6 IntroductionAlkaline Phosphatase(ALP) is an important enzyme exist in all human tissues as well as in bacterial tissues.Elevation or decrease in ALP levels produce the diseased condition.It also plays an important role in research.Reverse Transcriptase is a trademark of the Retrovirus family.RT due to its unique property is very powerful enzyme for viruses as well as in genetic research.
7 Alkaline PhosphataseAlkaline phosphatase (ALP) is a hydrolase enzyme responsible for dephosphorylation means from many types of molecules, including nucleotides, proteins, and alkaloids.As the name suggests, alkaline phosphatases are most effective in an alkaline environment.
8 Bacterial Alkaline Phosphatase Alkaline phosphatase located in bacterial periplasmic space is resistant to inactivation, denaturation, and degradation, and also has a higher rate of activity.Alkaline phosphatase is produced by the bacteria only during phosphate starvation and not when phosphate is plentiful suggests, it generate free phosphate groups for uptake and use.It also suggets that presence of phosphate groups usually prevents organic molecules from passing through the bacterial membrane, therefore dephosphorylating them may be important for bacterial uptake of organic compounds in the wild.In the laboratory, however, mutant Escherichia coli lacking alkaline phosphatase survive quite well than the mutants unable to shut off alkaline phosphatase production.
9 Human Alkaline Phosphatase In humans, alkaline phosphatase is present in all tissues throughout the entire body, but is particularly concentrated in liver, bile duct, kidney, bone, and the placenta.The optimal pH for the enzyme activity is pH=10.
10 Diagnostic Use of Human ALP Blood plasma (serum) levels of ALP are typically units per liter in normal adults.Elevated levels (hyperphosphatasemia) cause :(Liver ALP): Cholestasis, cholecystitis, cholangitis, cirrhosis, hepatitis, fatty liver, liver tumor, liver metastases, drug intoxication etc.(Bone ALP): Paget's disease, osteosarcoma, bone metastases of prostatic cancer, Other bone metastases, Fractured bone, Multiple myeloma etc.(Skeletal diseases): Osteomalacia, rickets, vitamin D deficiency, Malignant tumors, Renal disease etc.
11 HypophosphatasemiaLowered levels of ALP are less common than elevated levels.Hypophosphatasemia causes:Postmenopausal women – ostreoporosisMan with recent heart surgery – malnutrition, severe anemiaChildren – achondroplasia, cretinism
12 Industrial Use of ALPIt is used in the dairy industry as a marker of pasteurisation.ALP is denatured by elevated temperatures found during pasteurisation,which can be tested via colour change of a para-nitro-phenol phosphate substrate in a buffered solution.Raw milk would typically produce a yellow colouration within a couple of minutes, whereas properly pasteurised milk should show no change.There are of course exceptions to this in the case of heat stable alkaline phophatases produced by some bacteria.
13 Use of ALP In ResearchAlkaline phosphatase has become a useful tool in molecular biology laboratories.Problem arises when the cohesive ends of broken plasmids, instead of joining with foreign DNA, join the cohesive end of same DNA & get recircularized.To overcome this problem, the restricted plasmid is treated with alkaline phosphatase,that digests the terminal 5’ phosphoryl group.Removal of the phosphate groups allows radiolabeling in order to measure the presence of the labeled DNA through further steps in the process or experiment.For these purposes, the alkaline phosphatase from shrimp is the most useful, as it is the easiest to inactivate once it has done its job.Another important use of alkaline phosphatase is as a label for enzyme immunoassays.
14 List of ALPs Used In Research The most common alkaline phosphatases used in research are:Bacterial alkaline phosphatase (BAP), from Escherichia coli C4 cells.Shrimp alkaline phosphatase (SAP), from a species of arctic shrimp (Pandalus borealis).Calf intestine alkaline phosphatase (CIAP), from calf intestine.Placental alkaline phosphatase (PLAP) and its C terminally truncated version that lacks the last 24 amino acids (constituting the transmembrane domain) - the secreted alkaline phosphatase (SEAP).
15 Reverse Transcriptase Reverse transcriptase (RT) was discovered by Howard Temin and independently by David Baltimore in The two shared the 1975 ‘Nobel Prize in Physiology or Medicine’ with Renato Dulbecco for their discovery.Reverse transcriptase is a common name for an enzyme that functions as a RNA-dependent DNA polymerase.They are encoded by retroviruses, where they copy the viral RNA genome into DNA prior to its integration into host cells.These enzymes transcribes single-stranded DNA from double-stranded DNA.This complementary strand of DNA integrates into host cell DNA, leads to cause disease.
16 Commonly Used Reverse Transcriptase HIV-1 reverse transcriptase from the human immunodeficiency virus type 1 (PDB 1HMV).M-MLV reverse transcriptase from the Moloney murine leukemia virus.AMV reverse transcriptase from the avian myeloblastosis virus.Telomerase reverse transcriptase that maintains the telomeres of eukaryotic chromosomes.
17 Process of Reverse Transcription Reverse transcription is the process of making a double stranded DNA molecule from a single stranded RNA template.
18 Structure of HIV-1 RTThe enzyme is a heterodimer consisting of a full 66kDa RT monomer complete with RNaseH domain, and a 51kDa RT monomer from which the RNaseH domain has been cleaved.The 66kDa monomer has identifiable 'finger', 'palm', and 'thumb' components.
19 Structure of HIV-1 RTThe interface of the 66kDa and 51kDa monomers provides a track from the polymerase active site to the RNaseH active site, and allow for the concomitant destruction of the RNA primer template strand directly after it has been copied.In a complex of RT with a double stranded DNA molecule, the DNA close to the polymerase active site is in the A-form, while the rest is in the normal B-form.The 51kDa monomer may also provide a binding site for the lysine-tRNA that primes initial DNA synthesis.
20 Applications of RTReverse transcriptase is commonly used in research to apply the PCR technique to RNA called ‘reverse transcription polymerase chain reaction’ (RT-PCR).The classical PCR technique can only be applied to DNA strands, but with the help of reverse transcriptase, RNA can be transcribed into DNA, thus making PCR analysis of RNA molecules possible.Reverse transcriptase is also used to create cDNA libraries from mRNA.The commercial availability of reverse transcriptase greatly improved knowledge in the area of molecular biology as, along with other enzymes, it allowed scientists to clone, sequence and characterize DNA.
21 What We Learn………….ALP is an enzyme which removes the phosphate group from DNA or RNA, hence used in genetics experiment.ALP present in outer membrane of bacteria supposed to helps to satisfy the phosphate need of bacteria.Reverse Transcriptase catalyzes the synthesis of DNA from RNA & insert it into host DNA.It is used for constructing cDNA libraries & can make possible the PCR analysis of RNAs.