binding sites in human lipoprotein lipase using synthetic peptides.

Synthetic nonbasic peptides based on the type I repeats of thrombospondin (TSP) and four peptides corresponding to the predicted basic clusters in lipoprotein lipase (LPL) have been analyzed for heparin binding. In the present report we examine the structural requirement for the binding of these peptides to heparin-Sepharose column. The peptide containing the sequence Phe-Ser-Trp-Ser-Asp-Trp-Trp-Ser (residues 388-395 in lipoprotein lipase, which include the consensus TSP type I sequence) showed strong binding to heparin. Both the first and second Trp residues in this sequence were essential for tight heparin binding. Substitution of either of the Trp residues by an Ala resulted in the complete loss of heparin binding. The peptides representing the four basic cluster regions of lipoprotein lipase showed variable heparin binding. Strong retention was observed for peptides representing cluster 1 (residues 261-287) and cluster 3 (residues 147-151) peptides followed by cluster 2 (residues 290-302) peptide. A peptide corresponding to LPL cluster 4 (residues 405-414) did not show binding to heparin column. The present study confirms the presence of specific heparin-binding sites in LPL. Furthermore, this study also demonstrates the potential use of synthetic peptides to investigate the interaction between peptides and heparin as an alternative approach to site-directed mutagenesis in selected regions of large protein molecules. The affinity of these peptides toward heparin can be explored to block molecular interactions at these specific sites or to carry and deliver other coupled molecules at the site(s) of attachment of these peptides for therapeutic applications.

An efficient cost-effective protocol for automated fluorescent-DNA sequencing.

A simple and cost-effective method is described to sequence double-stranded DNA (< 100 ng) using a very small amount of fluorescent sequencing mix (one-tenth of manufacturer-recommended amount). The modification has significant advantages over conventional protocols and can be used to sequence, using a single sequencing kit, at least ten times the number of samples. This modified method also removes the post-reaction cleaning protocol and can be used for direct loading of samples on gels after completion of sequencing reactions. The accuracy of DNA sequencing data from analysis of 700 to 750 nucleotides is greater than 98.5%.

Homozygosity for two point mutations in the lipoprotein lipase (LPL) gene in a patient with familial LPL deficiency: LPL(Asp9-->Asn, Tyr262-->His).

Familial lipoprotein lipase (LPL) deficiency is an inherited disorder of lipoprotein metabolism characterized by hypertriglyceridemia and recurrent episodes of abdominal pain and pancreatitis. We have studied the genetic basis of LPL deficiency in a 62-year-old black male with undetectable pre- and post-heparin plasma LPL mass and activity, DNA sequence analysis of the patient's LPL cDNA and gene as well as digestion with Bcl I and Asu I revealed that the proband is a homozygote for two separate gene defects. One mutation changed a G to an A, resulting in the conversion of amino acid 9 of the mature protein, aspartic acid (GAC), to asparagine (AAC). The second substitution, a C for a T, replaced tyrosine (TAC) at residue 262 with histidine (CAC). Northern blot analysis of monocyte-derived macrophage RNA demonstrated the presence of LPL mRNA of approximately normal size and quantity when compared to control. Expression of both mutations separately (pCMV-9 and pCMV-262) or in combination (pCMV-9+262) in human embryonal kidney-293 cells demonstrated that LPL-9 had approximately 80% the specific activity of wild type LPL, but LPL-262 and LPL-9+262 had no enzymic activity, thus establishing the functional significance of the LPL-262 defect. Despite an absolute deficiency of LPL mass and activity demonstrated by analysis of patient post-heparin plasma, in vitro expression of both LPL mutants was normal, suggesting that the absence of LPL in patient post-heparin plasma was a result of altered in vivo processing. Analysis of the heparin binding properties of the mutant enzymes by heparin-Sepharose affinity chromatography indicated that most of the LPL-262 mass was present in an inactive peak, which like the normal LPL monomer, eluted at 0.8 M NaCl. Thus, the Tyr262 --> His mutation may alter the stability of the LPL dimer, leading to the formation of inactive LPL-262 monomer which exhibits reduced heparin affinity. Based on these results, we propose that, in vivo, enhanced formation of LPL-9+262 monomer leads to abnormal binding of the mutant lipase to endothelial glycosaminoglycans ultimately resulting in enhanced catabolism of the mutant enzyme and lower enzyme mass in post-heparin plasma.

Specific cleavage of synthetic renin substrate by mouse gamma-nerve growth factor.

Results of the present investigation indicate that mouse gamma-nerve growth factor (gamma-NGF), which belongs to the kallikrein family of proteins, specifically cleaves the Phe-His bond of a synthetic renin substrate and exhibits rat-tonin-like activity. Since other mouse kallikreins do not cleave this bond, gamma-NGF may play a regulatory role in the generation of antiogensin-II.

Isolation and characterization of a protein corresponding to mKlk-11 clone from male mouse submandibular gland.

A protein corresponding to the predicted genomic sequence of clone mKlk-11 has been characterized from mouse submandibular gland. This protein was purified by reverse-phase high-performance liquid chromatography from the submandibular glands of normal and hypertensive mice. The protein was not detected in the submandibular gland of mice selected for low blood pressure. It consists of three fragments starting at the residues 1, 98, and 141 of the predicted sequence of clone mKlk-11. The cleavage of beta-lactoglobulin (between residues 20 and 21, Tyr-Ser, and 40 and 41, Arg-Val) and a synthetic renin substrate tetradecapeptide (residues 4 and 5, Tyr-Ile) by the protein corresponding to clone mKlk-11 showed both tryptic- and chymotryptic-type cleavages. The possibility of this protein's involvement in the regulation of local blood flow is raised.

The complete primary structure of late lactation protein from quokka (Setonix brachyurus).

The complete primary structure of the late lactation protein from the milk of quokka (Setonix brachyurus) is presented. The amino acid sequence was established by N-terminal sequence analysis of high-performance liquid chromatography purified intact protein and peptides isolated from chemical and enzymatic digests of the protein. The protein contains 158 residues including four cysteines. The sequence comparison with the tamar wallaby (Macropus eugenii) late lactation protein shows only five differences. The protein is identified as a new member of a novel late lactation protein family present in the milk of marsupials.

Human lipoprotein lipase. Analysis of the catalytic triad by site-directed mutagenesis of Ser-132, Asp-156, and His-241.

Lipoprotein lipase (LPL) plays a central role in normal lipid metabolism as the key enzyme involved in the hydrolysis of triglycerides present in chylomicrons and very low density lipoproteins. LPL is a member of a family of hydrolytic enzymes that include hepatic lipase and pancreatic lipase. Based on primary sequence homology of LPL to pancreatic lipase, Ser-132, Asp-156, and His-241 have been proposed to be part of a domain required for normal enzymic activity. We have analyzed the role of these potential catalytic residues by site-directed mutagenesis and expression of the mutant LPL in human embryonic kidney-293 cells. Substitution of Ser-132, Asp-156, and His-241 by several different residues resulted in the expression of an enzyme that lacked both triolein and tributyrin esterase activities. Mutation of other conserved residues, including Ser-97, Ser-307, Asp-78, Asp-371, Asp-440, His-93, and His-439 resulted in the expression of active enzymes. Despite their effect on LPL activity, substitutions of Ser-132, Asp-156, and His-241 did not change either the heparin affinity or lipid binding properties of the mutant LPL. In summary, mutation of Ser-132, Asp-156, and His-241 specifically abolishes total hydrolytic activity without disrupting other important functional domains of LPL. These combined results strongly support the conclusion that Ser-132, Asp-156, and His-241 form the catalytic triad of LPL and are essential for LPL hydrolytic activity.

Identification of two separate allelic mutations in the lipoprotein lipase gene of a patient with the familial hyperchylomicronemia syndrome.

The molecular defects resulting in a deficiency of lipoprotein lipase activity in a patient with the familial hyperchylomicronemia syndrome have been identified. Increased lipoprotein lipase mass but undetectable lipoprotein lipase activity in the patient's post-heparin plasma indicate the presence of an inactive enzyme. No major gene rearrangements were identified by Southern blot analysis of the patient's lipoprotein lipase gene and Northern blot hybridization revealed an lipoprotein lipase mRNA of normal size. Sequence analysis of polymerase chain reaction-amplified lipoprotein lipase cDNA identified two separate allelic mutations. A T to C transition at nucleotide 836 results in the substitution of Ile194, located near the putative interfacial recognition site of lipoprotein lipase, to a Thr. A G to A mutation at base 983 leads to the substitution of a His for Arg243 and the loss of a HhaI restriction enzyme site. Arg243 is near His241, which has been postulated to be part of the catalytic triad of lipoprotein lipase. Direct sequencing of amplified cDNA and digestion with HhaI established that the proband is a compound heterozygote for each base substitution. Transient expression of each of the mutant lipoprotein lipase cDNAs in human embryonal kidney-293 cells resulted in the synthesis of enzymically inactive proteins, establishing the functional significance of the mutations. We conclude that the Ile194 to Thr194 and Arg243 to His243 substitutions occur in lipoprotein lipase regions essential for normal enzyme activity and each mutation results in the expression of a nonfunctional enzyme leading to the hyperchylomicronemia syndrome manifested in the proband.

Primary structure and functional properties of cobra (Naja naja naja) venom Kunitz-type trypsin inhibitor.

A trypsin inhibitor from the venom of the cobra Naja naja naja has been isolated by a single step of reverse-phase high-performance liquid chromatography. The protein strongly inhibits trypsin (Ki = 3.5 pM). The primary structure was determined by peptide analysis of the [14C]carboxymethylated inhibitor. The 57-residue polypeptide chain belongs to the family of Kunitz-type inhibitors, and exhibits 42% residue identity with bovine pancreatic trypsin inhibitor. The structure shows only 70% identity with the corresponding peptide from the Capa cobra (Naja nevia), establishing that the inhibitor molecule exhibits extensive variations. Functionally, a basic residue at position P3' correlates with strong inhibition.

Lipoprotein lipaseBethesda: a single amino acid substitution (Ala-176----Thr) leads to abnormal heparin binding and loss of enzymic activity.

The molecular defect that leads to a deficiency of lipoprotein lipase (LPL) activity in the proband from a Bethesda kindred has been identified. The pre- and post-heparin plasma LPL mass in the proband was elevated when compared to controls; however, there was no detectable LPL activity, indicating the presence of a defective enzyme (termed LPLBethesda). Analysis of the patient's post-heparin plasma by heparin-Sepharose affinity chromatography demonstrated that the mutant LPL had an altered affinity for heparin. Southern blot hybridization of the gene for LPLBethesda revealed no major rearrangements. Northern blot analysis of LPLBethesda mRNA from patient monocyte-derived macrophages revealed normal-sized mRNAs (3.4 and 3.7 kilobases) as well as normal cellular mRNA levels when compared to control macrophages. Sequence analysis of polymerase chain reaction-amplified LPL cDNA revealed a G----A substitution at position 781 of the normal LPL gene that resulted in the substitution of an alanine for a threonine at residue 176 and the loss of an SfaNI site present in the normal LPL gene. Amplification of cDNA by the PCR followed by digestion with SfaNI established that the patient was a true homozygote for the mutation. Expression of LPL cDNA in COS-7 cells resulted in the synthesis of a nonfunctional LPL enzyme establishing that the Ala----Thr substitution was the mutation responsible for the inactive LPL. The identification of this mutation in the LPL gene defines a region of the LPL enzyme, at Ala-176, that is essential for normal heparin-binding and catalytic activity. We propose that an amino acid substitution in this critical region of LPLBethesda results in the synthesis of a nonfunctional enzyme that leads to the chylomicronemia syndrome expressed in this proband.

Phospholipase A2 from cobra (Naja naja naja) venom. Primary structure and subspecies variation.

The primary structure of phospholipase A2 of the major race of Indian cobra has been determined. Together with previous data on other subforms, it establishes subspecies variations at no less than 20 of the 119 positions in the protein. These variations are large, not only in number but in several cases also regarding properties of the residues involved. Nevertheless, all structures are compatible with largely unaltered enzyme properties.

Characterization of two different peptides from the venom of the scorpion Buthus sindicus.

Two disulfide-rich, low-molecular mass peptides (approximately 3 kDa and approximately 4 kDa) have been isolated from Buthus sindicus venom using ion-exchange and reverse-phase HPLC. Peptide I has 35 residues with 8 half-cystine residues and is clearly related to four-disulfide core proteins of the neurophysin type and to toxins of other scorpion species (55-63% residue identity). Peptide II, present in low yield, has 28 residues with 6 half-cystine residues and a structure largely dissimilar from that of peptide I and other characterized toxins, although probably still a member of the disulfide core peptide type. Consequently, scorpion venom contains, in addition to toxins characterized before, toxin-like compounds with distant relationships.

Studies of rabbit serum transferrin.

Rabbit serum transferrin is isolated by a procedure designed to preserve its conformation and disulfide linkages. A progress report is presented on the determination of its amino acid sequence, as part of studies on its primary, secondary and tertiary structure. The sequence of 378 residues, of the approximately 680 residues in the molecule, are determined. Observations are made on the site of carbohydrate attachment, iron binding sites and half-cystine residue location. The results are discussed in relation to the X-ray crystallographic studies of human lactoferrin (lactotransferrin) and of rabbit serum transferrin being made in other laboratories.

Primary structure of the hemoglobin alpha-chain of rose-ringed parakeet (Psittacula krameri).

The structure of the hemoglobin alpha-chain of Rose-ringed Parakeet was determined by sequence degradations of the intact subunit, the CNBr fragments, and peptides obtained by digestion with staphylococcal Glu-specific protease and trypsin. Using this analysis, the complete alpha-chain structure of 21 avian species is known, permitting comparisons of the protein structure and of avian relationships. The structure exhibits differences from previously established avian alpha-chains at a total of 61 positions, five of which have residues unique to those of the parakeet (Ser-12, Gly-65, Ser-67, Ala-121, and Leu-134). The analysis defines hemoglobin variation within an additional avian order (Psittaciformes), demonstrates distant patterns for evaluation of relationships within other avian orders, and lends support to taxonomic conclusions from molecular data.

Characterization of a heterogeneous camel milk whey non-casein protein.

A milk protein, occurring in the whey fraction, has been characterized from camel milk. Determination of the primary structure reveals the existence of two related types of chain with residue differences in at least the N-terminal region. A fragment representing an N-terminal part of the protein was also recovered (heterogeneous at the same positions). The absence of cysteine residues in the protein shows that no disulphide bridges are present. The pattern of fragments and a parent protein resembles that for casein and its fragments, showing that fragments and a multiplicity of forms may be typical for different milk proteins.

A camel milk whey protein rich in half-cystine. Primary structure, assessment of variations, internal repeat patterns, and relationships with neurophysin and other active polypeptides.

The amino acid sequence of a recently isolated camel milk protein rich in half-cystine has been determined by peptide analyses. The 117-residue protein has 16 half-cystine residues, concluded to correspond to disulfide bridges and suggesting a tight conformation of the molecule. Comparisons of the structure with those of other proteins reveal several interesting relationships. The camel protein is clearly homologous with a previously reported rat whey phosphoprotein of possible importance for mammary gland growth regulation, and with a mouse protein of probable relationship to neurophysins. The camel, rat and mouse proteins may represent species variants from a rapidly evolving gene. Residue identities in pairwise comparisons are 40% for the camel/rat proteins and 33% for the camel/mouse proteins, with 38 positions conserved in all three forms. The camel protein also reveals an internal repeat pattern similar to that for the other two proteins. The homology between the three milk whey proteins has wide implications for further relationships. Thus, previously noticed similarities, involving either of the milk proteins, include limited similarities to casein phosphorylation sites for the camel protein, to neurophysins in repeat and half-cystine patterns for the mouse and rat proteins, and to an antiprotease for the rat protein. These similarities are reinforced by the camel protein structure and the recognition of the three whey proteins as related. Finally a few superficial similarities with the insulin family of peptides and with some other peptides of biological importance are noticed. Combined, the results relate the camel protein in a family of whey proteins, and extend suggestions of relationships with some binding proteins.